Conditions: Leukemia, Myeloid, Acute Myelodysplastic Syndromes
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 114
Healthy volunteers: no

Brief summary:
This clinical trial studies potential genetic markers which might be used to predict which patients with acute myeloid leukemia or myelodysplastic syndromes respond to decitabine. This study will contribute to the efforts to find effective and less toxic therapies to provide durable remissions in a significant proportion of elderly AML patients.

Detailed description:

Arms:
arm 1: Patients receive decitabine IV over 1 hour on days 1-10 of a 28-day cycle. Treatment continues for 2 cycles. Patients then receive decitabine IV over 1 hour on days 1-10, 1-5, or 1-3 (depending on response). Treatment continues in the absence of disease progression or unacceptable toxicity.

Interventions: intervention 1: decitabine
Intervention descriptions: intervention 1: None

Protocol document:
Prot_SAP_000.pdf: 
Genomic Predictors of Decitabine Response in AML/MDS 
 
 
NCT# NCT01687400 
 
 
Document Version Date:  11/15/2016 
 
IRB Approval Date:  11/28/2016 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 7 Version date: 11/15/2016  
Page 1 of 52 
 
 
Genomic Predictors of Decitabine Response in AML/MDS 
 
Washington University School of Medicine, Division of Oncology 
660 South Euclid Avenue, Box 8056, St. Louis, MO 63110 
 
Protocol #: 201210102 
Protocol Version Date: 11/15/2016 
 
Coordinating Center: Washington University School of Medicine 
 
Principal Investigator 
John Welch, M.D./Ph.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-2626, Fax Number: (314) 362-9333 
Email: jwelch@wustl.edu 
 
Sub-Investigator 
Timothy Ley, M.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-8832, Fax Number: (314) 362-9333 
Email: timley@wustl.edu 
 
Pathology 
Eric Duncavage, M.D. 
Department of Anatomic and Molecular Pathology 
Telephone: (314) 362-8832 
Email: eduncavage@wustl.edu  
 
Statistician 
Feng Gao, Ph.D. 
Division of Biostatistics 
Telephone: (314) 362-3682 
E-mail: feng@wustl.edu 
 
IND# 116548 //Clinical Trials.gov # NCT01687400 
 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to obtain 
informed consent, may not be disclosed to another party unless law or regulations require such disclosure.  Persons 
to whom the information is disclosed must be informed that the information is confidential and may not be further 
disclosed by them. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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Genomic Predictors of Decitabine Response in AML/MDS  
Principal Investigator Signature Page 
 
 
Principal Investigator: 
 
John Welch, M.D., Ph.D. 
 
 
 
 
 
Signature of Investigator 
 
Date 
 
 
 
Printed Name of Investigator 
 
 
 
By my signature, I agree to personally supervise the 
conduct of this study and to ensure its conduct in 
compliance with the protocol, informed consent, 
IRB/HRPO procedures, the Declaration of Helsinki, ICH 
Good Clinical Practices guidelines, and the applicable 
parts of the United States Code of Federal Regulations or 
local regulations governing the conduct of clinical 
studies. 
 
 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 7 Version date: 11/15/2016  
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Study Schema: 
 
 
Patients with progressive disease after two cycles (defined as a clear increase in bone marrow blast count, typically a 
> 50% increase in bone marrow blasts over baseline) should be removed from study. 
Patients with stable disease may continue on study and should undergo a diagnostic bone marrow biopsy at the end 
of even numbered cycles. 
Once AML patients achieve morphologic complete remission with incomplete blood count recovery (CRi) or MDS 
patients achieve a marrow complete response (Marrow CR) as defined in Section 9.0, decitabine 
administration may be decreased to 20 mg/m2/day for ten consecutive days every 6 weeks, to limit 
myelosuppressive toxicity and improve count recovery. After 2 cycles at 6 week intervals, the dose may be 
reduce to 20 mg/m2/day for five consecutive days in 6 week cycles, at the discretion of the treating physician. 
After two cycles of five consecutive days of treatment, if the patient meets the definition of CR with complete 
cytogenetic response as defined in Section 9.0, administration may further be decreased to three consecutive 
days per cycle at the discretion of the treating physician. 
Sample collection for correlative studies: 
Bone marrow biopsies should be performed at baseline, at Cycle 1 Day 10 ± 1, and at Day 28 ± 4 of Cycles 
1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be performed at the discretion of the 
treating physician to assess for relapse or progression.  
 
 
20 
mg/m
2/day  
Cycle 1 
20 
mg/m
2/day 
Cycle 2+ 
Day 1 
Day 28 
BM Bx 
PB 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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Genomic Predictors of Decitabine Response in AML/MDS 
Table of Contents 
Section 
 
Page 
1.0 INTRODUCTION .................................................................................. 7 
1.1 
Rationale ............................................................................................ 7 
1.2 
Decitabine and Hypomethylating agents in AML/MDS ................... 9 
1.2.1 
Pharmacokinetics and Metabolism of Decitabine ................... 11 
1.2.2 
Experience with extended dosing schedules ........................... 12 
1.3 
Correlative Studies .......................................................................... 13 
2.0 OBJECTIVES ....................................................................................... 15 
2.1 
Primary Objective ............................................................................ 15 
2.2 
Secondary Objectives ...................................................................... 15 
3.0 PATIENT SELECTION ....................................................................... 16 
3.1 
Inclusion Criteria ............................................................................. 16 
3.2 
Exclusion Criteria ............................................................................ 16 
3.3 
Inclusion of Women and Minorities ................................................ 17 
4.0 REGISTRATION PROCEDURES ...................................................... 18 
4.1 
Confirmation of Patient Eligibility .................................................. 18 
4.2 
Patient Registration in the Siteman Cancer Center OnCore 
Database .......................................................................................... 18 
4.3 
Assignment of UPN ......................................................................... 18 
5.0 INVESTIGATIONAL PLAN .............................................................. 20 
5.1 
Summary of Study Design............................................................... 20 
5.2 
Study Procedures ............................................................................. 21 
5.2.1 
Pre-study Procedures ............................................................... 21 
5.2.2 
Baseline Evaluation ................................................................. 21 
5.2.3 
Day 1 of Each Cycle ............................................................... 21 
5.2.4 
Cycle 1 Days 1-10 ................................................................... 21 
5.2.5 
Cycle 1 Day 10 ± 1 ................................................................. 22 
5.2.6 
Weekly .................................................................................... 22 
5.2.7 
Every Other Week ................................................................... 22 
5.2.8 
Cycles 1, 2, 4 and 6 Day 28 ± 4 days ...................................... 22 
5.2.9 
End-of-Study Procedures ........................................................ 22 
5.2.10 
Post-study Follow-up .............................................................. 23 
5.3 
Duration of Therapy ........................................................................ 23 


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5.4 
Concomitant Therapy and Supportive Care Guidelines .................. 23 
5.4.1 
Chemotherapy ......................................................................... 23 
5.4.2 
Growth Factors ........................................................................ 24 
5.4.3 
Transfusions ............................................................................ 24 
5.4.4 
Prophylactic Antimicrobial Agents ......................................... 24 
5.4.5 
Radiotherapy ........................................................................... 24 
6.0 DOSE MODIFICATIONS ................................................................... 25 
6.1 
Delay for Cytopenia or Infection ..................................................... 25 
6.2 
Delay for Organ Dysfunction .......................................................... 25 
6.3 
Modification in Dose or Schedule for Treatment Response ........... 25 
7.0 PHARMACEUTICAL INFORMATION ............................................ 26 
7.1 
Decitabine ........................................................................................ 26 
7.1.1 
Study Drug Preparation ........................................................... 26 
7.1.2 
Supplier ................................................................................... 26 
8.0 CORRELATIVE STUDIES SAMPLE COLLECTIONS .................... 27 
9.0 EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES28 
9.1 
Response Criteria - AML ................................................................ 28 
9.2 
Response Criteria - MDS................................................................. 29 
9.2.1 
Hematologic Improvement (HI) .............................................. 29 
9.3.1 Cytogenetic Response ....................................................................... 30 
9.3 
Guidelines for Evaluation of Disease .............................................. 30 
9.4 
Other Secondary Efficacy Measures ............................................... 30 
9.4.1 
Treatment Failure, Overall Survival and Event-free Survival 30 
9.4.2 
Duration of Remission and Relapse-free Survival for 
Patients Having Complete Remission ..................................... 31 
9.4.3 
Safety ...................................................................................... 31 
10.0 REGULATORY AND REPORTING REQUIREMENTS .................. 32 
10.1 Definitions ....................................................................................... 32 
10.1.1 
Adverse Events (AEs) ............................................................. 32 
10.1.2 
Serious Adverse Event (SAE) ................................................. 32 
10.1.3 
Unexpected Adverse Experience ............................................ 33 
10.1.4 
Life-Threatening Adverse Experience .................................... 33 
10.1.5 
Unanticipated Problems .......................................................... 33 
10.1.6 
Noncompliance ....................................................................... 33 
10.1.7 
Serious Noncompliance .......................................................... 34 
10.1.8 
Protocol Exceptions ................................................................ 34 
10.2 Reporting to the Human Research Protection Office (HRPO) at 
Washington University: ................................................................... 34 


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10.3 Reporting to the Quality Assurance and Safety Monitoring 
Committee (QASMC) at Washington University ........................... 34 
10.4 Reporting Requirements for Secondary Sites .................................. 35 
10.5 Reporting to Secondary Sites .......................................................... 35 
10.6 Reporting to the FDA ...................................................................... 35 
10.7 Timeframe for Tracking Reportable Events: ................................... 36 
11.0 DATA SAFETY MONITORING PLAN ............................................. 37 
12.0 AUDITING ........................................................................................... 39 
13.0 MULTICENTER REGULATORY REQUIREMENTS ...................... 40 
14.0 STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE ........ 41 
14.1 Analysis Populations ....................................................................... 41 
14.2 Statistical Analysis .......................................................................... 41 
14.2.1 
Descriptive Analyses ............................................................... 41 
14.2.2 
Primary Endpoint Analysis ..................................................... 41 
14.2.3 
Secondary Endpoint Analyses ................................................ 42 
14.2.4 
Safety Analysis ....................................................................... 43 
14.2.5 
Description of Planned Subgroup Analyses ............................ 44 
14.3 Sample Size ..................................................................................... 44 
15.0 DATA SUBMISSION SCHEDULE .................................................... 45 
16.0 REFERENCES ..................................................................................... 48 
  


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1.0 
INTRODUCTION 
 
1.1 
Rationale   
 
Personalized therapy for cancer requires that 1) patient-specific factors (e.g. patient-
specific mutations, expression profiles, or pharmacogenomics) influence treatment 
algorithms, and 2) once therapy is initiated, in vivo responsiveness is used to guide further 
therapy. The modern era of genomics has provided significant insights into cancer 
pathogenesis by describing patterns of germline and somatic variants, as well as defining 
disease-specific patterns of gene expression. However, the application and integration of 
this data with therapeutics remains a difficult task. 
 
Decitabine is a commonly used single-agent therapy for patients with acute myeloid 
leukemia (AML) and/or myelodysplastic syndrome (MDS). Historically, it has yielded 
clinical complete responses in approximately 25% of patients across diverse clinical trials, 
it is well tolerated, and it can be given as in the outpatient setting. However, the majority 
of patients do not respond to decitabine, the molecular etiology of decitabine sensitivity is 
unknown, and there are no algorithms to predict patient responsiveness a priori or even 
within one month of starting therapy.  
 
AML and MDS are two closely related hematological disorders characterized by 
ineffective hematopoiesis and malignant expansion of clonal myeloid cells.  Both AML 
and MDS frequently occur in elderly patients and some cases are secondary and/or related 
to long-term bone marrow damage after radiation or chemotherapy. Current therapy for 
AML consists of cytotoxic chemotherapy followed by consolidation chemotherapy or stem 
cell transplantation. Using these approaches, current cure rates may be as high as 60% for 
patients younger than 60 with good risk cytogenetics (e.g. t(15;17), t(8;21), inv(16)).[4]  
Outcomes in patients ≥ 60 years old and with relapsed disease remain poor with 2 year 
survival of only ~25%.[5] Historically, the available treatment for MDS has been 
symptomatic and has not been shown to be effective in producing sustained improvement 
in hematopoiesis or in delaying leukemic evolution[6, 7].  
 
Current AML therapy is poorly tolerated in elderly and pre-treated patients due to 
hematologic and non-hematologic toxicities; following such therapy, patients typically 
require 3-4 weeks of hospitalization for treatment of neutropenic fevers and for regular 
transfusions. Even among the relatively healthy patients enrolled in clinical trials, induction 
mortality in the elderly can be as high as 20-40% following standard induction therapy,[8-
11] and moderate to severe non-hematologic toxicity occurs in at least an additional 30% 
of patients.[9, 12-14] Consequently, many older AML patients are not offered traditional 
intensive chemotherapy, but receive supportive care alone.[15]  More effective and less 
toxic therapies are needed to provide durable remissions in a significant proportion of 
elderly AML in populations. 
 
Decitabine has been studied in MDS patients and in elderly and/or relapsed AML patients. 
It has been found to be reasonably well tolerated, can be given in the outpatient setting, 
and has overall response rates of 20-45%.[2, 3, 16-20] Our own experience has been of 


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CR/PR rates of 25%, [2] and this is typical of many other groups for both MDS and AML 
patients (reviewed in [21]  Decitabine is an S-phase depended drug.  [22-24] However, 
decitabine is usually administered in one hour daily infusions during 3 to 5 consecutive 
days (too short to expose decitabine to most AML blasts during at least 2 cycles of 
replication, which divide every 2 to 7 days).[25-27]  Recent experience with increasing the 
number of infusions per cycle from 5 consecutive days to 10 consecutive days has 
improved overall response rates to 67%.[3] Response to decitabine can be slow, sometimes 
requiring 3 to 5 cycles before maximum response is observed.[2] As a consequence, many 
patients are exposed to prolonged therapy, gaining little benefit, before determining 
resistance. There are currently no biomarkers that predict response to decitabine. 
 
Decitabine is a cytosine analogue that cannot be methylated. Incorporation of decitabine 
into DNA prevents DNA methylation at cytosine residues and derepresses epigenetically 
silenced gene expression. Therefore, the reduction in total genomic DNA methylcytosine 
content acts as a marker of effective decitabine dosing. Indeed, two groups have recently 
correlated effective methylcytosine reduction and gene expression derepression to 
response (Figure 1.1).[3, 28] 
 
 
 
   
Figure 1.1. From Shen et al JCO (2010) 28:611 and Blum et al JCO (2007) 25: 3884. 
 
We and others have recently used whole genome sequencing to identify recurrent, somatic 
mutations in AML and MDS patients in four genes that are involved in methylcytosine 
regulation: DNMT3A, IDH1, IDH2, and TET2.[29-37] Collectively, nearly half of all 
AML/MDS patients have a mutation in one of these four genes.  DNMT3A mutations are 
associated with an adverse outcome in AML patients treated with cytotoxic 
chemotherapy.[35, 38] However, it is not yet clear whether the presence of mutations in 
one or more of these genes is associated with response or resistance to decitabine. 
 
In this study, we will treat patients with the current state-of-the-art decitabine schedule.[3] 
We will perform exome sequencing on all patients and correlate response with mutation 
status, rate of mutation clearance, expression profile, plasma decitabine levels, and DNA 


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methylcytosine content reduction. This approach will comprehensively assess genomic and 
pharmacologic parameters that could clinically predict patient response to decitabine.  
 
In this study, we will combine analysis of patients with AML and MDS. Recent genomic 
analysis has shown that AML and MDS have shared mutations in many of the same genes, 
shared patterns of mutation acquisition, and shared patterns of mutation evolution.[39, 40] 
Furthermore, MDS, like AML, presents with clonal hematopoiesis, even when the 
morphologic blast count is zero.[36]Because this study will identify and track the rate of 
clearance of patient specific mutations, and because decitabine has been shown to have 
efficacy in both AML and MDS patients, [2, 3, 16-19, 41-45] combining analysis of AML 
and MDS patients is reasonable.  Asymptomatic MDS patients are excluded because these 
patients are typically followed with watchful-waiting rather than with chemotherapy. 
  
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
 
Methylation of cytosine in the CpG dinucleotide by DNA methyltransferase leads to 
transcriptional silencing of genes during normal development and has emerged as a 
significant mechanism for the loss of tumor suppressor gene expression in human cancer, 
including AML and MDS.[46, 47] Like azacitidine, decitabine (5-aza-2’-deoxycitidine) is 
incorporated into DNA during S phase and forms covalent adducts with DNA 
methyltransferase. The irreversible inhibition of this enzyme results in loss of methylation 
and is associated with differentiation.[48]  Decitabine is an approximately 5-fold more 
potent inhibitor of DNA methyltransferase than azacitidine, which might explain why 
decitabine can induce differentiation of myeloid progenitors in vitro at lower doses than 
azacitidine. In addition to their differentiation inducing activity at low doses, both agents 
have a direct cytotoxic effect at higher doses (1-2 µM).[49]  
 
Decitabine has been investigated in hematologic malignancies using a range of doses and 
schedules.  It is currently FDA approved for the treatment of adults with MDS at a dose of 
15 mg/m2 every 8 hours for 3 days, based on a Phase III study that demonstrated improved 
outcomes in the treatment arm compared to patients treated with supportive care alone.[15]  
However, the overall response rate in this trial was modest (17%). Of note, 18% of the 
patients in this trial had 20-30% bone marrow blasts, meeting the current WHO criteria for 
AML.  Subsequent phase I and II studies in AML, using a variety of low-dose decitabine 
regimens, have reported overall response rates of 22-49%.[2, 3, 16-19, 41-45] Comparisons 
of multiple low dose schedules have noted improved efficacy in the MDS population using 
a dose of 20 mg/m2 on Days 1-5 of a 28-day cycle.[50, 51] Because of these results, we 
have recently explored this dosing schedule in the treatment of elderly patients with 
AML.[52] In this study, we observed an overall response rate of 24%, and an additional 
44% of patients achieved stable disease during a median of 5 monthly cycles.[48] In this 
study, we noted that decitabine does not act quickly and the median time to response was 
3 months. These results are similar to the recently published experience in Germany 
treating 227 patients with 15 mg/m2 over 3 hours TID for 3 days every 6 weeks, which 
resulted in a CR/PR rate of 26%.[16]  
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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A recent study using a prolonged administration schedule (20 mg/m2 IV over 1 hour on 
consecutive days 1-10) showed improved response (64% CR/PR) and improved time to 
respond (50% of patients had bone marrow blasts < 5% after 1 cycle of therapy).[3]  These 
results are an improvement over previous experience by other groups using decitabine to 
treat elderly AML patients or patients with relapsed AML, and this experience is 
summarized below: 
Trials of decitabine in AML 
Status 
Other Therapy 
DAC Dose 
(mg x days) 
Evaluable 
CR 
de novo 
daunomycin1 
90 mg/m2 x 5 
6 
100% 
Salvage 
None4 
0.75-80 mg/kg 
16 
  
None5 
37-81 mg/kg 
6 
33% 
None7.  Ara-C 
Refractory 
Various 
4 
0 
AMSA7 
250-500 mg/m2 x 6 
10 
80% 
idarubicin or 
AMSA3 
250 mg/m2 x 6 
56 
39% 
Elderly 
None6 
270-360 mg/m2 x 3 
10 
30% 
None2 
45 mg/m2 x 3 
4 
50% 
None8 
20 mg/m2day 1-5,8-12 
50 
23% 
None9 
20 mg/m2 x 5 
55 
25% 
 
None10 
50 mg/m2 CIVI x 3 
29 
48% 
 
None11 
20 mg/m2 x 10 
53 
64% 
 
ATRA cycle 212 
15 mg/m2 TID x 3 
227 
26% 
1 Schwartsmann G.  Leukemia 1997; 11(Suppl 1):S28-31. 
2 Lubbert M. ASH 2002 Abstract #4601. 
3 Willemze R.  Leukemia.  1997; 11(Suppl 1):S24-27 
4 Rivard GE.  Leukemia Research 1981; 5:453-462. 
5 Momparler RL.  Pharmac Therapy 1985; 30:277-286.  
6 Petti MC.  Leukemia.  1993; 7(Suppl 1):36-41.  
7 Richel DJ.  Br. J. Cancer 1991; 64:144-148 
8 Issa, JP. Blood 2004. 103: 1635. 
9 Cashen AF. ASH Annual Meeting. 2008 abstract 560. 
10 Wijermans PW. Leukemia 1997. 11: Suppl 1, 19-23. 
11 Blum, W. PNAS 2010; 107:7473. 
12 Lubbert. Haematologica 2011. ePub 11/04/2011. 
 
In general, decitabine is well tolerated. Unlike conventional cytotoxic chemotherapy, 
decitabine can be administered in the outpatient setting. However, AML and MDS are 
aggressive hematologic malignancies. Cytopenias commonly result from AML/MDS 
inhibition of normal hematopoiesis, and often do not resolve until a complete response is 
achieved. Thus, neutropenic sepsis and thrombocytopenic bleeding remain significant 
problems regardless of therapy, especially during the first 4-8 weeks. In our experience 
treating elderly AML patients, neutropenia occurred in 24%, sepsis in 9%, and bacteremia 
in 7%.  Similar results have been observed in MDS patients.[20, 53, 54] As a result, death 
is not uncommon in the first 8 weeks of therapy, occurring in 15-17% of patients, and most 
commonly resulting from infection and cytopenias related to disease and therapy.[16, 52, 
55] While unsettling, these numbers are better than many historical patients treated with 
cytotoxic chemotherapy, and suggest a need for continued improvements in therapy.[9, 12]  


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1.2.1 Pharmacokinetics and Metabolism of Decitabine 
 
In human subjects, after IV administration, decitabine displayed a distribution phase 
with a half-life of 7 minutes and a terminal phase elimination half-life of 10-35 
minutes as measured by bioassay.[56, 57]   The short plasma half-life is due to rapid 
inactivation of decitabine by deamination by liver cytidine deaminase.[23] Total 
body clearance (Clp) in humans was high with a mean value of 126 mL/min/kg.[58]  
 
Among five adult patients with advanced solid tumors administered seven courses 
of 100 mg/m2 of decitabine by 1-hour infusions,[56] maximum plasma 
concentration ranged from 1 to 4 µM. Disappearance from plasma was biphasic 
with a rapid initial decline to less than 0.1 µM during the first hour post infusion 
and a slower decline over the next two hours to 6 nM, the level of detectability. 
Plasma half-life (meanSE) was estimated to be approximately 71 minutes in the 
initial phase (t½α) and 355 minutes in the second phase (t½β). The area under the 
plasma concentration-time curve was 40888 ng.hour/mL for these patients, with a 
peak plasma concentration of 2.010.44 µM (0.440.1 µg/mL), while mean volume 
of distribution (Vdss) and CLp was 4.591.42 L/kg and 12621 mL/min/kg, 
respectively. The time course of DAC concentrations was similar in 1 of 2 patients 
administered 75 mg/m2, while DAC was usually undetectable in the plasma of 
patients administered 25 to 60 mg/m2. 
 
Using a longer infusion time (up to 40 hours), Rivard et al.[57] reported a plasma 
concentration of 0.1 to 0.4 g/mL. With infusion times of 40-60 hours, at an infusion 
rate of 1 mg/kg/h, plasma concentrations of 0.43-0.76 µg/mL were achieved.[59]  The 
steady-state plasma concentration at an infusion rate of 1 mg/kg/h was estimated to 
be 0.2-0.5 µg/mL. The half-life after discontinuing the infusion was 12-20 min.[57]  
 
Urinary excretion of unchanged decitabine was low, ranging from less than 0.01% to 
0.9% of the total dose, and there was no relationship between excretion and dose or 
plasma drug levels. High clearance values and a total urinary excretion of <1% of the 
administered dose suggest that decitabine is eliminated rapidly and largely by 
metabolic processes, including extrahepatic metabolism.[60] 
 
The steady state plasma concentrations (Css) ranged from 0.31 to 0.39 µg/mL (1.27 
to 1.60 µM) during the infusion of 100 mg/m² decitabine over a 6-hour period.[61] 
The range of concentrations during a 600 mg/m2 infusion over an 8-hour period was 
0.41-16 g/mL(1.68 to 4.76 µM).[60] These plasma concentrations are in the same 
range as the in vitro concentrations that activated the expression of tumor 
suppressor gene p16 in three human non-small-cell lung cancer (NSCLC) cell 
lines.[61]  
 
Steady state decitabine levels of 0.1 to 0.2 µM were achieved in solid tumor patients 
dosed with 30 mg/m²/day and 40 mg/m²/day by continuous intravenous infusion 
over 72 hours.[62]  


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Summary of published steady state serum levels are below and are in proportion to 
infusion rate (R2 = 0.98, p < 0.0001):[63] 
 
Rate 
Serum 
Concentration 
mg/m2/hr 
ng/ml 
nM 
5 
55 
241 
7.5 
80 
351 
15 
180 
789 
21 
182.4 
800 
22.5 
350 
1535 
42 
456 
2000 
84 
1140 
5000 
 
In our experience treating primary patient blasts in vitro, concentrations of 100 – 
500 nM are required maximize methylcytosine reduction without causing cellular 
toxicity. Because decitabine incorporation into DNA is S-phase dependent, 
exposure of cells during cell division is necessary for efficacy. 
  
 
 
 
1.2.2 Experience with extended dosing schedules 
 
Blum et al recently published their experience treating elderly AML patients with 
a 10-day course of decitabine 20 mg/m2/day as a 1-hour infusion.[3] This prolonged 
exposure course was designed to increase the number of AML blast divisions 
exposed to decitabine per cycle. Toxicities were very similar to a similar regiment 
given as a 3 and 5 day course,[2, 16] but the overall response (CR/PR) was much 
improved (64% vs 25%). In all three studies, the toxicities were primarily myeloid 
suppression and the resultant infectious and bleeding complications associated with 
leukemic and pharmacologic hematopoietic suppression. Furthermore, Blum et al 
noted that the percent methylcytosine reduction was improved (22% vs 11%) as 


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was the time to blast clearance (50% of patients who ultimately achieved a CR had 
≤ 5% bone marrow blasts after 1 cycle vs 0% of patients after 2 cycles). Finally, 
like Shen et al, they observed a correlation between the extent of methylcytosine 
reduction and response.[28]  This study suggests that improvement in response 
rates and time to response can be achieved by prolonged administration of 
decitabine. Further, that DNA methylcytosine reduction can be achieved with 
prolonged exposure to decitabine and that this might be a patient-specific biological 
marker of adequate dosing. 
  
Scandura et al. recently published a study of infusional decitabine prior to 
cytarabine + daunorubicin induction therapy for AML.[64] They escalated the 
duration of infusional therapy and reached their planned goal of 20 mg/m2/day x 7 
days of continuous therapy given prior to cytarabine and daunorubicin. They noted 
similar toxicities (primarily hematopoietic and the associated neutropenic 
infections), but that these were not different from their experience giving cytarabine 
+ daunorubicin alone. 
 
1.3 
Correlative Studies 
 
All correlative studies will be collected under HRPO# 201011766 (“Tissue Acquisition for 
Analysis of Genetic Progression Factors in Hematologic Diseases”) that all patients will 
be concurrently enrolled in. The goals of the correlative studies include: 
 
1. To correlate patient-specific mutations with response to decitabine. 
We will perform exome sequencing of each patient’s pre-study bone marrow sample 
and skin sample. We will determine AML/MDS-associated variants found in the bone 
marrow, but not skin sample. We will correlate the presence of mutations with response 
(especially mutations in DNMT3A, IDH1, IDH2, and TET2). This study is powered to 
identify mutations with >80% sensitivity to predict decitabine response if used as a 
clinical assay. 
 
2. To correlate the velocity and depth of response with response to decitabine. 
We will assess tumor burden by allele frequency of patient-specific mutations in bone 
marrow samples collected pre study, Cycle 1 Day 10±1, Cycle 1 Day 28±4, and Cycle 
2 Day 28±4. We will assess mutations in at least 24 recurrently mutated genes 
(including, but not limited to: FLT3, DNMT3A, NPM1, TET2, WT1, IDH1, IDH2, 
RUNX1, NRAS, TP53, U2AF1, KRAS, PHF6, MSTP9, PTPN11, KIT, SMC3, PRUNE2, 
ETV6, CEBPA, SMC1A, ASXL1, STAG2, RAD21) as well as all patient-specific 
mutations discovered during exome sequencing. The leukemic allele frequency will be 
measured using the Illumnia MySeq or HiSeq platform using DNA capture by gene 
specific biotinylated-oligonucleotides. Patient bone marrow samples will be assessed 
pre-study, Cycle 1 Day 10 ± 1, Cycle 1 Day 28 ± 4, and Cycle 2 Day 28 ± 4 and 
compared with patient specific skin samples (normal control). The velocity of response 
will be calculated as the natural log rate of patient-specific mutation allele frequency 
clearance. Depth of response will be defined as the lowest patient-specific mutation 


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allele frequency in a bone marrow sample. The velocity and depth of tumor response 
will be correlated to: 
 Achievement of clinical CR, 
 Time to best response, 
 Mutational status, 
 Time to disease progression. 
Because all patient-specific exome-discovered mutations will be assessed a second 
time in the primary bone marrow sample as well as the  Cycle 1 Day 10 ± 1, Cycle 1 
Day 28 ± 4, and Cycle 2 Day 28 ± 4 bone marrow samples, this study will also serve 
to validate all exome-discovered mutations. This is important, because exome 
sequencing is associated with a 20-50% false-discovery rate depending on type I error 
tolerated (mutation-calling algorithms are typically set for greater sensitivity than 
specificity).  Thus, many mutations discovered during exome sequencing are incorrect.  
This second analysis of these mutations will server to exclude these false positive calls. 
 
3. To correlate the bone marrow expression profile from pre-study and Cycle 1 Day 10 ± 
1 with response to decitabine. 
We will correlate expression signatures, and changes in expression signatures, with: 
 Achievement of clinical CR, 
 Mutation status. 
 
4. To correlate the extent of bone marrow DNA hypomethylation on Cycle 1 Day 10 ± 1 
with response to decitabine. 
We will assess the percent reduction in bone marrow methylcytosine content using LC-
MS. Patients will be evaluated pre-study and Cycle 1 Day 10 ± 1. The change in DNA 
methylcytosine content will be correlated to: 
 Achievement of clinical CR, 
 Change in expression signature. 
This will determine whether inadequate blast exposure to decitabine correlates with 
drug resistance. 
 


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2.0 
OBJECTIVES 
 
2.1 
Primary Objective 
 
To determine whether patient-specific mutations correlate with clinical overall response to 
decitabine in patients with AML or MDS. 
 
2.2 
Secondary Objectives 
 
- 
To determine whether 10 consecutive days per cycle of decitabine improves outcomes 
compared with historical controls treated with 5 consecutive days per cycle of 
decitabine. 
 
- 
To determine whether other biomarkers correlate with clinical overall response to 
decitabine in patients with AML or MDS. These will include:  
 
1. The velocity and depth of patient-specific mutation clearance; 
2. The bone marrow expression profile and change in expression profile during 
decitabine treatment; 
3. The decrease in bone marrow methylcytosine content in response to decitabine.  
 
 


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3.0 
PATIENT SELECTION 
 
3.1 
Inclusion Criteria 
 
All of the following: 
 Patient must have non-M3 AML or MDS  
 An adverse risk karyotype defined by: 
-Complex karyotype by cytogenetics, or 
-Deletion of all or part of chromosome 5, 7, 12, or 17 defined by FISH or          
cytogenetics, or 
-Somatic TP53 mutation 
 
All of the following: 
1. Patient must have an ECOG performance status ≤ 2. 
2. Patient must have >10% disease burden measured by cytomorphology, flow 
cytometry, or cytogenetics. 
3. Patient must have peripheral white blood cell count < 50,000/mcl. 
4. Patient must have adequate organ function, defined as: 
a. 
Total bilirubin < 1.5 x ULN 
b. 
AST/ALT < 2.5 x ULN 
c. 
Serum creatinine < 2.0 x ULN  
5. Patient must have undergone ≤ 2 cycles of prior hypomethylating agent (decitabine 
or azacitidine). 
6. Patient must be enrolled in HRPO# 201011766 (“Tissue Acquisition for Analysis 
of Genetic Progression Factors in Hematologic Diseases”). 
7. Patient must be > 18 years of age. 
8. Patient must be able to understand and willing to sign an IRB-approved written 
informed consent document. 
 
3.2 
Exclusion Criteria 
 
1. Patient must not be pregnant or nursing. 
2. The patient must not have acute promyelocytic leukemia or t(15;17) observed by 
FISH. 
3. Patient must not have known CNS leukemia.  
4. Patient must not have a history of positive HIV serology.  
5. Patient must not have a history of positive Hepatitis C serology. 
6. Patient must not have undergone a prior allogeneic stem cell transplant. 
7. Patient must not have any uncontrolled intercurrent illness including, but not 
limited to, ongoing or active infection, ongoing or active GVHD, congestive heart 
failure of NYHA class 3 or 4, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situation that would limit compliance with study 
requirements. 
8. Patient must not have had radiation therapy within 14 days of enrollment. 
9. Patient must not have received any chemotherapy within 21 days of enrollment, 
and any acute treatment-related toxicities must have returned to baseline. Patients 


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may be receiving hydrea at time of enrollment.    
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible for this trial. 


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4.0 
REGISTRATION PROCEDURES 
 
Patients must not start any protocol intervention prior to registration through the Siteman 
Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility by Washington University 
2. Registration of patient in the Siteman Cancer Center database 
3. Assignment of unique patient number (UPN) 
 
Once the patient has been entered in the Siteman Cancer Center OnCore database, the WUSM 
coordinator will forward verification of enrollment and the UPN via email. 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility collecting the information listed below and scanning and 
emailing it to the research coordinator listed in the Siteman Cancer Center Clinical Trials 
Core Protocol Procedures for Secondary Sites packet at least one business day prior to 
registering patient: 
1. Your name and contact information (telephone number, fax number, and email 
address) 
2. Your site PI’s name, the registering MD’s name, and your institution name 
3. Patient’s race, sex, and DOB 
4. Three letters (or two letters and a dash) for the patient’s initials 
5. Currently approved protocol version date 
6. Copy of signed consent form (patient name may be blacked out) 
7. Planned date of enrollment 
8. Completed eligibility checklist, signed and dated by a member of the study team 
9. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center OnCore Database 
 
Registrations may be submitted Monday through Friday between 8am and 5pm CT. Urgent 
late afternoon or early morning enrollments should be planned in advance and coordinated 
with the Washington University research coordinator.  Registration will be confirmed by 
the research coordinator or his/her delegate by email within one business day. Verification 
of eligibility and registration should be kept in the patient chart. 
 
All patients at all sites must be registered through the Siteman Cancer Center OnCore 
database at Washington University. 
 
4.3 
Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  Patients 


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will also be identified by first, middle, and last initials.  If the patient has no middle initial, 
a dash will be used on the case report forms (CRFs).  All data will be recorded with this 
identification number on the appropriate CRFs. 
 


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5.0 
INVESTIGATIONAL PLAN 
 
5.1 
Summary of Study Design 
 
This is an open-label, single-arm, Phase II, biomarker study of patients with AML or MDS 
who are treated with 10 consecutive days of decitabine. FAB-M3 AML is excluded from 
this trial, because other well-tolerated options exist for the treatment of FAB-M3 acute 
promyelocytic leukemia. AML patients who are < 60 years old and have not received 
cytotoxic induction chemotherapy are also excluded, because cytotoxic therapy offers a 
curative possibility for these patients.  MDS patients without cytopenias are also excluded 
because these patients can be followed with watchful-waiting. 
 
All patients will undergo bone marrow biopsies pre-study, on Cycle 1 Day 10 ± 1, and on 
Day 28 ± 4 of Cycles 1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. 
Patients will provide a skin biopsy sample pre-study. See Section 8.0 for details on 
correlative studies.  
 
Patients will receive decitabine 20 mg/m2/day as a 1-hour infusion on consecutive Days 1-
10 in 28-day cycles.  The dose will be calculated based on actual patient weight at the 
beginning of each cycle. 
 
Once AML patients achieve morphologic complete remission with incomplete blood count 
recovery (CRi) or MDS patients achieve a marrow complete response (Marrow CR) as 
defined in Section 9.0, decitabine administration may be decreased to 20 mg/m2/day for 
ten consecutive days every 6 weeks, to limit myelosuppressive toxicity and improve count 
recovery. After 2 cycles at 6 week intervals, the dose may be reduce to 20 mg/m2/day for 
five consecutive days in 6 week cycles, at the discretion of the treating physician.  After 
two cycles of five consecutive days of treatment, if the patient meets the definition of CR 
with complete cytogenetic response as defined in Section 9.0, administration may further 
be decreased to three consecutive days per cycle at the discretion of the treating physician. 
 
It will be a logistical goal of this trial consistently to return preliminary exome results to 
the treating physician by the end of Cycle 1 and validated results by the end of Cycle 2. 
However, this logistical goal is not a primary or secondary aim of this study. Given the 
limited data regarding mutation presence and drug response, we do not anticipate that the 
results will directly influence therapeutic decisions made by the treating physician. 
However, the development of comprehensive genomic pipelines and integration of this 
data into a clinical trial setting will be required before personalized medicine can be 
realized. 
 
 
 


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5.2 
Study Procedures 
 
5.2.1 Pre-study Procedures 
 
Pre-study procedures must take place no more than 14 days prior to the first dose 
of decitabine except where noted below. 
 
1. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments performed 
on bone marrow will include correlative studies (collected under HRPO# 
201011766), morphologic assessment, cytogenetics, and FISH.  
2. Bone marrow or peripheral blood for flow cytometry 
3. Skin biopsy sample (collected under HRPO# 201011766). There is no required 
time frame for this sample; it may have been collected months or even years 
prior to the first dose of decitabine.  
 
5.2.2 Baseline Evaluation 
 
Baseline evaluations must take place no more than 7 days prior to the first dose of 
decitabine.  
 
1. Medical history 
2. Physical exam including height, weight, and ECOG performance score. 
3. Record transfusion requirements for 8 weeks before first dose of study drug 
4. CBC with differential and platelets 
5. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
6. Urine βHCG pregnancy test if appropriate 
7. Clinical pathology should be carefully reviewed to rule-out acute 
promyelocytic leukemia (M3-AML), and discussion between the pathologist 
and treating physician should take place if warranted by morphology or 
immunophenotype. 
 
5.2.3 Day 1 of Each Cycle 
 
1. Physical exam, including weight  
2. CBC with differential and platelets 
3. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
 
5.2.4 Cycle 1 Days 1-10 
 
Decitabine administration, 20 mg/m2 per day given as a 1-hour continuous 
intravenous infusion.  The dose will be calculated based on actual patient weight at 


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the beginning of each cycle.  Patients may be premedicated with lorazepam, 
prochlorperazine, palonosetron, or ondansetron at the treating physician’s 
discretion. 
 
The clinical research coordinator should monitor for final FISH results to confirm 
that PML/RARA screening is negative. 
 
5.2.5 Cycle 1 Day 10 ± 1 
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH. 
 
5.2.6 Weekly 
 
CBC with differential and platelets. After the first cycle, this may be changed to 
every other week at the discretion of the treating physician. 
 
5.2.7 Every Other Week 
 
Serum chemistries, including bicarbonate, calcium, glucose, total protein, albumin, 
BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, ALT, alkaline 
phosphatase, LDH. After the first cycle, this may be changed to every month at the 
discretion of the treating physician. 
 
5.2.8 Cycles 1, 2, 4 and 6 Day 28 ± 4 days  
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include: correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH.  
 
Patients who achieve with clinical response (CRi or better) and are receiving cycles 
every 6 weeks, will undergo bone marrow biopsies on day 42 ± 4.  
 
5.2.9 End-of-Study Procedures  
 
1. Physical exam 
2. CBC with differential and platelets.  
3. Serum chemistries including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, and LDH.  
4. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments performed 
on bone marrow will include morphologic assessment, cytogenetics, and FISH.  


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5.2.10 Post-study Follow-up 
 
Patients are to be followed according to standard patient care procedures for 30 
days following the date of the last decitabine dose to monitor for toxicity, survival, 
and response. Thereafter, the investigator or designee will record information on 
subsequent therapy, disease progression, and/or death every 6 months for 2 years 
after last dose of study drug or until the patient is lost to follow-up or dies. 
 
5.3 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to the 
patient’s health and/or the patient no longer wishes to continue protocol therapy, the 
protocol therapy should be discontinued and the reason(s) for discontinuation documented 
in the case report forms. 
 
In the absence of treatment delays due to adverse events, treatment may continue 
indefinitely or until one of the following criteria applies: 
 
 Documented and confirmed disease progression 
 Death 
 Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
 General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
 Suspected pregnancy 
 Serious non-compliance with the study protocol 
 Lost to follow-up 
 Patient withdraws consent 
 Investigator removes the patient from study 
 The Siteman Cancer Center decides to close the study 
 
Patients who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar. 
 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
 
5.4.1 Chemotherapy 
 
Patients cannot receive any chemotherapy within 21 days of study enrollment and 
must have recovered from any treatment-related toxicities. However, patients may 
be receiving hydrea at enrollment and may continue on hydrea through Cycle 2 of 
decitabine. An indication for hydrea following 2 cycles of decitabine is evidence 
that the patient is not responding and decitabine should be discontinued. Other than 
decitabine, patients cannot receive chemotherapy during this trial.  
 


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5.4.2 Growth Factors 
 
Erythroid growth factors should not be routinely administered to patients during the 
study. 
 
Thrombopoietin receptor agonists should not be routinely administered to patients 
during the study. 
 
Filgrastim (G-CSF) or sargramostim (GM-CSF) may be administered to patients 
with septic shock or febrile neutropenia at the discretion of the treating physician. 
 
5.4.3 Transfusions 
 
 Packed red blood cells (RBCs) transfusion:   
RBC transfusions will be administered at the discretion of the treating 
physician.  
 
 Platelet transfusion:   
Platelet transfusions will be administered at the discretion of the treating 
physician  
 
5.4.4 Prophylactic Antimicrobial Agents  
 
Antimicrobial agents will be administered at the discretion of the treating physician. 
Common antimicrobial prophylaxis during cycles 1 and 2 include acyclovir, 
ciprofloxacin, and fluconazole. 
 
5.4.5 Radiotherapy 
 
Patients cannot receive radiotherapy within 14 days prior to study enrollment or 
while on study. 
 


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6.0 
DOSE MODIFICATIONS 
 
A criterion for discontinuation of the study is a delay of > 8 weeks between any two cycles.  
 
6.1 
Delay for Cytopenia or Infection 
 
As cytopenias are a defining feature of AML and MDS, their presence alone does not 
generally warrant dose modifications or delays. Patients will receive four cycles of therapy 
without adjustment for any cytopenia. Following Cycle 4, dose modifications or delays for 
cytopenias will be made at the discretion of the investigator.  
 
Patients with febrile neutropenia (temperature ≥ 38.5° with ANC < 1,000/l) or systemic 
infections should have treatment held until clinical resolution of the infection.  
 
6.2 
Delay for Organ Dysfunction 
 
If renal dysfunction (creatinine ≥ 3 X institutional upper limit of normal) or hepatic 
dysfunction (bilirubin ≥ 2.5 mg/dl or ALT/AST ≥ 3 X institutional upper limit of normal) 
occurs, therapy may not resume until the abnormalities have resolved to below the limits 
stated above. If renal or hepatic dysfunction occurs during a treatment course, the next dose 
of decitabine should be held, and serum chemistries should be checked twice per week.  
Decitabine can be resumed at full dose when creatinine, bilirubin, ALT and AST are below 
the limits listed above.  
 
For grade 3-4 non-hematologic toxicities related to decitabine, decitabine treatment should 
be delayed until recovery to baseline or to grade ≤ 1. Decitabine may be reintroduced with 
a 50% dose reduction, or at full dose at the discretion of the investigator. 
 
6.3 
Modification in Dose or Schedule for Treatment Response 
 
Once AML patients achieve morphologic complete remission with incomplete blood count 
recovery (CRi) or MDS patients achieve a marrow complete response (Marrow CR) as 
defined in Section 9.0, decitabine administration may be decreased to 20 mg/m2/day for 
ten consecutive days, and the cycles may be administered every 6 weeks to limit 
myelosuppressive toxicity, at the discretion of the treating physician. After two cycles at 
six week intervals, the dose may be reduced to 20 mg/m2/day for five consecutive days of 
treatment in six week intervals. If the patient meets the definition of CR with complete 
cytogenetic response as defined in Section 9.0, administration may further be decreased to 
three  consecutive days per cycle at the discretion of the treating physician. 
 


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7.0 
PHARMACEUTICAL INFORMATION 
 
7.1 
Decitabine 
 
Decitabine is an antimetabolite that can replace cytosine in DNA but, unlike cytosine, it 
cannot be methylated. The exact mechanism of decitabine activity has not been determined 
and may involve multiple pathways. Decitabine is thought primarily to act by correcting 
DNA methylation, a major mechanism for gene expression. In some cancer cells, 
hypermethylation blocks the activity of tumor suppressor genes, which regulate cell 
division and differentiation to prevent malignant transformation. When suppressor gene 
activity is blocked, cell division becomes unregulated, leading to the formation of 
neoplastic cells. Decitabine regulates DNA methylation (specifically targets the 
methyltransferase, DNMT1), effectively demethylating and reactivating different tumor 
suppressor genes. Because hypermethylation occurs early in the malignant transformation 
of cells, decitabine may have activity in chemoprevention. Other possible mechanisms for 
decitabine activity include cytotoxicity due to incorporation into DNA similar to other 
antimetabolites, methylation-independent induction of gene expression, and degradation of 
DNA methyltransferase 1. 
 
7.1.1 Study Drug Preparation  
 
OSHA Guidelines for handling cytotoxic drugs outlined in the American Journal 
of Hospital Pharmacy must be followed. 
 
Decitabine (5-aza-2’-deoxycytidine) is supplied as a lyophilized powder for 
injection, 50 mg in 20-ml vials. When reconstituted with 10 ml of sterile water for 
injection, each ml will contain 5 mg of decitabine and 6.8 mg of KH2PO4. The 
reconstituted solution should be diluted to 0.1-1.0 mg/mL with pre-chilled 0.9% 
sodium chloride intravenous infusion, which has been cooled to 2-8oC. 
Reconstitution of the powder results in a rapidly decomposing solution. Unless used 
within 15 minutes of reconstitution, the diluted solution must be prepared using 
cold (2—8 degrees C) infusion fluids and stored at 2—8 degrees C (36—46 degrees 
F) for a maximum of 7 hours. 
Decitabine will be administered per dosing schedule every 28 ± 4 days.  
 
Decitabine may be administered either as an inpatient or an outpatient.  
 
7.1.2 Supplier 
 
Decitabine is available commercially. 
 
 
  
 
  
 


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8.0 
CORRELATIVE STUDIES SAMPLE COLLECTIONS 
 
Bone marrow samples will be collected pre-study, on Cycle 1 Day 10 ± 1, and on Day 28 ± 4 of 
Cycle 1, 2, 4, and 6 for correlative studies. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. The bone 
marrow samples will be collected under HRPO# 201011766 (“Tissue Acquisition for Analysis of 
Genetic Progression Factors in Hematologic Diseases”) that all patients will be concurrently 
enrolled in.  
 
Skin biopsy sample will be collected as part of the patient’s participation in HRPO# 201011766 
(“Tissue Acquisition for Analysis of Genetic Progression Factors in Hematologic Malignancies”). 
There is no required time frame for this sample, and it may have been collected months or even 
years prior to the first dose of decitabine.  
 
 
 


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9.0 
EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
 
A secondary endpoint of this study is to estimate morphologic complete remission rate. 
  
9.1 
Response Criteria - AML 
 
AML Patients will be assessed for response according to the IWG criteria:1  
 
Morphologic complete remission (CR) – Defined as morphologic leukemia-free state, 
defined as less than 5% blasts in aspirate with marrow spicules and a count of ≥200 
nucleated cells and no blasts with Auer rods or any persistence of extramedullary disease. 
When erythroid precursors constitute less than 50% of bone marrow nucleated cells, the 
percentage of blasts is based on all nucleated cells; when there are 50% or more erythroid 
cells, the percentage blasts should be based on the nonerythroid cells. The presence of mild 
megaloblastoid changes may be permitted if they are thought to be consistent with 
treatment effect. However, persistence of pretreatment abnormalities (eg, pseudo-Pelger-
Hüet cells, ringed sideroblasts, dysplastic megakaryocytes) are not consistent with CR.  In 
addition, ANC >1000/μl and platelet count of ≥100,000/μl. Patient must be independent of 
transfusions. No duration requirement for this designation. 
 
Cytogenetic complete remission (CCR) – Defined as morphologic complete remission plus 
a reversion to a normal karyotype. For this study, reversion of a normal karyotype is 
defined as no clonal abnormalities detected in a minimum of 20 mitotic cells. 
 
Morphologic complete remission with incomplete blood count recovery (CRi) – Defined as 
morphologic complete recovery with the exception of neutropenia <1000/μl or 
thrombocytopenia <100,000/μl.  
 
Partial remission (PR) – Defined as meeting the above criteria for complete remission, 
with the following exceptions: decrease of ≥50% in the percentage of blast to 5-25% in the 
bone marrow aspirate. A value of 5% blasts in bone marrow with Auer rods present may 
also be considered a partial remission.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, CRi, PR, PD, or 
recurrence/morphologic relapse.   
 
Progressive disease (PD) – Defined as a clear increase in bone marrow blast count, 
typically a greater than 50% increase compared with baseline, or new extramedullary 
disease. Patients with a > 50% increase in peripheral blast count with an increase of the 
total peripheral white blood cell count to > 10,000/l should undergo evaluation by bone 
marrow biopsy to access for progressive disease.  
 
Recurrence/morphologic relapse - Defined as relapse following complete remission is 
defined as reappearance of blasts in the blood or the finding of ≥5% blasts in the bone 
marrow, not attributable to any other cause. New dysplastic changes is considered relapse. 


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If there are no blasts in the peripheral blood and 5-20% blasts in the bone marrow, bone 
marrow biopsy should be repeated in > 1 week to confirm relapse. 
 
9.2 
Response Criteria - MDS 
 
MDS Patients will be assessed for response according to the IWG criteria:69 
 
Complete remission (CR) – Defined as < 5% myeloblasts with normal maturation of all 
cell lines in the bone marrow and peripheral blood values of Hgb > 11g/dL, Platelets > 100 
x 109/L, Neutrophils > 1.0 x 109/L, and 0% blasts. Persistent dysplasia does not exclude 
CR but will be noted.  
 
Marrow Complete Response (Marrow CR) – Defined as < 5% myeloblasts in the bone 
marrow and a decrease by > 50% from pretreatment values, but not meeting the definition 
of CR above.  
 
Partial Remission (PR) – Defined as meeting the definition of CR above with a decrease 
of myeloeblasts in the bone marrow by > 50% from pretreatment values, but absolute 
myeloblasts still >5%.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, Marrow CR, PR, SD, 
PD, or recurrence/morphologic relapse.   
 
Progressive Disease//Relapse (PD) – Defined as ≥ 50% increase in blasts to > 5% blasts 
(for patients with less than 5% blasts at baseline only), ≥ 50% increase to > 10% blasts (for 
patients with 5-10% blasts at baseline only), ≥ 50% increase to > 20% blasts (for patients 
with l0-20% blasts at baseline only), ≥ 50% increase to > 30% blasts (for patients with 20-
30% blasts at baseline only) or any of the following: At least 50% decrement from 
maximum remission/response in granulocytes or platelets, reduction in Hgb by ≥ 2 g/dL, 
or New or worsened transfusion dependence not related to study drug toxicity. Or for 
patients with a CR, Marrow CR, or PR as defined above and subsequently development of 
one of the following: Return to pretreatment bone marrow blast percentage, decrement of 
≥ 50% from maximum remission/response levels in granulocytes or platelets, or reduction 
in Hgb concentration by ≥ 1.5 g/dL or transfusion dependence. 
 
9.2.1 Hematologic Improvement (HI) 
 
 
 
Progressive disease as defined above nullifies hematologic improvement.  
 
Erythroid response requires all of the following (only required if pretreatment HgB 
<11 g/dL): 
 Hgb increase by ≥ 1.5 g/dL 
 Relevant reduction of units of RBC transfusions by an absolute number of at 
least 4 RBC transfusions/8 wk compared with the pretreatment transfusion 
number in the previous 8 wk. Only RBC transfusions given for a Hgb of ≤ 9.0 
g/dL pretreatment will count in the RBC transfusion response evaluation 


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Platelet response requires one of the following (only required if pretreatment 
platelets < 100 x 109/L):  
 Absolute increase of ≥ 30 x 109/L (for patients starting with > 20 x 109/L 
platelets) 
 Increase from < 20 x 109/L to > 20 x 109/L and absolute increase > 100% (for 
patients starting with < 20 x 109/L) 
 
Neutrophil response requires the following (only required if pretreatment ANC 
<1.0 x 109/L):  
 At least 100% increase and an absolute increase > 0.5 x 109/L 
 
9.3.1 Cytogenetic Response  
 
Complete Cytogenetic Response – Defined as reversion to a normal karyotype. For 
this study, reversion of a normal karyotype is defined as no clonal abnormalities 
detected in a minimum of 20 mitotic cells. Progressive disease as defined above 
nullifies cytogenetic response.  
 
9.3 
Guidelines for Evaluation of Disease 
 
Bone marrow biopsy should be performed on Day 28 ± 4 of cycles 1, 2, 4 and 6 for clinical 
disease assessment. Following Cycle 6, bone marrow biopsies should be performed at the 
discretion of the treating physician to assess for relapse or progression. 
 
9.4 
Other Secondary Efficacy Measures 
 
9.4.1 Treatment Failure, Overall Survival and Event-free Survival 
 
Treatment failure is defined as failure to achieve a complete or partial remission 
following 4 cycles of therapy.  
 
Overall survival is defined as the date of first dose of study drug to the date of death 
from any cause.  Patients still alive at the time of the final analysis will be right-
censored. 
 
Event-free survival is defined as the interval from the date of first dose of study 
drug to date of treatment failure, recurrence, death due to any cause, or loss to 
follow up. 
 
 


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9.4.2 Duration of Remission and Relapse-free Survival for Patients Having 
Complete Remission 
 
Duration of complete remission is defined as the interval from the date that 
complete remission is documented to the date of recurrence, death due to any cause, 
or loss to follow-up.  
 
Relapse-free survival is defined only for those patients achieving a complete 
remission. It is defined as the interval from the date of first documentation of a 
leukemia-free state to the date of recurrence, death due to any cause, or loss to 
follow up.   
  
9.4.3 Safety 
 
Safety evaluation will include assessments of adverse experiences, medical history, 
physical examinations, vital signs, and laboratory assessments at baseline and 
throughout the study period. Local laboratories may be used for this study.  
 


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10.0 
REGULATORY AND REPORTING REQUIREMENTS 
 
The entities providing oversight of safety and compliance with the protocol require reporting as 
outlined below. 
 
The Washington University Human Research Protection Office (HRPO) requires that all events 
meeting the definition of unanticipated problem or serious noncompliance be reported as outlined 
in Section 10.2. 
 
The FDA requires that all serious and unexpected adverse events be reported as outlined in Section 
10.6.  In addition, any fatal or life-threatening adverse experiences where there is a reasonable 
possibility of relationship to study intervention must be reported. 
 
10.1 
Definitions 
 
10.1.1 Adverse Events (AEs) 
 
Definition:  any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading:  the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for 
all toxicity reporting.  A copy of the CTCAE version 4.0 can be downloaded from 
the CTEP web site:  http://ctep.cancer.gov/reporting/ctc.html.  Grade 1-2 events 
will be recorded for the first fifty enrolled patients. Subsequently, only events 
graded as 3 or greater will be recorded. Fifty patients should be sufficient to 
estimate the true incidence of any adverse event to within ± 14% with 95% 
confidence intervals. 
 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the 
terms listed that should be used are those provided by the Department of Health 
and Human Services’ Office for Human Research Protections (OHRP).  A copy of 
this 
guidance 
can 
be 
found 
on 
OHRP’s 
website: 
(http://www.hhs.gov/ohrp/policy/AdvEvntGuid.htm). 
 
10.1.2 Serious Adverse Event (SAE) 
 
Definition:  any adverse drug experience occurring at any dose that results in any 
of the following outcomes: 
o Death 
o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial disruption 
of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 


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o Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed above 
 
All unexpected SAEs must be reported to the FDA. 
 
10.1.3 Unexpected Adverse Experience 
 
Definition: any adverse drug experience, the specificity or severity of which is not 
consistent with the current investigator brochure (or risk information, if an IB is not 
required or available). 
 
Events that are both serious AND unexpected must be reported to the FDA.  
 
10.1.4 Life-Threatening Adverse Experience  
 
Definition: any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that, had it occurred in a more severe form, might have caused 
death. 
 
Life-threatening adverse experiences must be reported to the FDA. 
 
10.1.5 Unanticipated Problems 
 
Definition: 
 
 unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the 
IRB-approved research protocol and informed consent document; and (b) 
the characteristics of the subject population being studied; 
 related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the 
incident, experience, or outcome may have been caused by the procedures 
involved in the research); and 
 suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was 
previously known or recognized. 
 
10.1.6 Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that 
govern human subjects research or failure to follow the determinations of the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate 
choice to ignore regulations, institutional policies, or determinations of the IRB. 
 


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10.1.7 Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial 
harm to subjects or others, or that materially compromises the rights or welfare of 
participants. 
 
10.1.8 Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under the 
research team’s control. Exceptions apply only to a single participant or a singular 
situation. 
 
Local IRB Pre-approval of all protocol exceptions must be obtained prior to the 
event.  For secondary sites, the Washington University PI will issue approval of the 
exception, but it must also be submitted to the local IRB with documentation of 
approval forwarded to Washington University.  Washington University IRB 
approval is not required for protocol exceptions occurring at secondary sites. 
 
10.2 
Reporting to the Human Research Protection Office (HRPO) at Washington 
University: 
 
The PI is required to promptly notify the IRB of the following events: 
 
 Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct of 
the study. 
 Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 
 Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence of the 
event or notification to the PI of the event.  The death of a research participant that qualifies 
as a reportable event should be reported within 1 working day of the occurrence of the 
event or notification to the PI of the event. 
 
10.3 
Reporting to the Quality Assurance and Safety Monitoring Committee 
(QASMC) at Washington University 
 
The PI is required to notify the QASMC of any unanticipated problem occurring at WU or 
any BJH or SLCH institution that has been reported to and acknowledged by HRPO as 
reportable.  (Unanticipated problems reported to HRPO and withdrawn during the review 
process need not be reported to QASMC.) 
 
QASMC must be notified within 10 days of receipt of IRB acknowledgment via email to 
a QASMC auditor. 


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10.4 
Reporting Requirements for Secondary Sites 
 
The research team at each secondary site is required to promptly notify the Washington 
University PI and research coordinator of all reportable events (as described in Section 7.6) 
within 1 working day of the occurrence of the event or notification of the secondary site’s 
PI of the event.  This notification may take place via email if there is not yet enough 
information for a formal written report (using either an FDA MedWatch form if required 
or an institutional SAE reporting form if not).  A formal written report must be sent to the 
Washington University PI and research coordinator within 10 working days of the 
occurrence of the event or notification of the secondary site’s PI of the event.  The death 
of a research participant that qualifies as a reportable event should be reported within 1 
working day of the occurrence of the event or notification of the secondary site’s PI of the 
event. 
 
The research team at a secondary site is responsible for following its site’s guidelines for 
reporting applicable events to its site’s IRB according to its own institutional guidelines.  
The research team at Washington University is responsible for reporting all applicable 
events to the FDA.  
 
10.5 
Reporting to Secondary Sites 
 
The Washington University PI (or designee) will notify the research team at each secondary 
site of all reportable events that have occurred at other sites within 10 working days of the 
occurrence of the event or notification of the PI of the event.  This includes events that take 
place both at Washington University and at other secondary sites, if applicable. 
 
10.6 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER 
FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the Washington University principal investigator to report any 
unanticipated problem to the FDA as follows: 
 
 Report any unexpected fatal or life-threatening adverse experiences (Section 
10.1.4) associated with use of the drug by telephone or fax no later than 7 calendar 
days after initial receipt of the information.   
 Report any serious, unexpected adverse experiences (Section 10.1.2), as well as 
results from animal studies that suggest significant clinical risk within 15 calendar 
days after initial receipt of this information.   
 
All MedWatch forms will be sent by the investigator or investigator’s team to the FDA at 
the following address or by fax: 
 
Food and Drug Administration 


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Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
Secondary sites must submit a completed MedWatch form to the Washington University 
PI and research coordinator within 4 calendar days (for fatal or life-threatening adverse 
experiences) or 11 calendar days (for serious, unexpected adverse experiences).  The 
Washington University PI will be responsible for submitting all MedWatch forms from 
secondary sites to the FDA within the timeframes specified above. 
 
10.7 
Timeframe for Tracking Reportable Events:  
 
Adverse events will be tracked for 30 days following the last day of study treatment, or 
until the start of a new chemotherapy/treatment, whichever comes first. 


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11.0 
DATA SAFETY MONITORING PLAN 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, the 
Data and Safety Monitoring Committee (DSMC) will meet to review toxicity data at least every 6 
months following the activation of the first secondary site.  The report will be prepared by the 
statistician and will be submitted to the Quality Assurance and Safety Monitoring Committee 
(QASMC).  This report will include: 
 
 HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
 Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of study 
 History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol exceptions, 
error, or breach of confidentiality including start/stop dates and reason 
 Study-wide target accrual and study-wide actual accrual including numbers from 
participating sites 
 Protocol activation date at each participating site 
 Average rate of accrual observed in year 1, year 2, and subsequent years at each 
participating site 
 Expected accrual end date and accrual by site 
 Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
 Measures of efficacy 
 Early stopping rules with supporting data and list the number of participants who have 
met the early stopping rules 
 Summary of toxicities at all participating sites 
 Abstract submissions/publications 
 Summary of any recent literature that may affect the safety or ethics of the study  
 
Until such a time as the first secondary site activates this protocol, a semi-annual DSM report to 
be prepared by the study team will be submitted to the QASM Committee beginning 6 months 
after study activation at Washington University. 
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC according 
to institutional guidelines. 
 
A DSMC will consist of no fewer than 3 members including 2 clinical investigators and a 
biostatistician.  Like investigators, DSMC members are subject to the Washington University 
School of Medicine policies regarding standards of conduct. Individuals invited to serve on the 
DSMC will disclose any potential conflicts of interest to the trial principal investigator and/or 
appropriate university officials, in accordance with institution policies. Potential conflicts that 
develop during a trial or a member’s tenure on a DSMC must also be disclosed.  


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Refer to the Washington University Quality Assurance and Data Safety Monitoring Committee 
Policies and Procedures for full details on the responsibilities of the DSMC at 
http://www.siteman.wustl.edu/uploadedFiles/Research_Programs/Clinical_Research_Resources/
Protocol_Review_and_Monitoring_Committee/QASMCQualityAssurcance.pdf  
 
 


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12.0 
AUDITING 
 
Since Washington University is the coordinating center, each site will be audited annually by 
Siteman Cancer Center personnel (QASMC) unless the outside institution has an auditing 
mechanism in place and can provide a report. The outside sites will be asked to send copies of all 
audit materials, including source documentation.  The audit notification will be sent to the 
Washington University Research Patient Coordinator, who will obtain the audit materials from the 
participating institution. 
 
Notification of an upcoming audit will be sent to the research team one month ahead of the audit. 
Once accrual numbers are confirmed, and approximately 30 days prior to the audit, a list of the 
cases selected for review (up to 10 for each site) will be sent to the research team. However, if 
during the audit the need arises to review cases not initially selected, the research team will be 
asked to provide the additional charts within two working days. 
 
Additional details regarding the Auditing Policies and Procedures can be found at 
https://siteman.wustl.edu/wp-content/uploads/2015/QASMC-Policies-and-Procedures-
03.31.2015.pdf  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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13.0 
MULTICENTER REGULATORY REQUIREMENTS 
 
Washington University requires that each participating site sends its informed consent document 
to be reviewed and approved by the Washington University Regulatory Coordinator (or designee) 
prior to IRB/IEC submission.    
 
Site activation is defined as when the secondary site has received official written documentation 
from the coordinating center that the site has been approved to begin enrollment.  At a minimum, 
each participating institution must have the following documents on file at Washington University 
prior to study activation: 
 
 Documentation of IRB approval of the study in the form of a letter or other official 
document from the participating institution’s IRB.  This documentation must show 
which version of the protocol was approved by the IRB. 
 Documentation of IRB approval of an informed consent form. The consent must 
include a statement that data will be shared with Washington University, including the 
Quality Assurance and Safety Monitoring Committee (QASMC), the DSMC (if 
applicable), and the Washington University study team. 
 Documentation of FWA, signed FDA Form 1572 (if applicable), and the CVs of all 
participating investigators. 
 Protocol signature page signed and dated by the investigator at each participating site. 
 
The coordinating center Principal Investigator (or designee) is responsible for disseminating to the 
participating sites all study updates, amendments, reportable adverse events, etc.  Protocol/consent 
modifications and IB updates will be forwarded electronically to the secondary sites within 4 
weeks of obtaining Washington University IRB approval.  Activated secondary sites are expected 
to submit protocol/consent/IB modifications to their local IRBs within 4 weeks of receipt unless 
otherwise noted.  Upon the secondary sites obtaining local IRB approval, documentation of such 
shall be sent to the Washington University study team within 2 weeks of receipt of approval. 
 
Documentation of participating sites’ IRB approval of annual continuing reviews, protocol 
amendments or revisions, all SAE reports, and all protocol violations/deviations/exceptions must 
be kept on file at Washington University. 
 
The investigator or a designee from each institution must participate in a regular conference call 
to update and inform regarding the progress of the trial. 
 
 


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14.0 
STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
 
14.1 
Analysis Populations 
 
Intent-to-Treat - The intent-to-treat (ITT) population will be defined as all patients who 
receive at least one dose of decitabine.  
 
Efficacy-Evaluable - The efficacy-evaluable (EE) population will be defined as all patients 
who receive at least one cycle of treatment.   
 
Safety - The safety population will be defined as all patients with at least one dose of 
decitabine. 
 
14.2 
Statistical Analysis 
 
14.2.1 Descriptive Analyses 
 
Patient Disposition: The number of patients discontinued, the reasons for 
discontinuation, and the number of cycles administered will be summarized. 
 
Demographics and Baseline Characteristics: Descriptive summary statistics will 
be provided for demographic and important baseline characteristics.  For 
continuous variables, the number of patients, mean, standard deviation, median, 
minimum and maximum will be provided.  For categorical variables, the number 
and percent of patients in each demographic/characteristic category will be 
summarized. 
 
14.2.2 Primary Endpoint Analysis 
 
The primary end-point of this phase II study is the correlation biomarkers with 
overall response in AML/MDS patients to decitabine. 
 
We will correlate the presence of patient specific mutations (especially mutations 
in DNMT3A, IDH1, IDH2, and TET2) with overall response rates. Mutation rates 
in these genes and in other patient-specific genes will be correlated with overall 
response and their respective 95% confidence intervals will be provided. 
 
Statistical considerations. The relationship between AML/MDS-associated 
mutations and the responsiveness to decitabine will be described by contingency 
tables and assessed using Chi-square test or Fisher’s exact test as appropriate. The 
differentially expressed genes between responders and non-responders will be 
determined using the significance analysis of microarray (SAM). Multivariate 
logistic regression will also be fit to adjust the potential confounding effects of other 
demographic and clinical characteristics. The resultant critical P values will be 
corrected for multiple testing.  Since the Bonferroni correction is known to be too 
conservative, a permutation analysis (e.g., 10,000 random assignments for 


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responsive status) will be used to derive an empirical critical P value.  The new 
critical p-value is the number of permutation tests which yield a lower p-value than 
the original test divided by the total number of permutation tests.[61]   
 
A power analysis was performed using Chi-square test for the association between 
mutation and response to decitabine. The frequency of DNMT3A, IDH1/2, and 
TET2 mutations in AML and MDS patients are each between 15% and 30% in 
multiple cohorts.[29-37] Table 3 shows the minimal detectable differences of 
response rates between mutant and wild-type cases, assuming an expected overall 
45% CR/CRi rate and 80% power. For an average of 20% frequency of a mutated 
gene, for example, 100 patients will provide 80% power to detect 30% difference 
at a 2-sided 0.1 alpha error. The purpose of this study is to determine whether any 
of these mutations result in a 
clinically 
significant 
association 
that 
would 
warrant pre-treatment testing. 
We believe that a reasonable 
cut off would be if at least 
30% of clinical response 
could be attributed to the 
presence 
of 
a 
specific 
mutation. 
However, 
we 
anticipate that more power 
could be achieved as we 
assess patient groups with 
combined characteristics (e.g. 
DNMT3A mutation or loss of 
DNMT3A 
expression). 
In 
addition, we expect that the 
majority of the enrolled 125 
patients can be used in the analysis (since all of our molecular information will be 
acquired within 30 days of enrollment, we anticipate a drop-out rate of 20% or less). 
 
14.2.3 Secondary Endpoint Analyses 
 
The overall response rate (CR/CRi/PR) and complete response rate (CR/CRi) will 
be compared with historical controls[2], and the 95% confidence intervals will be 
provided. 
 
The rate of patient specific mutation clearance after 10, 28, and 56 days of therapy 
will be determined using an Illumina based platform. We will compare the rate of 
mutation clearance between the patients who achieve a CR/CRi after 4 cycles and 
those who do not. We anticipate that we will identify between 6 and 25 exonic 
SNVs per case (the range obtained with >200 AML samples sequenced to date with 
whole genome or exome sequencing), and that the expected 95% confidence 
interval of the mean variant allele frequency will be ±2.56% and ±1.25%, 
Table 3. Detectable mutation associated response rates (80% power, α = 0.05) 
 
Total 
N 
% of patients 
with a given 
mutation  
Detectable 
response 
difference 
Sensitivity of mutation to 
predict response at minimum 
detectable response difference* 
100 
15%  
20%  
30% 
40% 
50% 
0.40 
0.35 
0.30 
0.27 
0.26 
80% 
75% 
67% 
61% 
58% 
* Assuming the overall response  rate (CR/Cri) of 45%   


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respectively, if we assume an anticipated standard deviation of 3.2% within the 
same clone. The change of tumor burden over time and its association with clinical 
response will  be assessed using 2-way ANOVA for repeated measurement data, 
followed by the ad-hoc multiple comparisons for between-group differences (e.g., 
responders vs. non-responders) at given time points as well as the between-time 
differences within each group. The resultant critical P values will also be corrected 
using permutation analysis for multiple testing. 
 
The percent decrease in bone marrow methylcytosine will be calculated on day 
10±1. We will compare this reduction with previously published data and between 
the patients who achieve CR/CRi and those who do not. The correlation of peak 
plasma levels of decitabine with response will be assessed using 2-sample t-test or 
Mann-Whitney non-parametric rank-sum test as appropriate. The change of total 
bone marrow DNA methylcytosine from baseline and its association with both 
steady-state plasma drug levels and response will be assessed using 2-way ANOVA 
for repeated measurement data, followed by the ad-hoc multiple comparisons for 
between-group differences (e.g., responders vs. non-responders) and between-time 
differences. Similar analyses will be performed for the correlation of expression 
profiles and SNP analysis with pharmacokinetic data.   
 
Statistical power justification.  A power analysis was performed using two-sample 
t-test for between-group difference and paired t-test for the change over time. For 
tumor clearance, this will provide > 99% power to detect a 10% difference in the 
means at two separate time points for clones with 6 or more variants, and > 98% 
power to detect a 15% difference if there are 3 or more variants per clone with α = 
0.05 (power will decrease to 91% and 82%, respectively, in cases where the 
standard deviation is 4.7%, the maximum we have observed thus far). For 
pharmacologic correlations, we anticipate 85% power to determine a 25 ng/ml 
difference between the average Cmax of 45 evaluable responders and 55 evaluable 
non-responders with an anticipated average Cmax of 70 ng/ml and a standard 
deviation of 40 ng/ml within the entire cohort, and with α = 0.05.[22] We believe a 
less significant difference would not be sufficient to warrant application in clinical 
decision making (i.e. whether to continue therapy for more than 1 cycle). We 
anticipate 99% power to determine a difference of 20% in the reduction of bone 
marrow methylcytosine between 45 evaluable responders and 55 evaluable non-
responders with an expected standard deviation of 15% and α = 0.05.[28, 66-68] 
The difference in methylcytosine reduction previously observed was 30% between 
patients achieving CR/PR and those with only hematologic improvement.[28]  
 
Time to relapse and time to death will also be listed for each patient. 
 
14.2.4 Safety Analysis 
 
Extent of Exposure – Duration (number of cycles/infusions) of decitabine doses 
(cumulative and intensity) will be summarized. 
 


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Adverse Events - All adverse events will be coded using NCI CTCAE v. 4.0.  The 
incidence of treatment-emergent adverse events (number and percent of patients 
reporting the adverse event at least once during the study) will be summarized.  In 
addition, adverse events will be summarized by investigator attribution of 
relationship to study medication and by grade.  Similar summaries will be provided 
by treatment cycle. 
 
Clinical Laboratory Evaluation – Applicable laboratory parameters will be graded 
according to NCI CTCAE version 4.0. The incidence of maximum grade (number 
and percent of patients experiencing the maximum grade during the study) will be 
summarized for each laboratory parameter.  Similar summaries will be provided by 
treatment cycle.  Graphical displays of select hematological parameters will also be 
provided. 
 
Vital Signs, Physical Findings, and Other Safety Observations – Vital signs, 
findings from physical examinations, ECOG scores and weights will be presented 
in listing format. 
 
14.2.5 Description of Planned Subgroup Analyses 
 
The following subgroups (if sufficient numbers of patients) will be explored with 
respect to the primary and overall survival endpoints. 
 Age (60-69 years old vs ≥ 70 years old) 
 Cytogenetics 
 Patients with AML-associated mutations in DNMT3A, TET2, IDH1, or 
IDH2 vs those who do not have somatic variants in these genes. 
 
14.3 
Sample Size 
 
We anticipate initially enrolling 25 patients at Washington University. Once we establish 
a routine exome analysis with validation and calculation of the rate of patient-specific 
mutation clearance, we will enroll an additional 100 patients from at least 2 additional 
centers.  
 


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15.0 
DATA SUBMISSION SCHEDULE 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to registration 
Patient Registration Form 
Disease Diagnosis Form 
Inclusion Criteria Worksheet 
Exclusion Criteria Worksheet 
 
Prior to starting treatment 
Treatment Record Form 
To be maintained throughout a patient’s treatment 
cycle.  The complete record is due at completion of 
treatment. 
Bone Marrow Sample Collection 
Form 
To be maintained throughout the course of the study 
at designated bone marrow sample collections time 
points (See Appendix 2). 
Pathology: Bone Marrow Biopsy 
To be completed upon the return of pathology 
report for each bone marrow biopsy. 
Study Calendar Form 
To be completed with the date of each time point 
upon occurrence. 
Labs Form 
To be completed upon the return of laboratory 
reports for each study time point (See Appendix 2) 
Dose Modifications 
To be maintained throughout the course of the study 
if dose modifications are performed. 
Therapy Response Form 
To be filled out upon remission (prior to, and/or on 
therapy) 
End of Study Form 
 
Due at the completion of the EOS visit, at the time 
of death, and/or at the time that the patient is lost to 
follow-up 
Follow Up Form 
 30 days post date of last decitabine dose. 
 Every 6 months for 2 years after the last dose of 
study drug. 
Record of Adverse Events 
At the time of any AE 
 
Any queries generated by Washington University must be responded to within 28 days of receipt 
by the participating site.  The Washington University research team will conduct a regular review 
of data status at all secondary sites, with appropriate corrective action to be requested as needed. 
 


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Appendix 1 
ECOG Performance Scale 
Activity Status 
Description 
0 
Asymptomatic, fully active, and able to carry on all predisease 
performance without restrictions. 
 
1 
Symptomatic, fully ambulatory but restricted in physically strenuous 
activity and able to carry out performance of a light or sedentary 
nature, eg, light housework, office work. 
 
2 
Symptomatic, ambulatory and capable of all self-care but unable to 
carry out any work activities.  Up and about more than 50% of 
waking hours: in bed less than 50% of day. 
 
3 
Symptomatic, capable of only limited self-care, confined to bed or 
chair more than 50% of waking hours, but not bedridden. 
 
4 
Completely disabled.  Cannot carry on any self-care.   Totally 
bedridden. 
 
5 
Dead 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 7 Version date: 11/15/2016  
Page 47 of 52 
Appendix 2 
Study Flow Chart 
Study Flow Chart 
Test 
Pre-
study1 
Base-
line2 
All 
Cycles 
Day 1 
All Cycles 
Days 1-103 
Cycle 1 Day 
4 ± 1 
 
Cycle 1 
Day 10 ± 1 
and 28 ± 4 
Every 
week 
Every 
2 
weeks 
Even 
cycles 
Day 28±4 
End of 
Study 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Medical History 
 
X 
 
 
 
 
 
 
 
 
Physical Exam 
 
Xa 
Xb 
 
 
 
 
 
 
X 
Transfusion 
Requirementsc 
 
Xc 
Ongoing 
Bone Marrow 
Aspirate and Biopsy 
Xd 
 
 
 
 
Xd 
 
 
Xd 
Xd 
Flow Cytometrye 
X 
 
 
 
 
 
 
 
 
 
Pregnancy test 
 
X 
 
 
 
 
 
 
 
 
CBC with differential 
and platelets 
 
X 
X 
 
 
 
Xf 
 
 
X 
Serum Chemistriesg 
 
X 
X 
 
 
 
 
Xf 
 
X 
Decitabine 
administrationh 
 
 
 
X 
 
 
 
 
 
 
Adverse Events  
 
 
Ongoing 
Post-study follow-up 
 
 
 
 
 
 
 
 
 
Xi 
1 To be performed within 28 days of the first dose of decitabine.  
2 To be performed within 7 days of the first dose of decitabine.  
3 Decitabine may be reduced to Days 1-5 or Days 1-3. See Section 6.3 for details.  
a To include height, weight, and ECOG. 
b To include weight. 
c Care should be given to record the frequency of transfusions from 8 weeks prior to first dose of study drug through 
the duration of the study. 
d Bone marrow biopsy and aspirate should be performed at baseline, at Cycle 1 Day 10 ± 1, and at  Day 28 ± 4 of Cycles 
1, 2, 4 and 6 for clinical disease assessment. Patients who achieve with clinical response (CRi or better) and are receiving 
cycles every 6 weeks, will undergo bone marrow biopsies on day 42 ± 4. Following Cycle 6, bone marrow biopsies 
should be performed at the discretion of the treating physician to assess for relapse or progression. Standard bone marrow 
procurement procedures will be followed for collection of the tissue. To include: correlative studies (collected under 
HRPO# 201011766), morphologic assessment, cytogenetics and FISH at all time points. A skin biopsy sample (collected 
under HRPO# 201011766) is required at pre-study, but may have been collected months or even years prior to first dose 
of decitabine.  
e On bone marrow or peripheral blood.  
f After Cycle 1, these labs may be obtained every other week (CBC) or once a month (Chemistries) at the discretion of 
the treating physician. 
g Serum chemistries include bicarbonate, calcium, glucose, total protein, albumin, BUN, potassium, chloride, sodium, 
creatinine, total bilirubin, AST, ALT, alkaline phosphatase, and LDH. 
h Decitabine treatment will be 20 mg/m2 over a 1-hour IV infusion on consecutive Days 1-10 every 28-day cycle.   The 
dose will be calculated based on actual patient weight at the beginning of each cycle. Patients may be premedicated 
with lorazepam, prochlorperazine, palonosetron, or ondansetron at the treating physician’s discretion. 
i Patients will be followed for  30 days following the date of the last decitabine dose to monitor for toxicity, survival 
and response. Thereafter, the investigator or designee will record information on subsequent therapy, disease 
progression, and death, every 6 months for 2 years after first dose of study drug or until the patient is lost to follow-up.


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 7 Version date: 11/15/2016  
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16.0 
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Genomic Predictors of Decitabine Response in AML/MDS 
 
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Pathogenesis of Malignant Evolution. ASH Annual Meeting Abstracts, 2008. 
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62. 
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Genomic Predictors of Decitabine Response in AML/MDS 
 
 
NCT# NCT01687400 
 
 
Document Version Date:  07/21/2016 
 
IRB Approval Date:  08/17/2016 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 6 Version date: 07/21/2016  
Page 1 of 52 
 
 
Genomic Predictors of Decitabine Response in AML/MDS 
 
Washington University School of Medicine, Division of Oncology 
660 South Euclid Avenue, Box 8056, St. Louis, MO 63110 
 
Protocol #: 201210102 
Protocol Version Date: 07/21/2016 
 
Coordinating Center: Washington University School of Medicine 
 
Principal Investigator 
John Welch, M.D./Ph.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-2626, Fax Number: (314) 362-9333 
Email: jwelch@wustl.edu 
 
Sub-Investigator 
Timothy Ley, M.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-8832, Fax Number: (314) 362-9333 
Email: timley@wustl.edu 
 
Pathology 
Eric Duncavage, M.D. 
Department of Anatomic and Molecular Pathology 
Telephone: (314) 362-8832 
Email: eduncavage@wustl.edu  
 
Statistician 
Feng Gao, Ph.D. 
Division of Biostatistics 
Telephone: (314) 362-3682 
E-mail: feng@wustl.edu 
 
IND# 116548 //Clinical Trials.gov # NCT01687400 
 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to obtain 
informed consent, may not be disclosed to another party unless law or regulations require such disclosure.  Persons 
to whom the information is disclosed must be informed that the information is confidential and may not be further 
disclosed by them. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 6 Version date: 07/21/2016  
Page 2 of 52 
Genomic Predictors of Decitabine Response in AML/MDS  
Principal Investigator Signature Page 
 
 
Principal Investigator: 
 
John Welch, M.D., Ph.D. 
 
 
 
 
 
Signature of Investigator 
 
Date 
 
 
 
Printed Name of Investigator 
 
 
 
By my signature, I agree to personally supervise the 
conduct of this study and to ensure its conduct in 
compliance with the protocol, informed consent, 
IRB/HRPO procedures, the Declaration of Helsinki, ICH 
Good Clinical Practices guidelines, and the applicable 
parts of the United States Code of Federal Regulations or 
local regulations governing the conduct of clinical 
studies. 
 
 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 6 Version date: 07/21/2016  
Page 3 of 52 
 
Study Schema: 
 
 
Patients with progressive disease after two cycles (defined as a clear increase in bone marrow blast count, typically a 
> 50% increase in bone marrow blasts over baseline) should be removed from study. 
Patients with stable disease may continue on study and should undergo a diagnostic bone marrow biopsy at the end 
of even numbered cycles. 
Once AML patients achieve morphologic complete remission with incomplete blood count recovery (CRi) or MDS 
patients achieve a marrow complete response (Marrow CR) as defined in Section 9.0, decitabine 
administration may be decreased to 20 mg/m2/day for ten consecutive days every 6 weeks, to limit 
myelosuppressive toxicity and improve count recovery. After 2 cycles at 6 week intervals, the dose may be 
reduce to 20 mg/m2/day for five consecutive days in 6 week cycles, at the discretion of the treating physician. 
After two cycles of five consecutive days of treatment, if the patient meets the definition of CR with complete 
cytogenetic response as defined in Section 9.0, administration may further be decreased to three consecutive 
days per cycle at the discretion of the treating physician. 
Sample collection for correlative studies: 
Bone marrow biopsies should be performed at baseline, at Cycle 1 Day 10 ± 1, and at Day 28 ± 4 of Cycles 
1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be performed at the discretion of the 
treating physician to assess for relapse or progression.  
 
 
20 
mg/m
2/day  
Cycle 1 
20 
mg/m
2/day 
Cycle 2+ 
Day 1 
Day 28 
BM Bx 
PB 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 6 Version date: 07/21/2016  
Page 4 of 52 
 
Genomic Predictors of Decitabine Response in AML/MDS 
Table of Contents 
Section 
 
Page 
1.0 INTRODUCTION .................................................................................. 7 
1.1 
Rationale ............................................................................................ 7 
1.2 
Decitabine and Hypomethylating agents in AML/MDS ................... 9 
1.2.1 
Pharmacokinetics and Metabolism of Decitabine ................... 11 
1.2.2 
Experience with extended dosing schedules ........................... 12 
1.3 
Correlative Studies .......................................................................... 13 
2.0 OBJECTIVES ....................................................................................... 15 
2.1 
Primary Objective ............................................................................ 15 
2.2 
Secondary Objectives ...................................................................... 15 
3.0 PATIENT SELECTION ....................................................................... 16 
3.1 
Inclusion Criteria ............................................................................. 16 
3.2 
Exclusion Criteria ............................................................................ 16 
3.3 
Inclusion of Women and Minorities ................................................ 17 
4.0 REGISTRATION PROCEDURES ...................................................... 18 
4.1 
Confirmation of Patient Eligibility .................................................. 18 
4.2 
Patient Registration in the Siteman Cancer Center OnCore 
Database .......................................................................................... 18 
4.3 
Assignment of UPN ......................................................................... 18 
5.0 INVESTIGATIONAL PLAN .............................................................. 20 
5.1 
Summary of Study Design............................................................... 20 
5.2 
Study Procedures ............................................................................. 21 
5.2.1 
Pre-study Procedures ............................................................... 21 
5.2.2 
Baseline Evaluation ................................................................. 21 
5.2.3 
Day 1 of Each Cycle ............................................................... 21 
5.2.4 
Cycle 1 Days 1-10 ................................................................... 21 
5.2.5 
Cycle 1 Day 10 ± 1 ................................................................. 22 
5.2.6 
Weekly .................................................................................... 22 
5.2.7 
Every Other Week ................................................................... 22 
5.2.8 
Cycles 1, 2, 4 and 6 Day 28 ± 4 days ...................................... 22 
5.2.9 
End-of-Study Procedures ........................................................ 22 
5.2.10 
Post-study Follow-up .............................................................. 23 
5.3 
Duration of Therapy ........................................................................ 23 


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5.4 
Concomitant Therapy and Supportive Care Guidelines .................. 23 
5.4.1 
Chemotherapy ......................................................................... 23 
5.4.2 
Growth Factors ........................................................................ 24 
5.4.3 
Transfusions ............................................................................ 24 
5.4.4 
Prophylactic Antimicrobial Agents ......................................... 24 
5.4.5 
Radiotherapy ........................................................................... 24 
6.0 DOSE MODIFICATIONS ................................................................... 25 
6.1 
Delay for Cytopenia or Infection ..................................................... 25 
6.2 
Delay for Organ Dysfunction .......................................................... 25 
6.3 
Modification in Dose or Schedule for Treatment Response ........... 25 
7.0 PHARMACEUTICAL INFORMATION ............................................ 26 
7.1 
Decitabine ........................................................................................ 26 
7.1.1 
Study Drug Preparation ........................................................... 26 
7.1.2 
Supplier ................................................................................... 26 
8.0 CORRELATIVE STUDIES SAMPLE COLLECTIONS .................... 27 
9.0 EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES28 
9.1 
Response Criteria - AML ................................................................ 28 
9.2 
Response Criteria - MDS................................................................. 29 
9.2.1 
Hematologic Improvement (HI) .............................................. 29 
9.3.1 Cytogenetic Response ....................................................................... 30 
9.3 
Guidelines for Evaluation of Disease .............................................. 30 
9.4 
Other Secondary Efficacy Measures ............................................... 30 
9.4.1 
Treatment Failure, Overall Survival and Event-free Survival 30 
9.4.2 
Duration of Remission and Relapse-free Survival for 
Patients Having Complete Remission ..................................... 31 
9.4.3 
Safety ...................................................................................... 31 
10.0 REGULATORY AND REPORTING REQUIREMENTS .................. 32 
10.1 Definitions ....................................................................................... 32 
10.1.1 
Adverse Events (AEs) ............................................................. 32 
10.1.2 
Serious Adverse Event (SAE) ................................................. 32 
10.1.3 
Unexpected Adverse Experience ............................................ 33 
10.1.4 
Life-Threatening Adverse Experience .................................... 33 
10.1.5 
Unanticipated Problems .......................................................... 33 
10.1.6 
Noncompliance ....................................................................... 33 
10.1.7 
Serious Noncompliance .......................................................... 34 
10.1.8 
Protocol Exceptions ................................................................ 34 
10.2 Reporting to the Human Research Protection Office (HRPO) at 
Washington University: ................................................................... 34 


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10.3 Reporting to the Quality Assurance and Safety Monitoring 
Committee (QASMC) at Washington University ........................... 34 
10.4 Reporting Requirements for Secondary Sites .................................. 35 
10.5 Reporting to Secondary Sites .......................................................... 35 
10.6 Reporting to the FDA ...................................................................... 35 
10.7 Timeframe for Tracking Reportable Events: ................................... 36 
11.0 DATA SAFETY MONITORING PLAN ............................................. 37 
12.0 AUDITING ........................................................................................... 39 
13.0 MULTICENTER REGULATORY REQUIREMENTS ...................... 40 
14.0 STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE ........ 41 
14.1 Analysis Populations ....................................................................... 41 
14.2 Statistical Analysis .......................................................................... 41 
14.2.1 
Descriptive Analyses ............................................................... 41 
14.2.2 
Primary Endpoint Analysis ..................................................... 41 
14.2.3 
Secondary Endpoint Analyses ................................................ 42 
14.2.4 
Safety Analysis ....................................................................... 43 
14.2.5 
Description of Planned Subgroup Analyses ............................ 44 
14.3 Sample Size ..................................................................................... 44 
15.0 DATA SUBMISSION SCHEDULE .................................................... 45 
16.0 REFERENCES ..................................................................................... 48 
  


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1.0 
INTRODUCTION 
 
1.1 
Rationale   
 
Personalized therapy for cancer requires that 1) patient-specific factors (e.g. patient-
specific mutations, expression profiles, or pharmacogenomics) influence treatment 
algorithms, and 2) once therapy is initiated, in vivo responsiveness is used to guide further 
therapy. The modern era of genomics has provided significant insights into cancer 
pathogenesis by describing patterns of germline and somatic variants, as well as defining 
disease-specific patterns of gene expression. However, the application and integration of 
this data with therapeutics remains a difficult task. 
 
Decitabine is a commonly used single-agent therapy for patients with acute myeloid 
leukemia (AML) and/or myelodysplastic syndrome (MDS). Historically, it has yielded 
clinical complete responses in approximately 25% of patients across diverse clinical trials, 
it is well tolerated, and it can be given as in the outpatient setting. However, the majority 
of patients do not respond to decitabine, the molecular etiology of decitabine sensitivity is 
unknown, and there are no algorithms to predict patient responsiveness a priori or even 
within one month of starting therapy.  
 
AML and MDS are two closely related hematological disorders characterized by 
ineffective hematopoiesis and malignant expansion of clonal myeloid cells.  Both AML 
and MDS frequently occur in elderly patients and some cases are secondary and/or related 
to long-term bone marrow damage after radiation or chemotherapy. Current therapy for 
AML consists of cytotoxic chemotherapy followed by consolidation chemotherapy or stem 
cell transplantation. Using these approaches, current cure rates may be as high as 60% for 
patients younger than 60 with good risk cytogenetics (e.g. t(15;17), t(8;21), inv(16)).[4]  
Outcomes in patients ≥ 60 years old and with relapsed disease remain poor with 2 year 
survival of only ~25%.[5] Historically, the available treatment for MDS has been 
symptomatic and has not been shown to be effective in producing sustained improvement 
in hematopoiesis or in delaying leukemic evolution[6, 7].  
 
Current AML therapy is poorly tolerated in elderly and pre-treated patients due to 
hematologic and non-hematologic toxicities; following such therapy, patients typically 
require 3-4 weeks of hospitalization for treatment of neutropenic fevers and for regular 
transfusions. Even among the relatively healthy patients enrolled in clinical trials, induction 
mortality in the elderly can be as high as 20-40% following standard induction therapy,[8-
11] and moderate to severe non-hematologic toxicity occurs in at least an additional 30% 
of patients.[9, 12-14] Consequently, many older AML patients are not offered traditional 
intensive chemotherapy, but receive supportive care alone.[15]  More effective and less 
toxic therapies are needed to provide durable remissions in a significant proportion of 
elderly AML in populations. 
 
Decitabine has been studied in MDS patients and in elderly and/or relapsed AML patients. 
It has been found to be reasonably well tolerated, can be given in the outpatient setting, 
and has overall response rates of 20-45%.[2, 3, 16-20] Our own experience has been of 


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CR/PR rates of 25%, [2] and this is typical of many other groups for both MDS and AML 
patients (reviewed in [21]  Decitabine is an S-phase depended drug.  [22-24] However, 
decitabine is usually administered in one hour daily infusions during 3 to 5 consecutive 
days (too short to expose decitabine to most AML blasts during at least 2 cycles of 
replication, which divide every 2 to 7 days).[25-27]  Recent experience with increasing the 
number of infusions per cycle from 5 consecutive days to 10 consecutive days has 
improved overall response rates to 67%.[3] Response to decitabine can be slow, sometimes 
requiring 3 to 5 cycles before maximum response is observed.[2] As a consequence, many 
patients are exposed to prolonged therapy, gaining little benefit, before determining 
resistance. There are currently no biomarkers that predict response to decitabine. 
 
Decitabine is a cytosine analogue that cannot be methylated. Incorporation of decitabine 
into DNA prevents DNA methylation at cytosine residues and derepresses epigenetically 
silenced gene expression. Therefore, the reduction in total genomic DNA methylcytosine 
content acts as a marker of effective decitabine dosing. Indeed, two groups have recently 
correlated effective methylcytosine reduction and gene expression derepression to 
response (Figure 1.1).[3, 28] 
 
 
 
   
Figure 1.1. From Shen et al JCO (2010) 28:611 and Blum et al JCO (2007) 25: 3884. 
 
We and others have recently used whole genome sequencing to identify recurrent, somatic 
mutations in AML and MDS patients in four genes that are involved in methylcytosine 
regulation: DNMT3A, IDH1, IDH2, and TET2.[29-37] Collectively, nearly half of all 
AML/MDS patients have a mutation in one of these four genes.  DNMT3A mutations are 
associated with an adverse outcome in AML patients treated with cytotoxic 
chemotherapy.[35, 38] However, it is not yet clear whether the presence of mutations in 
one or more of these genes is associated with response or resistance to decitabine. 
 
In this study, we will treat patients with the current state-of-the-art decitabine schedule.[3] 
We will perform exome sequencing on all patients and correlate response with mutation 
status, rate of mutation clearance, expression profile, plasma decitabine levels, and DNA 


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methylcytosine content reduction. This approach will comprehensively assess genomic and 
pharmacologic parameters that could clinically predict patient response to decitabine.  
 
In this study, we will combine analysis of patients with AML and MDS. Recent genomic 
analysis has shown that AML and MDS have shared mutations in many of the same genes, 
shared patterns of mutation acquisition, and shared patterns of mutation evolution.[39, 40] 
Furthermore, MDS, like AML, presents with clonal hematopoiesis, even when the 
morphologic blast count is zero.[36]Because this study will identify and track the rate of 
clearance of patient specific mutations, and because decitabine has been shown to have 
efficacy in both AML and MDS patients, [2, 3, 16-19, 41-45] combining analysis of AML 
and MDS patients is reasonable.  Asymptomatic MDS patients are excluded because these 
patients are typically followed with watchful-waiting rather than with chemotherapy. 
  
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
 
Methylation of cytosine in the CpG dinucleotide by DNA methyltransferase leads to 
transcriptional silencing of genes during normal development and has emerged as a 
significant mechanism for the loss of tumor suppressor gene expression in human cancer, 
including AML and MDS.[46, 47] Like azacitidine, decitabine (5-aza-2’-deoxycitidine) is 
incorporated into DNA during S phase and forms covalent adducts with DNA 
methyltransferase. The irreversible inhibition of this enzyme results in loss of methylation 
and is associated with differentiation.[48]  Decitabine is an approximately 5-fold more 
potent inhibitor of DNA methyltransferase than azacitidine, which might explain why 
decitabine can induce differentiation of myeloid progenitors in vitro at lower doses than 
azacitidine. In addition to their differentiation inducing activity at low doses, both agents 
have a direct cytotoxic effect at higher doses (1-2 µM).[49]  
 
Decitabine has been investigated in hematologic malignancies using a range of doses and 
schedules.  It is currently FDA approved for the treatment of adults with MDS at a dose of 
15 mg/m2 every 8 hours for 3 days, based on a Phase III study that demonstrated improved 
outcomes in the treatment arm compared to patients treated with supportive care alone.[15]  
However, the overall response rate in this trial was modest (17%). Of note, 18% of the 
patients in this trial had 20-30% bone marrow blasts, meeting the current WHO criteria for 
AML.  Subsequent phase I and II studies in AML, using a variety of low-dose decitabine 
regimens, have reported overall response rates of 22-49%.[2, 3, 16-19, 41-45] Comparisons 
of multiple low dose schedules have noted improved efficacy in the MDS population using 
a dose of 20 mg/m2 on Days 1-5 of a 28-day cycle.[50, 51] Because of these results, we 
have recently explored this dosing schedule in the treatment of elderly patients with 
AML.[52] In this study, we observed an overall response rate of 24%, and an additional 
44% of patients achieved stable disease during a median of 5 monthly cycles.[48] In this 
study, we noted that decitabine does not act quickly and the median time to response was 
3 months. These results are similar to the recently published experience in Germany 
treating 227 patients with 15 mg/m2 over 3 hours TID for 3 days every 6 weeks, which 
resulted in a CR/PR rate of 26%.[16]  
 


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A recent study using a prolonged administration schedule (20 mg/m2 IV over 1 hour on 
consecutive days 1-10) showed improved response (64% CR/PR) and improved time to 
respond (50% of patients had bone marrow blasts < 5% after 1 cycle of therapy).[3]  These 
results are an improvement over previous experience by other groups using decitabine to 
treat elderly AML patients or patients with relapsed AML, and this experience is 
summarized below: 
Trials of decitabine in AML 
Status 
Other Therapy 
DAC Dose 
(mg x days) 
Evaluable 
CR 
de novo 
daunomycin1 
90 mg/m2 x 5 
6 
100% 
Salvage 
None4 
0.75-80 mg/kg 
16 
  
None5 
37-81 mg/kg 
6 
33% 
None7.  Ara-C 
Refractory 
Various 
4 
0 
AMSA7 
250-500 mg/m2 x 6 
10 
80% 
idarubicin or 
AMSA3 
250 mg/m2 x 6 
56 
39% 
Elderly 
None6 
270-360 mg/m2 x 3 
10 
30% 
None2 
45 mg/m2 x 3 
4 
50% 
None8 
20 mg/m2day 1-5,8-12 
50 
23% 
None9 
20 mg/m2 x 5 
55 
25% 
 
None10 
50 mg/m2 CIVI x 3 
29 
48% 
 
None11 
20 mg/m2 x 10 
53 
64% 
 
ATRA cycle 212 
15 mg/m2 TID x 3 
227 
26% 
1 Schwartsmann G.  Leukemia 1997; 11(Suppl 1):S28-31. 
2 Lubbert M. ASH 2002 Abstract #4601. 
3 Willemze R.  Leukemia.  1997; 11(Suppl 1):S24-27 
4 Rivard GE.  Leukemia Research 1981; 5:453-462. 
5 Momparler RL.  Pharmac Therapy 1985; 30:277-286.  
6 Petti MC.  Leukemia.  1993; 7(Suppl 1):36-41.  
7 Richel DJ.  Br. J. Cancer 1991; 64:144-148 
8 Issa, JP. Blood 2004. 103: 1635. 
9 Cashen AF. ASH Annual Meeting. 2008 abstract 560. 
10 Wijermans PW. Leukemia 1997. 11: Suppl 1, 19-23. 
11 Blum, W. PNAS 2010; 107:7473. 
12 Lubbert. Haematologica 2011. ePub 11/04/2011. 
 
In general, decitabine is well tolerated. Unlike conventional cytotoxic chemotherapy, 
decitabine can be administered in the outpatient setting. However, AML and MDS are 
aggressive hematologic malignancies. Cytopenias commonly result from AML/MDS 
inhibition of normal hematopoiesis, and often do not resolve until a complete response is 
achieved. Thus, neutropenic sepsis and thrombocytopenic bleeding remain significant 
problems regardless of therapy, especially during the first 4-8 weeks. In our experience 
treating elderly AML patients, neutropenia occurred in 24%, sepsis in 9%, and bacteremia 
in 7%.  Similar results have been observed in MDS patients.[20, 53, 54] As a result, death 
is not uncommon in the first 8 weeks of therapy, occurring in 15-17% of patients, and most 
commonly resulting from infection and cytopenias related to disease and therapy.[16, 52, 
55] While unsettling, these numbers are better than many historical patients treated with 
cytotoxic chemotherapy, and suggest a need for continued improvements in therapy.[9, 12]  


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1.2.1 Pharmacokinetics and Metabolism of Decitabine 
 
In human subjects, after IV administration, decitabine displayed a distribution phase 
with a half-life of 7 minutes and a terminal phase elimination half-life of 10-35 
minutes as measured by bioassay.[56, 57]   The short plasma half-life is due to rapid 
inactivation of decitabine by deamination by liver cytidine deaminase.[23] Total 
body clearance (Clp) in humans was high with a mean value of 126 mL/min/kg.[58]  
 
Among five adult patients with advanced solid tumors administered seven courses 
of 100 mg/m2 of decitabine by 1-hour infusions,[56] maximum plasma 
concentration ranged from 1 to 4 µM. Disappearance from plasma was biphasic 
with a rapid initial decline to less than 0.1 µM during the first hour post infusion 
and a slower decline over the next two hours to 6 nM, the level of detectability. 
Plasma half-life (meanSE) was estimated to be approximately 71 minutes in the 
initial phase (t½α) and 355 minutes in the second phase (t½β). The area under the 
plasma concentration-time curve was 40888 ng.hour/mL for these patients, with a 
peak plasma concentration of 2.010.44 µM (0.440.1 µg/mL), while mean volume 
of distribution (Vdss) and CLp was 4.591.42 L/kg and 12621 mL/min/kg, 
respectively. The time course of DAC concentrations was similar in 1 of 2 patients 
administered 75 mg/m2, while DAC was usually undetectable in the plasma of 
patients administered 25 to 60 mg/m2. 
 
Using a longer infusion time (up to 40 hours), Rivard et al.[57] reported a plasma 
concentration of 0.1 to 0.4 g/mL. With infusion times of 40-60 hours, at an infusion 
rate of 1 mg/kg/h, plasma concentrations of 0.43-0.76 µg/mL were achieved.[59]  The 
steady-state plasma concentration at an infusion rate of 1 mg/kg/h was estimated to 
be 0.2-0.5 µg/mL. The half-life after discontinuing the infusion was 12-20 min.[57]  
 
Urinary excretion of unchanged decitabine was low, ranging from less than 0.01% to 
0.9% of the total dose, and there was no relationship between excretion and dose or 
plasma drug levels. High clearance values and a total urinary excretion of <1% of the 
administered dose suggest that decitabine is eliminated rapidly and largely by 
metabolic processes, including extrahepatic metabolism.[60] 
 
The steady state plasma concentrations (Css) ranged from 0.31 to 0.39 µg/mL (1.27 
to 1.60 µM) during the infusion of 100 mg/m² decitabine over a 6-hour period.[61] 
The range of concentrations during a 600 mg/m2 infusion over an 8-hour period was 
0.41-16 g/mL(1.68 to 4.76 µM).[60] These plasma concentrations are in the same 
range as the in vitro concentrations that activated the expression of tumor 
suppressor gene p16 in three human non-small-cell lung cancer (NSCLC) cell 
lines.[61]  
 
Steady state decitabine levels of 0.1 to 0.2 µM were achieved in solid tumor patients 
dosed with 30 mg/m²/day and 40 mg/m²/day by continuous intravenous infusion 
over 72 hours.[62]  


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Summary of published steady state serum levels are below and are in proportion to 
infusion rate (R2 = 0.98, p < 0.0001):[63] 
 
Rate 
Serum 
Concentration 
mg/m2/hr 
ng/ml 
nM 
5 
55 
241 
7.5 
80 
351 
15 
180 
789 
21 
182.4 
800 
22.5 
350 
1535 
42 
456 
2000 
84 
1140 
5000 
 
In our experience treating primary patient blasts in vitro, concentrations of 100 – 
500 nM are required maximize methylcytosine reduction without causing cellular 
toxicity. Because decitabine incorporation into DNA is S-phase dependent, 
exposure of cells during cell division is necessary for efficacy. 
  
 
 
 
1.2.2 Experience with extended dosing schedules 
 
Blum et al recently published their experience treating elderly AML patients with 
a 10-day course of decitabine 20 mg/m2/day as a 1-hour infusion.[3] This prolonged 
exposure course was designed to increase the number of AML blast divisions 
exposed to decitabine per cycle. Toxicities were very similar to a similar regiment 
given as a 3 and 5 day course,[2, 16] but the overall response (CR/PR) was much 
improved (64% vs 25%). In all three studies, the toxicities were primarily myeloid 
suppression and the resultant infectious and bleeding complications associated with 
leukemic and pharmacologic hematopoietic suppression. Furthermore, Blum et al 
noted that the percent methylcytosine reduction was improved (22% vs 11%) as 


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was the time to blast clearance (50% of patients who ultimately achieved a CR had 
≤ 5% bone marrow blasts after 1 cycle vs 0% of patients after 2 cycles). Finally, 
like Shen et al, they observed a correlation between the extent of methylcytosine 
reduction and response.[28]  This study suggests that improvement in response 
rates and time to response can be achieved by prolonged administration of 
decitabine. Further, that DNA methylcytosine reduction can be achieved with 
prolonged exposure to decitabine and that this might be a patient-specific biological 
marker of adequate dosing. 
  
Scandura et al. recently published a study of infusional decitabine prior to 
cytarabine + daunorubicin induction therapy for AML.[64] They escalated the 
duration of infusional therapy and reached their planned goal of 20 mg/m2/day x 7 
days of continuous therapy given prior to cytarabine and daunorubicin. They noted 
similar toxicities (primarily hematopoietic and the associated neutropenic 
infections), but that these were not different from their experience giving cytarabine 
+ daunorubicin alone. 
 
1.3 
Correlative Studies 
 
All correlative studies will be collected under HRPO# 201011766 (“Tissue Acquisition for 
Analysis of Genetic Progression Factors in Hematologic Diseases”) that all patients will 
be concurrently enrolled in. The goals of the correlative studies include: 
 
1. To correlate patient-specific mutations with response to decitabine. 
We will perform exome sequencing of each patient’s pre-study bone marrow sample 
and skin sample. We will determine AML/MDS-associated variants found in the bone 
marrow, but not skin sample. We will correlate the presence of mutations with response 
(especially mutations in DNMT3A, IDH1, IDH2, and TET2). This study is powered to 
identify mutations with >80% sensitivity to predict decitabine response if used as a 
clinical assay. 
 
2. To correlate the velocity and depth of response with response to decitabine. 
We will assess tumor burden by allele frequency of patient-specific mutations in bone 
marrow samples collected pre study, Cycle 1 Day 10±1, Cycle 1 Day 28±4, and Cycle 
2 Day 28±4. We will assess mutations in at least 24 recurrently mutated genes 
(including, but not limited to: FLT3, DNMT3A, NPM1, TET2, WT1, IDH1, IDH2, 
RUNX1, NRAS, TP53, U2AF1, KRAS, PHF6, MSTP9, PTPN11, KIT, SMC3, PRUNE2, 
ETV6, CEBPA, SMC1A, ASXL1, STAG2, RAD21) as well as all patient-specific 
mutations discovered during exome sequencing. The leukemic allele frequency will be 
measured using the Illumnia MySeq or HiSeq platform using DNA capture by gene 
specific biotinylated-oligonucleotides. Patient bone marrow samples will be assessed 
pre-study, Cycle 1 Day 10 ± 1, Cycle 1 Day 28 ± 4, and Cycle 2 Day 28 ± 4 and 
compared with patient specific skin samples (normal control). The velocity of response 
will be calculated as the natural log rate of patient-specific mutation allele frequency 
clearance. Depth of response will be defined as the lowest patient-specific mutation 


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allele frequency in a bone marrow sample. The velocity and depth of tumor response 
will be correlated to: 
 Achievement of clinical CR, 
 Time to best response, 
 Mutational status, 
 Time to disease progression. 
Because all patient-specific exome-discovered mutations will be assessed a second 
time in the primary bone marrow sample as well as the  Cycle 1 Day 10 ± 1, Cycle 1 
Day 28 ± 4, and Cycle 2 Day 28 ± 4 bone marrow samples, this study will also serve 
to validate all exome-discovered mutations. This is important, because exome 
sequencing is associated with a 20-50% false-discovery rate depending on type I error 
tolerated (mutation-calling algorithms are typically set for greater sensitivity than 
specificity).  Thus, many mutations discovered during exome sequencing are incorrect.  
This second analysis of these mutations will server to exclude these false positive calls. 
 
3. To correlate the bone marrow expression profile from pre-study and Cycle 1 Day 10 ± 
1 with response to decitabine. 
We will correlate expression signatures, and changes in expression signatures, with: 
 Achievement of clinical CR, 
 Mutation status. 
 
4. To correlate the extent of bone marrow DNA hypomethylation on Cycle 1 Day 10 ± 1 
with response to decitabine. 
We will assess the percent reduction in bone marrow methylcytosine content using LC-
MS. Patients will be evaluated pre-study and Cycle 1 Day 10 ± 1. The change in DNA 
methylcytosine content will be correlated to: 
 Achievement of clinical CR, 
 Change in expression signature. 
This will determine whether inadequate blast exposure to decitabine correlates with 
drug resistance. 
 


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2.0 
OBJECTIVES 
 
2.1 
Primary Objective 
 
To determine whether patient-specific mutations correlate with clinical overall response to 
decitabine in patients with AML or MDS. 
 
2.2 
Secondary Objectives 
 
- 
To determine whether 10 consecutive days per cycle of decitabine improves outcomes 
compared with historical controls treated with 5 consecutive days per cycle of 
decitabine. 
 
- 
To determine whether other biomarkers correlate with clinical overall response to 
decitabine in patients with AML or MDS. These will include:  
 
1. The velocity and depth of patient-specific mutation clearance; 
2. The bone marrow expression profile and change in expression profile during 
decitabine treatment; 
3. The decrease in bone marrow methylcytosine content in response to decitabine.  
 
 


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3.0 
PATIENT SELECTION 
 
3.1 
Inclusion Criteria 
 
All of the following: 
 Patient must have non-M3 AML or MDS  
 An adverse risk karyotype defined by: 
-Complex karyotype by cytogenetics, or 
-Deletion of all or part of chromosome 5, 7, 12, or 17 defined by FISH or          
cytogenetics, or 
-Somatic TP53 mutation 
 
All of the following: 
1. Patient must have an ECOG performance status ≤ 2. 
2. Patient must have >10% disease burden measured by cytomorphology, flow 
cytometry, or cytogenetics. 
3. Patient must have peripheral white blood cell count < 50,000/mcl. 
4. Patient must have adequate organ function, defined as: 
a. 
Total bilirubin < 1.5 x ULN 
b. 
AST/ALT < 2.5 x ULN 
c. 
Serum creatinine < 2.0 x ULN  
5. Patient must have undergone ≤ 2 cycles of prior hypomethylating agent (decitabine 
or azacitidine). 
6. Patient must be enrolled in HRPO# 201011766 (“Tissue Acquisition for Analysis 
of Genetic Progression Factors in Hematologic Diseases”). 
7. Patient must be > 18 years of age. 
8. Patient must be able to understand and willing to sign an IRB-approved written 
informed consent document. 
 
3.2 
Exclusion Criteria 
 
1. Patient must not be pregnant or nursing. 
2. The patient must not have acute promyelocytic leukemia or t(15;17) observed by 
FISH. 
3. Patient must not have known CNS leukemia.  
4. Patient must not have a history of positive HIV serology.  
5. Patient must not have a history of positive Hepatitis C serology. 
6. Patient must not have undergone a prior allogeneic stem cell transplant. 
7. Patient must not have any uncontrolled intercurrent illness including, but not 
limited to, ongoing or active infection, ongoing or active GVHD, congestive heart 
failure of NYHA class 3 or 4, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situation that would limit compliance with study 
requirements. 
8. Patient must not have had radiation therapy within 14 days of enrollment. 
9. Patient must not have received any chemotherapy within 21 days of enrollment, 
and any acute treatment-related toxicities must have returned to baseline. Patients 


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may be receiving hydrea at time of enrollment.    
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible for this trial. 


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4.0 
REGISTRATION PROCEDURES 
 
Patients must not start any protocol intervention prior to registration through the Siteman 
Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility by Washington University 
2. Registration of patient in the Siteman Cancer Center database 
3. Assignment of unique patient number (UPN) 
 
Once the patient has been entered in the Siteman Cancer Center OnCore database, the WUSM 
coordinator will forward verification of enrollment and the UPN via email. 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility collecting the information listed below and scanning and 
emailing it to the research coordinator listed in the Siteman Cancer Center Clinical Trials 
Core Protocol Procedures for Secondary Sites packet at least one business day prior to 
registering patient: 
1. Your name and contact information (telephone number, fax number, and email 
address) 
2. Your site PI’s name, the registering MD’s name, and your institution name 
3. Patient’s race, sex, and DOB 
4. Three letters (or two letters and a dash) for the patient’s initials 
5. Currently approved protocol version date 
6. Copy of signed consent form (patient name may be blacked out) 
7. Planned date of enrollment 
8. Completed eligibility checklist, signed and dated by a member of the study team 
9. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center OnCore Database 
 
Registrations may be submitted Monday through Friday between 8am and 5pm CT. Urgent 
late afternoon or early morning enrollments should be planned in advance and coordinated 
with the Washington University research coordinator.  Registration will be confirmed by 
the research coordinator or his/her delegate by email within one business day. Verification 
of eligibility and registration should be kept in the patient chart. 
 
All patients at all sites must be registered through the Siteman Cancer Center OnCore 
database at Washington University. 
 
4.3 
Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  Patients 


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will also be identified by first, middle, and last initials.  If the patient has no middle initial, 
a dash will be used on the case report forms (CRFs).  All data will be recorded with this 
identification number on the appropriate CRFs. 
 


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5.0 
INVESTIGATIONAL PLAN 
 
5.1 
Summary of Study Design 
 
This is an open-label, single-arm, Phase II, biomarker study of patients with AML or MDS 
who are treated with 10 consecutive days of decitabine. FAB-M3 AML is excluded from 
this trial, because other well-tolerated options exist for the treatment of FAB-M3 acute 
promyelocytic leukemia. AML patients who are < 60 years old and have not received 
cytotoxic induction chemotherapy are also excluded, because cytotoxic therapy offers a 
curative possibility for these patients.  MDS patients without cytopenias are also excluded 
because these patients can be followed with watchful-waiting. 
 
All patients will undergo bone marrow biopsies pre-study, on Cycle 1 Day 10 ± 1, and on 
Day 28 ± 4 of Cycles 1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. 
Patients will provide a skin biopsy sample pre-study. See Section 8.0 for details on 
correlative studies.  
 
Patients will receive decitabine 20 mg/m2/day as a 1-hour infusion on consecutive Days 1-
10 in 28-day cycles.  The dose will be calculated based on actual patient weight at the 
beginning of each cycle. 
 
Once AML patients achieve morphologic complete remission with incomplete blood count 
recovery (CRi) or MDS patients achieve a marrow complete response (Marrow CR) as 
defined in Section 9.0, decitabine administration may be decreased to 20 mg/m2/day for 
ten consecutive days every 6 weeks, to limit myelosuppressive toxicity and improve count 
recovery. After 2 cycles at 6 week intervals, the dose may be reduce to 20 mg/m2/day for 
five consecutive days in 6 week cycles, at the discretion of the treating physician.  After 
two cycles of five consecutive days of treatment, if the patient meets the definition of CR 
with complete cytogenetic response as defined in Section 9.0, administration may further 
be decreased to three consecutive days per cycle at the discretion of the treating physician. 
 
It will be a logistical goal of this trial consistently to return preliminary exome results to 
the treating physician by the end of Cycle 1 and validated results by the end of Cycle 2. 
However, this logistical goal is not a primary or secondary aim of this study. Given the 
limited data regarding mutation presence and drug response, we do not anticipate that the 
results will directly influence therapeutic decisions made by the treating physician. 
However, the development of comprehensive genomic pipelines and integration of this 
data into a clinical trial setting will be required before personalized medicine can be 
realized. 
 
 
 


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5.2 
Study Procedures 
 
5.2.1 Pre-study Procedures 
 
Pre-study procedures must take place no more than 14 days prior to the first dose 
of decitabine except where noted below. 
 
1. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments performed 
on bone marrow will include correlative studies (collected under HRPO# 
201011766), morphologic assessment, cytogenetics, and FISH.  
2. Bone marrow or peripheral blood for flow cytometry 
3. Skin biopsy sample (collected under HRPO# 201011766). There is no required 
time frame for this sample; it may have been collected months or even years 
prior to the first dose of decitabine.  
 
5.2.2 Baseline Evaluation 
 
Baseline evaluations must take place no more than 7 days prior to the first dose of 
decitabine.  
 
1. Medical history 
2. Physical exam including height, weight, and ECOG performance score. 
3. Record transfusion requirements for 8 weeks before first dose of study drug 
4. CBC with differential and platelets 
5. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
6. Urine βHCG pregnancy test if appropriate 
7. Clinical pathology should be carefully reviewed to rule-out acute 
promyelocytic leukemia (M3-AML), and discussion between the pathologist 
and treating physician should take place if warranted by morphology or 
immunophenotype. 
 
5.2.3 Day 1 of Each Cycle 
 
1. Physical exam, including weight  
2. CBC with differential and platelets 
3. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
 
5.2.4 Cycle 1 Days 1-10 
 
Decitabine administration, 20 mg/m2 per day given as a 1-hour continuous 
intravenous infusion.  The dose will be calculated based on actual patient weight at 


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the beginning of each cycle.  Patients may be premedicated with lorazepam, 
prochlorperazine, palonosetron, or ondansetron at the treating physician’s 
discretion. 
 
The clinical research coordinator should monitor for final FISH results to confirm 
that PML/RARA screening is negative. 
 
5.2.5 Cycle 1 Day 10 ± 1 
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH. 
 
5.2.6 Weekly 
 
CBC with differential and platelets. After the first cycle, this may be changed to 
every other week at the discretion of the treating physician. 
 
5.2.7 Every Other Week 
 
Serum chemistries, including bicarbonate, calcium, glucose, total protein, albumin, 
BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, ALT, alkaline 
phosphatase, LDH. After the first cycle, this may be changed to every month at the 
discretion of the treating physician. 
 
5.2.8 Cycles 1, 2, 4 and 6 Day 28 ± 4 days  
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include: correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH.  
 
Patients who achieve with clinical response (CRi or better) and are receiving cycles 
every 6 weeks, will undergo bone marrow biopsies on day 42 ± 4.  
 
5.2.9 End-of-Study Procedures  
 
1. Physical exam 
2. CBC with differential and platelets.  
3. Serum chemistries including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, and LDH.  
4. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments performed 
on bone marrow will include morphologic assessment, cytogenetics, and FISH.  


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5.2.10 Post-study Follow-up 
 
Patients are to be followed according to standard patient care procedures for 30 
days following the date of the last decitabine dose to monitor for toxicity, survival, 
and response. Thereafter, the investigator or designee will record information on 
subsequent therapy, disease progression, and/or death every 6 months for 2 years 
after last dose of study drug or until the patient is lost to follow-up or dies. 
 
5.3 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to the 
patient’s health and/or the patient no longer wishes to continue protocol therapy, the 
protocol therapy should be discontinued and the reason(s) for discontinuation documented 
in the case report forms. 
 
In the absence of treatment delays due to adverse events, treatment may continue 
indefinitely or until one of the following criteria applies: 
 
 Documented and confirmed disease progression 
 Death 
 Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
 General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
 Suspected pregnancy 
 Serious non-compliance with the study protocol 
 Lost to follow-up 
 Patient withdraws consent 
 Investigator removes the patient from study 
 The Siteman Cancer Center decides to close the study 
 
Patients who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar. 
 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
 
5.4.1 Chemotherapy 
 
Patients cannot receive any chemotherapy within 21 days of study enrollment and 
must have recovered from any treatment-related toxicities. However, patients may 
be receiving hydrea at enrollment and may continue on hydrea through Cycle 2 of 
decitabine. An indication for hydrea following 2 cycles of decitabine is evidence 
that the patient is not responding and decitabine should be discontinued. Other than 
decitabine, patients cannot receive chemotherapy during this trial.  
 


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5.4.2 Growth Factors 
 
Erythroid growth factors should not be routinely administered to patients during the 
study. 
 
Thrombopoietin receptor agonists should not be routinely administered to patients 
during the study. 
 
Filgrastim (G-CSF) or sargramostim (GM-CSF) may be administered to patients 
with septic shock or febrile neutropenia at the discretion of the treating physician. 
 
5.4.3 Transfusions 
 
 Packed red blood cells (RBCs) transfusion:   
RBC transfusions will be administered at the discretion of the treating 
physician.  
 
 Platelet transfusion:   
Platelet transfusions will be administered at the discretion of the treating 
physician  
 
5.4.4 Prophylactic Antimicrobial Agents  
 
Antimicrobial agents will be administered at the discretion of the treating physician. 
Common antimicrobial prophylaxis during cycles 1 and 2 include acyclovir, 
ciprofloxacin, and fluconazole. 
 
5.4.5 Radiotherapy 
 
Patients cannot receive radiotherapy within 14 days prior to study enrollment or 
while on study. 
 


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6.0 
DOSE MODIFICATIONS 
 
A criterion for discontinuation of the study is a delay of > 8 weeks between any two cycles.  
 
6.1 
Delay for Cytopenia or Infection 
 
As cytopenias are a defining feature of AML and MDS, their presence alone does not 
generally warrant dose modifications or delays. Patients will receive four cycles of therapy 
without adjustment for any cytopenia. Following Cycle 4, dose modifications or delays for 
cytopenias will be made at the discretion of the investigator.  
 
Patients with febrile neutropenia (temperature ≥ 38.5° with ANC < 1,000/l) or systemic 
infections should have treatment held until clinical resolution of the infection.  
 
6.2 
Delay for Organ Dysfunction 
 
If renal dysfunction (creatinine ≥ 3 X institutional upper limit of normal) or hepatic 
dysfunction (bilirubin ≥ 2.5 mg/dl or ALT/AST ≥ 3 X institutional upper limit of normal) 
occurs, therapy may not resume until the abnormalities have resolved to below the limits 
stated above. If renal or hepatic dysfunction occurs during a treatment course, the next dose 
of decitabine should be held, and serum chemistries should be checked twice per week.  
Decitabine can be resumed at full dose when creatinine, bilirubin, ALT and AST are below 
the limits listed above.  
 
For grade 3-4 non-hematologic toxicities related to decitabine, decitabine treatment should 
be delayed until recovery to baseline or to grade ≤ 1. Decitabine may be reintroduced with 
a 50% dose reduction, or at full dose at the discretion of the investigator. 
 
6.3 
Modification in Dose or Schedule for Treatment Response 
 
Once AML patients achieve morphologic complete remission with incomplete blood count 
recovery (CRi) or MDS patients achieve a marrow complete response (Marrow CR) as 
defined in Section 9.0, decitabine administration may be decreased to 20 mg/m2/day for 
ten consecutive days, and the cycles may be administered every 6 weeks to limit 
myelosuppressive toxicity, at the discretion of the treating physician. After two cycles at 
six week intervals, the dose may be reduced to 20 mg/m2/day for five consecutive days of 
treatment in six week intervals. If the patient meets the definition of CR with complete 
cytogenetic response as defined in Section 9.0, administration may further be decreased to 
three  consecutive days per cycle at the discretion of the treating physician. 
 


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7.0 
PHARMACEUTICAL INFORMATION 
 
7.1 
Decitabine 
 
Decitabine is an antimetabolite that can replace cytosine in DNA but, unlike cytosine, it 
cannot be methylated. The exact mechanism of decitabine activity has not been determined 
and may involve multiple pathways. Decitabine is thought primarily to act by correcting 
DNA methylation, a major mechanism for gene expression. In some cancer cells, 
hypermethylation blocks the activity of tumor suppressor genes, which regulate cell 
division and differentiation to prevent malignant transformation. When suppressor gene 
activity is blocked, cell division becomes unregulated, leading to the formation of 
neoplastic cells. Decitabine regulates DNA methylation (specifically targets the 
methyltransferase, DNMT1), effectively demethylating and reactivating different tumor 
suppressor genes. Because hypermethylation occurs early in the malignant transformation 
of cells, decitabine may have activity in chemoprevention. Other possible mechanisms for 
decitabine activity include cytotoxicity due to incorporation into DNA similar to other 
antimetabolites, methylation-independent induction of gene expression, and degradation of 
DNA methyltransferase 1. 
 
7.1.1 Study Drug Preparation  
 
OSHA Guidelines for handling cytotoxic drugs outlined in the American Journal 
of Hospital Pharmacy must be followed. 
 
Decitabine (5-aza-2’-deoxycytidine) is supplied as a lyophilized powder for 
injection, 50 mg in 20-ml vials. When reconstituted with 10 ml of sterile water for 
injection, each ml will contain 5 mg of decitabine and 6.8 mg of KH2PO4. The 
reconstituted solution should be diluted to 0.1-1.0 mg/mL with pre-chilled 0.9% 
sodium chloride intravenous infusion, which has been cooled to 2-8oC. 
Reconstitution of the powder results in a rapidly decomposing solution. Unless used 
within 15 minutes of reconstitution, the diluted solution must be prepared using 
cold (2—8 degrees C) infusion fluids and stored at 2—8 degrees C (36—46 degrees 
F) for a maximum of 7 hours. 
Decitabine will be administered per dosing schedule every 28 ± 4 days.  
 
Decitabine may be administered either as an inpatient or an outpatient.  
 
7.1.2 Supplier 
 
Decitabine is available commercially. 
 
 
  
 
  
 


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8.0 
CORRELATIVE STUDIES SAMPLE COLLECTIONS 
 
Bone marrow samples will be collected pre-study, on Cycle 1 Day 10 ± 1, and on Day 28 ± 4 of 
Cycle 1, 2, 4, and 6 for correlative studies. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. The bone 
marrow samples will be collected under HRPO# 201011766 (“Tissue Acquisition for Analysis of 
Genetic Progression Factors in Hematologic Diseases”) that all patients will be concurrently 
enrolled in.  
 
Skin biopsy sample will be collected as part of the patient’s participation in HRPO# 201011766 
(“Tissue Acquisition for Analysis of Genetic Progression Factors in Hematologic Malignancies”). 
There is no required time frame for this sample, and it may have been collected months or even 
years prior to the first dose of decitabine.  
 
 
 


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9.0 
EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
 
A secondary endpoint of this study is to estimate morphologic complete remission rate. 
  
9.1 
Response Criteria - AML 
 
AML Patients will be assessed for response according to the IWG criteria:1  
 
Morphologic complete remission (CR) – Defined as morphologic leukemia-free state, 
defined as less than 5% blasts in aspirate with marrow spicules and a count of ≥200 
nucleated cells and no blasts with Auer rods or any persistence of extramedullary disease. 
When erythroid precursors constitute less than 50% of bone marrow nucleated cells, the 
percentage of blasts is based on all nucleated cells; when there are 50% or more erythroid 
cells, the percentage blasts should be based on the nonerythroid cells. The presence of mild 
megaloblastoid changes may be permitted if they are thought to be consistent with 
treatment effect. However, persistence of pretreatment abnormalities (eg, pseudo-Pelger-
Hüet cells, ringed sideroblasts, dysplastic megakaryocytes) are not consistent with CR.  In 
addition, ANC >1000/μl and platelet count of ≥100,000/μl. Patient must be independent of 
transfusions. No duration requirement for this designation. 
 
Cytogenetic complete remission (CCR) – Defined as morphologic complete remission plus 
a reversion to a normal karyotype. For this study, reversion of a normal karyotype is 
defined as no clonal abnormalities detected in a minimum of 20 mitotic cells. 
 
Morphologic complete remission with incomplete blood count recovery (CRi) – Defined as 
morphologic complete recovery with the exception of neutropenia <1000/μl or 
thrombocytopenia <100,000/μl.  
 
Partial remission (PR) – Defined as meeting the above criteria for complete remission, 
with the following exceptions: decrease of ≥50% in the percentage of blast to 5-25% in the 
bone marrow aspirate. A value of 5% blasts in bone marrow with Auer rods present may 
also be considered a partial remission.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, CRi, PR, PD, or 
recurrence/morphologic relapse.   
 
Progressive disease (PD) – Defined as a clear increase in bone marrow blast count, 
typically a greater than 50% increase compared with baseline, or new extramedullary 
disease. Patients with a > 50% increase in peripheral blast count with an increase of the 
total peripheral white blood cell count to > 10,000/l should undergo evaluation by bone 
marrow biopsy to access for progressive disease.  
 
Recurrence/morphologic relapse - Defined as relapse following complete remission is 
defined as reappearance of blasts in the blood or the finding of ≥5% blasts in the bone 
marrow, not attributable to any other cause. New dysplastic changes is considered relapse. 


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If there are no blasts in the peripheral blood and 5-20% blasts in the bone marrow, bone 
marrow biopsy should be repeated in > 1 week to confirm relapse. 
 
9.2 
Response Criteria - MDS 
 
MDS Patients will be assessed for response according to the IWG criteria:69 
 
Complete remission (CR) – Defined as < 5% myeloblasts with normal maturation of all 
cell lines in the bone marrow and peripheral blood values of Hgb > 11g/dL, Platelets > 100 
x 109/L, Neutrophils > 1.0 x 109/L, and 0% blasts. Persistent dysplasia does not exclude 
CR but will be noted.  
 
Marrow Complete Response (Marrow CR) – Defined as < 5% myeloblasts in the bone 
marrow and a decrease by > 50% from pretreatment values, but not meeting the definition 
of CR above.  
 
Partial Remission (PR) – Defined as meeting the definition of CR above with a decrease 
of myeloeblasts in the bone marrow by > 50% from pretreatment values, but absolute 
myeloblasts still >5%.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, Marrow CR, PR, SD, 
PD, or recurrence/morphologic relapse.   
 
Progressive Disease//Relapse (PD) – Defined as ≥ 50% increase in blasts to > 5% blasts 
(for patients with less than 5% blasts at baseline only), ≥ 50% increase to > 10% blasts (for 
patients with 5-10% blasts at baseline only), ≥ 50% increase to > 20% blasts (for patients 
with l0-20% blasts at baseline only), ≥ 50% increase to > 30% blasts (for patients with 20-
30% blasts at baseline only) or any of the following: At least 50% decrement from 
maximum remission/response in granulocytes or platelets, reduction in Hgb by ≥ 2 g/dL, 
or New or worsened transfusion dependence not related to study drug toxicity. Or for 
patients with a CR, Marrow CR, or PR as defined above and subsequently development of 
one of the following: Return to pretreatment bone marrow blast percentage, decrement of 
≥ 50% from maximum remission/response levels in granulocytes or platelets, or reduction 
in Hgb concentration by ≥ 1.5 g/dL or transfusion dependence. 
 
9.2.1 Hematologic Improvement (HI) 
 
 
 
Progressive disease as defined above nullifies hematologic improvement.  
 
Erythroid response requires all of the following (only required if pretreatment HgB 
<11 g/dL): 
 Hgb increase by ≥ 1.5 g/dL 
 Relevant reduction of units of RBC transfusions by an absolute number of at 
least 4 RBC transfusions/8 wk compared with the pretreatment transfusion 
number in the previous 8 wk. Only RBC transfusions given for a Hgb of ≤ 9.0 
g/dL pretreatment will count in the RBC transfusion response evaluation 


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Platelet response requires one of the following (only required if pretreatment 
platelets < 100 x 109/L):  
 Absolute increase of ≥ 30 x 109/L (for patients starting with > 20 x 109/L 
platelets) 
 Increase from < 20 x 109/L to > 20 x 109/L and absolute increase > 100% (for 
patients starting with < 20 x 109/L) 
 
Neutrophil response requires the following (only required if pretreatment ANC 
<1.0 x 109/L):  
 At least 100% increase and an absolute increase > 0.5 x 109/L 
 
9.3.1 Cytogenetic Response  
 
Complete Cytogenetic Response – Defined as reversion to a normal karyotype. For 
this study, reversion of a normal karyotype is defined as no clonal abnormalities 
detected in a minimum of 20 mitotic cells. Progressive disease as defined above 
nullifies cytogenetic response.  
 
9.3 
Guidelines for Evaluation of Disease 
 
Bone marrow biopsy should be performed on Day 28 ± 4 of cycles 1, 2, 4 and 6 for clinical 
disease assessment. Following Cycle 6, bone marrow biopsies should be performed at the 
discretion of the treating physician to assess for relapse or progression. 
 
9.4 
Other Secondary Efficacy Measures 
 
9.4.1 Treatment Failure, Overall Survival and Event-free Survival 
 
Treatment failure is defined as failure to achieve a complete or partial remission 
following 4 cycles of therapy.  
 
Overall survival is defined as the date of first dose of study drug to the date of death 
from any cause.  Patients still alive at the time of the final analysis will be right-
censored. 
 
Event-free survival is defined as the interval from the date of first dose of study 
drug to date of treatment failure, recurrence, death due to any cause, or loss to 
follow up. 
 
 


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9.4.2 Duration of Remission and Relapse-free Survival for Patients Having 
Complete Remission 
 
Duration of complete remission is defined as the interval from the date that 
complete remission is documented to the date of recurrence, death due to any cause, 
or loss to follow-up.  
 
Relapse-free survival is defined only for those patients achieving a complete 
remission. It is defined as the interval from the date of first documentation of a 
leukemia-free state to the date of recurrence, death due to any cause, or loss to 
follow up.   
  
9.4.3 Safety 
 
Safety evaluation will include assessments of adverse experiences, medical history, 
physical examinations, vital signs, and laboratory assessments at baseline and 
throughout the study period. Local laboratories may be used for this study.  
 


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10.0 
REGULATORY AND REPORTING REQUIREMENTS 
 
The entities providing oversight of safety and compliance with the protocol require reporting as 
outlined below. 
 
The Washington University Human Research Protection Office (HRPO) requires that all events 
meeting the definition of unanticipated problem or serious noncompliance be reported as outlined 
in Section 10.2. 
 
The FDA requires that all serious and unexpected adverse events be reported as outlined in Section 
10.6.  In addition, any fatal or life-threatening adverse experiences where there is a reasonable 
possibility of relationship to study intervention must be reported. 
 
10.1 
Definitions 
 
10.1.1 Adverse Events (AEs) 
 
Definition:  any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading:  the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for 
all toxicity reporting.  A copy of the CTCAE version 4.0 can be downloaded from 
the CTEP web site:  http://ctep.cancer.gov/reporting/ctc.html.  Grade 1-2 events 
will be recorded for the first fifty enrolled patients. Subsequently, only events 
graded as 3 or greater will be recorded. Fifty patients should be sufficient to 
estimate the true incidence of any adverse event to within ± 14% with 95% 
confidence intervals. 
 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the 
terms listed that should be used are those provided by the Department of Health 
and Human Services’ Office for Human Research Protections (OHRP).  A copy of 
this 
guidance 
can 
be 
found 
on 
OHRP’s 
website: 
(http://www.hhs.gov/ohrp/policy/AdvEvntGuid.htm). 
 
10.1.2 Serious Adverse Event (SAE) 
 
Definition:  any adverse drug experience occurring at any dose that results in any 
of the following outcomes: 
o Death 
o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial disruption 
of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 


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o Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed above 
 
All unexpected SAEs must be reported to the FDA. 
 
10.1.3 Unexpected Adverse Experience 
 
Definition: any adverse drug experience, the specificity or severity of which is not 
consistent with the current investigator brochure (or risk information, if an IB is not 
required or available). 
 
Events that are both serious AND unexpected must be reported to the FDA.  
 
10.1.4 Life-Threatening Adverse Experience  
 
Definition: any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that, had it occurred in a more severe form, might have caused 
death. 
 
Life-threatening adverse experiences must be reported to the FDA. 
 
10.1.5 Unanticipated Problems 
 
Definition: 
 
 unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the 
IRB-approved research protocol and informed consent document; and (b) 
the characteristics of the subject population being studied; 
 related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the 
incident, experience, or outcome may have been caused by the procedures 
involved in the research); and 
 suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was 
previously known or recognized. 
 
10.1.6 Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that 
govern human subjects research or failure to follow the determinations of the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate 
choice to ignore regulations, institutional policies, or determinations of the IRB. 
 


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10.1.7 Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial 
harm to subjects or others, or that materially compromises the rights or welfare of 
participants. 
 
10.1.8 Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under the 
research team’s control. Exceptions apply only to a single participant or a singular 
situation. 
 
Local IRB Pre-approval of all protocol exceptions must be obtained prior to the 
event.  For secondary sites, the Washington University PI will issue approval of the 
exception, but it must also be submitted to the local IRB with documentation of 
approval forwarded to Washington University.  Washington University IRB 
approval is not required for protocol exceptions occurring at secondary sites. 
 
10.2 
Reporting to the Human Research Protection Office (HRPO) at Washington 
University: 
 
The PI is required to promptly notify the IRB of the following events: 
 
 Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct of 
the study. 
 Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 
 Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence of the 
event or notification to the PI of the event.  The death of a research participant that qualifies 
as a reportable event should be reported within 1 working day of the occurrence of the 
event or notification to the PI of the event. 
 
10.3 
Reporting to the Quality Assurance and Safety Monitoring Committee 
(QASMC) at Washington University 
 
The PI is required to notify the QASMC of any unanticipated problem occurring at WU or 
any BJH or SLCH institution that has been reported to and acknowledged by HRPO as 
reportable.  (Unanticipated problems reported to HRPO and withdrawn during the review 
process need not be reported to QASMC.) 
 
QASMC must be notified within 10 days of receipt of IRB acknowledgment via email to 
a QASMC auditor. 


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10.4 
Reporting Requirements for Secondary Sites 
 
The research team at each secondary site is required to promptly notify the Washington 
University PI and research coordinator of all reportable events (as described in Section 7.6) 
within 1 working day of the occurrence of the event or notification of the secondary site’s 
PI of the event.  This notification may take place via email if there is not yet enough 
information for a formal written report (using either an FDA MedWatch form if required 
or an institutional SAE reporting form if not).  A formal written report must be sent to the 
Washington University PI and research coordinator within 10 working days of the 
occurrence of the event or notification of the secondary site’s PI of the event.  The death 
of a research participant that qualifies as a reportable event should be reported within 1 
working day of the occurrence of the event or notification of the secondary site’s PI of the 
event. 
 
The research team at a secondary site is responsible for following its site’s guidelines for 
reporting applicable events to its site’s IRB according to its own institutional guidelines.  
The research team at Washington University is responsible for reporting all applicable 
events to the FDA.  
 
10.5 
Reporting to Secondary Sites 
 
The Washington University PI (or designee) will notify the research team at each secondary 
site of all reportable events that have occurred at other sites within 10 working days of the 
occurrence of the event or notification of the PI of the event.  This includes events that take 
place both at Washington University and at other secondary sites, if applicable. 
 
10.6 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER 
FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the Washington University principal investigator to report any 
unanticipated problem to the FDA as follows: 
 
 Report any unexpected fatal or life-threatening adverse experiences (Section 
10.1.4) associated with use of the drug by telephone or fax no later than 7 calendar 
days after initial receipt of the information.   
 Report any serious, unexpected adverse experiences (Section 10.1.2), as well as 
results from animal studies that suggest significant clinical risk within 15 calendar 
days after initial receipt of this information.   
 
All MedWatch forms will be sent by the investigator or investigator’s team to the FDA at 
the following address or by fax: 
 
Food and Drug Administration 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
Secondary sites must submit a completed MedWatch form to the Washington University 
PI and research coordinator within 4 calendar days (for fatal or life-threatening adverse 
experiences) or 11 calendar days (for serious, unexpected adverse experiences).  The 
Washington University PI will be responsible for submitting all MedWatch forms from 
secondary sites to the FDA within the timeframes specified above. 
 
10.7 
Timeframe for Tracking Reportable Events:  
 
Adverse events will be tracked for 30 days following the last day of study treatment, or 
until the start of a new chemotherapy/treatment, whichever comes first. 


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11.0 
DATA SAFETY MONITORING PLAN 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, the 
Data and Safety Monitoring Committee (DSMC) will meet to review toxicity data at least every 6 
months following the activation of the first secondary site.  The report will be prepared by the 
statistician and will be submitted to the Quality Assurance and Safety Monitoring Committee 
(QASMC).  This report will include: 
 
 HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
 Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of study 
 History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol exceptions, 
error, or breach of confidentiality including start/stop dates and reason 
 Study-wide target accrual and study-wide actual accrual including numbers from 
participating sites 
 Protocol activation date at each participating site 
 Average rate of accrual observed in year 1, year 2, and subsequent years at each 
participating site 
 Expected accrual end date and accrual by site 
 Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
 Measures of efficacy 
 Early stopping rules with supporting data and list the number of participants who have 
met the early stopping rules 
 Summary of toxicities at all participating sites 
 Abstract submissions/publications 
 Summary of any recent literature that may affect the safety or ethics of the study  
 
Until such a time as the first secondary site activates this protocol, a semi-annual DSM report to 
be prepared by the study team will be submitted to the QASM Committee beginning 6 months 
after study activation at Washington University. 
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC according 
to institutional guidelines. 
 
A DSMC will consist of no fewer than 3 members including 2 clinical investigators and a 
biostatistician.  Like investigators, DSMC members are subject to the Washington University 
School of Medicine policies regarding standards of conduct. Individuals invited to serve on the 
DSMC will disclose any potential conflicts of interest to the trial principal investigator and/or 
appropriate university officials, in accordance with institution policies. Potential conflicts that 
develop during a trial or a member’s tenure on a DSMC must also be disclosed.  


Genomic Predictors of Decitabine Response in AML/MDS 
 
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Refer to the Washington University Quality Assurance and Data Safety Monitoring Committee 
Policies and Procedures for full details on the responsibilities of the DSMC at 
http://www.siteman.wustl.edu/uploadedFiles/Research_Programs/Clinical_Research_Resources/
Protocol_Review_and_Monitoring_Committee/QASMCQualityAssurcance.pdf  
 
 


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12.0 
AUDITING 
 
Since Washington University is the coordinating center, each site will be audited annually by 
Siteman Cancer Center personnel (QASMC) unless the outside institution has an auditing 
mechanism in place and can provide a report. The outside sites will be asked to send copies of all 
audit materials, including source documentation.  The audit notification will be sent to the 
Washington University Research Patient Coordinator, who will obtain the audit materials from the 
participating institution. 
 
Notification of an upcoming audit will be sent to the research team one month ahead of the audit. 
Once accrual numbers are confirmed, and approximately 30 days prior to the audit, a list of the 
cases selected for review (up to 10 for each site) will be sent to the research team. However, if 
during the audit the need arises to review cases not initially selected, the research team will be 
asked to provide the additional charts within two working days. 
 
Additional details regarding the Auditing Policies and Procedures can be found at 
https://siteman.wustl.edu/wp-content/uploads/2015/QASMC-Policies-and-Procedures-
03.31.2015.pdf  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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13.0 
MULTICENTER REGULATORY REQUIREMENTS 
 
Washington University requires that each participating site sends its informed consent document 
to be reviewed and approved by the Washington University Regulatory Coordinator (or designee) 
prior to IRB/IEC submission.    
 
Site activation is defined as when the secondary site has received official written documentation 
from the coordinating center that the site has been approved to begin enrollment.  At a minimum, 
each participating institution must have the following documents on file at Washington University 
prior to study activation: 
 
 Documentation of IRB approval of the study in the form of a letter or other official 
document from the participating institution’s IRB.  This documentation must show 
which version of the protocol was approved by the IRB. 
 Documentation of IRB approval of an informed consent form. The consent must 
include a statement that data will be shared with Washington University, including the 
Quality Assurance and Safety Monitoring Committee (QASMC), the DSMC (if 
applicable), and the Washington University study team. 
 Documentation of FWA, signed FDA Form 1572 (if applicable), and the CVs of all 
participating investigators. 
 Protocol signature page signed and dated by the investigator at each participating site. 
 
The coordinating center Principal Investigator (or designee) is responsible for disseminating to the 
participating sites all study updates, amendments, reportable adverse events, etc.  Protocol/consent 
modifications and IB updates will be forwarded electronically to the secondary sites within 4 
weeks of obtaining Washington University IRB approval.  Activated secondary sites are expected 
to submit protocol/consent/IB modifications to their local IRBs within 4 weeks of receipt unless 
otherwise noted.  Upon the secondary sites obtaining local IRB approval, documentation of such 
shall be sent to the Washington University study team within 2 weeks of receipt of approval. 
 
Documentation of participating sites’ IRB approval of annual continuing reviews, protocol 
amendments or revisions, all SAE reports, and all protocol violations/deviations/exceptions must 
be kept on file at Washington University. 
 
The investigator or a designee from each institution must participate in a regular conference call 
to update and inform regarding the progress of the trial. 
 
 


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14.0 
STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
 
14.1 
Analysis Populations 
 
Intent-to-Treat - The intent-to-treat (ITT) population will be defined as all patients who 
receive at least one dose of decitabine.  
 
Efficacy-Evaluable - The efficacy-evaluable (EE) population will be defined as all patients 
who receive at least one cycle of treatment.   
 
Safety - The safety population will be defined as all patients with at least one dose of 
decitabine. 
 
14.2 
Statistical Analysis 
 
14.2.1 Descriptive Analyses 
 
Patient Disposition: The number of patients discontinued, the reasons for 
discontinuation, and the number of cycles administered will be summarized. 
 
Demographics and Baseline Characteristics: Descriptive summary statistics will 
be provided for demographic and important baseline characteristics.  For 
continuous variables, the number of patients, mean, standard deviation, median, 
minimum and maximum will be provided.  For categorical variables, the number 
and percent of patients in each demographic/characteristic category will be 
summarized. 
 
14.2.2 Primary Endpoint Analysis 
 
The primary end-point of this phase II study is the correlation biomarkers with 
overall response in AML/MDS patients to decitabine. 
 
We will correlate the presence of patient specific mutations (especially mutations 
in DNMT3A, IDH1, IDH2, and TET2) with overall response rates. Mutation rates 
in these genes and in other patient-specific genes will be correlated with overall 
response and their respective 95% confidence intervals will be provided. 
 
Statistical considerations. The relationship between AML/MDS-associated 
mutations and the responsiveness to decitabine will be described by contingency 
tables and assessed using Chi-square test or Fisher’s exact test as appropriate. The 
differentially expressed genes between responders and non-responders will be 
determined using the significance analysis of microarray (SAM). Multivariate 
logistic regression will also be fit to adjust the potential confounding effects of other 
demographic and clinical characteristics. The resultant critical P values will be 
corrected for multiple testing.  Since the Bonferroni correction is known to be too 
conservative, a permutation analysis (e.g., 10,000 random assignments for 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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responsive status) will be used to derive an empirical critical P value.  The new 
critical p-value is the number of permutation tests which yield a lower p-value than 
the original test divided by the total number of permutation tests.[61]   
 
A power analysis was performed using Chi-square test for the association between 
mutation and response to decitabine. The frequency of DNMT3A, IDH1/2, and 
TET2 mutations in AML and MDS patients are each between 15% and 30% in 
multiple cohorts.[29-37] Table 3 shows the minimal detectable differences of 
response rates between mutant and wild-type cases, assuming an expected overall 
45% CR/CRi rate and 80% power. For an average of 20% frequency of a mutated 
gene, for example, 100 patients will provide 80% power to detect 30% difference 
at a 2-sided 0.1 alpha error. The purpose of this study is to determine whether any 
of these mutations result in a 
clinically 
significant 
association 
that 
would 
warrant pre-treatment testing. 
We believe that a reasonable 
cut off would be if at least 
30% of clinical response 
could be attributed to the 
presence 
of 
a 
specific 
mutation. 
However, 
we 
anticipate that more power 
could be achieved as we 
assess patient groups with 
combined characteristics (e.g. 
DNMT3A mutation or loss of 
DNMT3A 
expression). 
In 
addition, we expect that the 
majority of the enrolled 125 
patients can be used in the analysis (since all of our molecular information will be 
acquired within 30 days of enrollment, we anticipate a drop-out rate of 20% or less). 
 
14.2.3 Secondary Endpoint Analyses 
 
The overall response rate (CR/CRi/PR) and complete response rate (CR/CRi) will 
be compared with historical controls[2], and the 95% confidence intervals will be 
provided. 
 
The rate of patient specific mutation clearance after 10, 28, and 56 days of therapy 
will be determined using an Illumina based platform. We will compare the rate of 
mutation clearance between the patients who achieve a CR/CRi after 4 cycles and 
those who do not. We anticipate that we will identify between 6 and 25 exonic 
SNVs per case (the range obtained with >200 AML samples sequenced to date with 
whole genome or exome sequencing), and that the expected 95% confidence 
interval of the mean variant allele frequency will be ±2.56% and ±1.25%, 
Table 3. Detectable mutation associated response rates (80% power, α = 0.05) 
 
Total 
N 
% of patients 
with a given 
mutation  
Detectable 
response 
difference 
Sensitivity of mutation to 
predict response at minimum 
detectable response difference* 
100 
15%  
20%  
30% 
40% 
50% 
0.40 
0.35 
0.30 
0.27 
0.26 
80% 
75% 
67% 
61% 
58% 
* Assuming the overall response  rate (CR/Cri) of 45%   


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respectively, if we assume an anticipated standard deviation of 3.2% within the 
same clone. The change of tumor burden over time and its association with clinical 
response will  be assessed using 2-way ANOVA for repeated measurement data, 
followed by the ad-hoc multiple comparisons for between-group differences (e.g., 
responders vs. non-responders) at given time points as well as the between-time 
differences within each group. The resultant critical P values will also be corrected 
using permutation analysis for multiple testing. 
 
The percent decrease in bone marrow methylcytosine will be calculated on day 
10±1. We will compare this reduction with previously published data and between 
the patients who achieve CR/CRi and those who do not. The correlation of peak 
plasma levels of decitabine with response will be assessed using 2-sample t-test or 
Mann-Whitney non-parametric rank-sum test as appropriate. The change of total 
bone marrow DNA methylcytosine from baseline and its association with both 
steady-state plasma drug levels and response will be assessed using 2-way ANOVA 
for repeated measurement data, followed by the ad-hoc multiple comparisons for 
between-group differences (e.g., responders vs. non-responders) and between-time 
differences. Similar analyses will be performed for the correlation of expression 
profiles and SNP analysis with pharmacokinetic data.   
 
Statistical power justification.  A power analysis was performed using two-sample 
t-test for between-group difference and paired t-test for the change over time. For 
tumor clearance, this will provide > 99% power to detect a 10% difference in the 
means at two separate time points for clones with 6 or more variants, and > 98% 
power to detect a 15% difference if there are 3 or more variants per clone with α = 
0.05 (power will decrease to 91% and 82%, respectively, in cases where the 
standard deviation is 4.7%, the maximum we have observed thus far). For 
pharmacologic correlations, we anticipate 85% power to determine a 25 ng/ml 
difference between the average Cmax of 45 evaluable responders and 55 evaluable 
non-responders with an anticipated average Cmax of 70 ng/ml and a standard 
deviation of 40 ng/ml within the entire cohort, and with α = 0.05.[22] We believe a 
less significant difference would not be sufficient to warrant application in clinical 
decision making (i.e. whether to continue therapy for more than 1 cycle). We 
anticipate 99% power to determine a difference of 20% in the reduction of bone 
marrow methylcytosine between 45 evaluable responders and 55 evaluable non-
responders with an expected standard deviation of 15% and α = 0.05.[28, 66-68] 
The difference in methylcytosine reduction previously observed was 30% between 
patients achieving CR/PR and those with only hematologic improvement.[28]  
 
Time to relapse and time to death will also be listed for each patient. 
 
14.2.4 Safety Analysis 
 
Extent of Exposure – Duration (number of cycles/infusions) of decitabine doses 
(cumulative and intensity) will be summarized. 
 


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Adverse Events - All adverse events will be coded using NCI CTCAE v. 4.0.  The 
incidence of treatment-emergent adverse events (number and percent of patients 
reporting the adverse event at least once during the study) will be summarized.  In 
addition, adverse events will be summarized by investigator attribution of 
relationship to study medication and by grade.  Similar summaries will be provided 
by treatment cycle. 
 
Clinical Laboratory Evaluation – Applicable laboratory parameters will be graded 
according to NCI CTCAE version 4.0. The incidence of maximum grade (number 
and percent of patients experiencing the maximum grade during the study) will be 
summarized for each laboratory parameter.  Similar summaries will be provided by 
treatment cycle.  Graphical displays of select hematological parameters will also be 
provided. 
 
Vital Signs, Physical Findings, and Other Safety Observations – Vital signs, 
findings from physical examinations, ECOG scores and weights will be presented 
in listing format. 
 
14.2.5 Description of Planned Subgroup Analyses 
 
The following subgroups (if sufficient numbers of patients) will be explored with 
respect to the primary and overall survival endpoints. 
 Age (60-69 years old vs ≥ 70 years old) 
 Cytogenetics 
 Patients with AML-associated mutations in DNMT3A, TET2, IDH1, or 
IDH2 vs those who do not have somatic variants in these genes. 
 
14.3 
Sample Size 
 
We anticipate initially enrolling 25 patients at Washington University. Once we establish 
a routine exome analysis with validation and calculation of the rate of patient-specific 
mutation clearance, we will enroll an additional 100 patients from at least 2 additional 
centers.  
 


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15.0 
DATA SUBMISSION SCHEDULE 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to registration 
Patient Registration Form 
Disease Diagnosis Form 
Inclusion Criteria Worksheet 
Exclusion Criteria Worksheet 
 
Prior to starting treatment 
Treatment Record Form 
To be maintained throughout a patient’s treatment 
cycle.  The complete record is due at completion of 
treatment. 
Bone Marrow Sample Collection 
Form 
To be maintained throughout the course of the study 
at designated bone marrow sample collections time 
points (See Appendix 2). 
Pathology: Bone Marrow Biopsy 
To be completed upon the return of pathology 
report for each bone marrow biopsy. 
Study Calendar Form 
To be completed with the date of each time point 
upon occurrence. 
Labs Form 
To be completed upon the return of laboratory 
reports for each study time point (See Appendix 2) 
Dose Modifications 
To be maintained throughout the course of the study 
if dose modifications are performed. 
Therapy Response Form 
To be filled out upon remission (prior to, and/or on 
therapy) 
End of Study Form 
 
Due at the completion of the EOS visit, at the time 
of death, and/or at the time that the patient is lost to 
follow-up 
Follow Up Form 
 30 days post date of last decitabine dose. 
 Every 6 months for 2 years after the last dose of 
study drug. 
Record of Adverse Events 
At the time of any AE 
 
Any queries generated by Washington University must be responded to within 28 days of receipt 
by the participating site.  The Washington University research team will conduct a regular review 
of data status at all secondary sites, with appropriate corrective action to be requested as needed. 
 


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Appendix 1 
ECOG Performance Scale 
Activity Status 
Description 
0 
Asymptomatic, fully active, and able to carry on all predisease 
performance without restrictions. 
 
1 
Symptomatic, fully ambulatory but restricted in physically strenuous 
activity and able to carry out performance of a light or sedentary 
nature, eg, light housework, office work. 
 
2 
Symptomatic, ambulatory and capable of all self-care but unable to 
carry out any work activities.  Up and about more than 50% of 
waking hours: in bed less than 50% of day. 
 
3 
Symptomatic, capable of only limited self-care, confined to bed or 
chair more than 50% of waking hours, but not bedridden. 
 
4 
Completely disabled.  Cannot carry on any self-care.   Totally 
bedridden. 
 
5 
Dead 
 
 


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Appendix 2 
Study Flow Chart 
Study Flow Chart 
Test 
Pre-
study1 
Base-
line2 
All 
Cycles 
Day 1 
All Cycles 
Days 1-103 
Cycle 1 Day 
4 ± 1 
 
Cycle 1 
Day 10 ± 1 
and 28 ± 4 
Every 
week 
Every 
2 
weeks 
Even 
cycles 
Day 28±4 
End of 
Study 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Medical History 
 
X 
 
 
 
 
 
 
 
 
Physical Exam 
 
Xa 
Xb 
 
 
 
 
 
 
X 
Transfusion 
Requirementsc 
 
Xc 
Ongoing 
Bone Marrow 
Aspirate and Biopsy 
Xd 
 
 
 
 
Xd 
 
 
Xd 
Xd 
Flow Cytometrye 
X 
 
 
 
 
 
 
 
 
 
Pregnancy test 
 
X 
 
 
 
 
 
 
 
 
CBC with differential 
and platelets 
 
X 
X 
 
 
 
Xf 
 
 
X 
Serum Chemistriesg 
 
X 
X 
 
 
 
 
Xf 
 
X 
Decitabine 
administrationh 
 
 
 
X 
 
 
 
 
 
 
Adverse Events  
 
 
Ongoing 
Post-study follow-up 
 
 
 
 
 
 
 
 
 
Xi 
1 To be performed within 14 days of the first dose of decitabine.  
2 To be performed within 7 days of the first dose of decitabine.  
3 Decitabine may be reduced to Days 1-5 or Days 1-3. See Section 6.3 for details.  
a To include height, weight, and ECOG. 
b To include weight. 
c Care should be given to record the frequency of transfusions from 8 weeks prior to first dose of study drug through 
the duration of the study. 
d Bone marrow biopsy and aspirate should be performed at baseline, at Cycle 1 Day 10 ± 1, and at  Day 28 ± 4 of Cycles 
1, 2, 4 and 6 for clinical disease assessment. Patients who achieve with clinical response (CRi or better) and are receiving 
cycles every 6 weeks, will undergo bone marrow biopsies on day 42 ± 4. Following Cycle 6, bone marrow biopsies 
should be performed at the discretion of the treating physician to assess for relapse or progression. Standard bone marrow 
procurement procedures will be followed for collection of the tissue. To include: correlative studies (collected under 
HRPO# 201011766), morphologic assessment, cytogenetics and FISH at all time points. A skin biopsy sample (collected 
under HRPO# 201011766) is required at pre-study, but may have been collected months or even years prior to first dose 
of decitabine.  
e On bone marrow or peripheral blood.  
f After Cycle 1, these labs may be obtained every other week (CBC) or once a month (Chemistries) at the discretion of 
the treating physician. 
g Serum chemistries include bicarbonate, calcium, glucose, total protein, albumin, BUN, potassium, chloride, sodium, 
creatinine, total bilirubin, AST, ALT, alkaline phosphatase, and LDH. 
h Decitabine treatment will be 20 mg/m2 over a 1-hour IV infusion on consecutive Days 1-10 every 28-day cycle.   The 
dose will be calculated based on actual patient weight at the beginning of each cycle. Patients may be premedicated 
with lorazepam, prochlorperazine, palonosetron, or ondansetron at the treating physician’s discretion. 
i Patients will be followed for  30 days following the date of the last decitabine dose to monitor for toxicity, survival 
and response. Thereafter, the investigator or designee will record information on subsequent therapy, disease 
progression, and death, every 6 months for 2 years after first dose of study drug or until the patient is lost to follow-up.


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16.0 
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Cashen, A.F., et al., Pharmacokinetics of decitabine administered as a 3-h 
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23. 
Chabot, G.G., J. Bouchard, and R.L. Momparler, Kinetics of deamination of 5-
aza-2'-deoxycytidine and cytosine arabinoside by human liver cytidine deaminase 
and its inhibition by 3-deazauridine, thymidine or uracil arabinoside. Biochem 
Pharmacol, 1983. 32(7): p. 1327-8. 
24. 
Momparler, R.L., et al., Cell cycle effects and cellular pharmacology of 5-aza-2'-
deoxycytidine. Cancer Chemother Pharmacol, 1984. 13(3): p. 191-4. 
25. 
Raza, A., et al., Biological significance of cell cycle kinetics in 128 standard risk 
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26. 
Brons, P.P., et al., Proliferation patterns in acute myeloid leukemia: leukemic 
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225-33. 
27. 
Raza, A., et al., Direct relationship between remission duration in acute myeloid 
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28. 
Shen, L., et al., DNA methylation predicts survival and response to therapy in 
patients with myelodysplastic syndromes. J Clin Oncol, 2010. 28(4): p. 605-13. 
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Tefferi, A., et al., IDH1 and IDH2 mutation studies in 1473 patients with chronic-
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30. 
Ley, T.J., et al., DNMT3A mutations in acute myeloid leukemia. N Engl J Med, 
2010. 363(25): p. 2424-33. 
31. 
Mardis, E.R., et al., Recurring mutations found by sequencing an acute myeloid 
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Genomic Predictors of Decitabine Response in AML/MDS 
 
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32. 
Abdel-Wahab, O., J. Patel, and R.L. Levine, Clinical Implications of Novel 
Mutations in Epigenetic Modifiers in AML. Hematol Oncol Clin North Am, 2011. 
25(6): p. 1119-33. 
33. 
Reuther, G.W., Recurring mutations in myeloproliferative neoplasms alter 
epigenetic regulation of gene expression. Am J Cancer Res, 2011. 1(6): p. 752-62. 
34. 
Paschka, P., et al., IDH1 and IDH2 mutations are frequent genetic alterations in 
acute myeloid leukemia and confer adverse prognosis in cytogenetically normal 
acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem 
duplication. J Clin Oncol, 2010. 28(22): p. 3636-43. 
35. 
Shen, Y., et al., Gene mutation patterns and their prognostic impact in a cohort of 
1185 patients with acute myeloid leukemia. Blood, 2011. 118(20): p. 5593-603. 
36. 
Tefferi, A., et al., Detection of mutant TET2 in myeloid malignancies other than 
myeloproliferative neoplasms: CMML, MDS, MDS/MPN and AML. Leukemia, 
2009. 23(7): p. 1343-5. 
37. 
Walter, M.J., et al., Recurrent DNMT3A mutations in patients with 
myelodysplastic syndromes. Leukemia, 2011. 25(7): p. 1153-8. 
38. 
Lin, J., et al., Recurrent DNMT3A R882 Mutations in Chinese Patients with Acute 
Myeloid Leukemia and Myelodysplastic Syndrome. PLoS One, 2011. 6(10): p. 
e26906. 
39. 
Bejar, R., et al., Clinical effect of point mutations in myelodysplastic syndromes. 
N Engl J Med, 2011. 364(26): p. 2496-506. 
40. 
Walter, M.J., et al., Clonal architecture of secondary acute myeloid leukemia. N 
Engl J Med, 2012. 366(12): p. 1090-8. 
41. 
Kantarjian, H.M., et al., Survival advantage with decitabine versus intensive 
chemotherapy in patients with higher risk myelodysplastic syndrome: comparison 
with historical experience. Cancer, 2007. 109(6): p. 1133-7. 
42. 
Lubbert, M., et al., Continued Low-Dose Decitabine (DAC) Is an Active First-
Line Treatment in All Cytogenetic Subgroups of Older AML Patients: Results of 
the FR00331 Multicenter Phase II Study. ASH Annual Meeting Abstracts, 2007. 
110(11): p. 300-. 
43. 
Blum, W., et al., Phase I Study of Decitabine Alone or in Combination With 
Valproic Acid in Acute Myeloid Leukemia. J Clin Oncol, 2007. 25(25): p. 3884-
3891. 
44. 
Issa, J.-P.J., et al., Phase 1 study of low-dose prolonged exposure schedules of the 
hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in hematopoietic 
malignancies. Blood, 2004. 103(5): p. 1635-1640. 
45. 
Garcia-Manero, G., et al., Phase 1/2 study of the combination of 5-aza-2'-
deoxycytidine with valproic acid in patients with leukemia. Blood, 2006. 108(10): 
p. 3271-3279. 
46. 
Issa, J.P., S.B. Baylin, and J.G. Herman, DNA methylation changes in 
hematologic malignancies: biologic and clinical implications. Leukemia, 1997. 
11 Suppl 1: p. S7-11. 
47. 
Ganetzky, R.D., et al., Differences Between Normal and Leukemic Stem Cell-
Specific Methylome Indicates Aberrantly Silenced Genes Involved in the 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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Pathogenesis of Malignant Evolution. ASH Annual Meeting Abstracts, 2008. 
112(11): p. 599-. 
48. 
Christman, J.K., 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA 
methylation: mechanistic studies and their implications for cancer therapy. 
Oncogene, 2002. 21(35): p. 5483-95. 
49. 
Lubbert, M., DNA methylation inhibitors in the treatment of leukemias, 
myelodysplastic syndromes and hemoglobinopathies: clinical results and possible 
mechanisms of action. Curr Top Microbiol Immunol, 2000. 249: p. 135-64. 
50. 
Kantarjian, H., et al., Results of a randomized study of 3 schedules of low-dose 
decitabine in higher-risk myelodysplastic syndrome and chronic myelomonocytic 
leukemia. Blood, 2007. 109(1): p. 52-57. 
51. 
Steensma, D.P., et al., Multicenter Study of Decitabine Administered Daily for 5 
Days Every 4 Weeks to Adults With Myelodysplastic Syndromes: The Alternative 
Dosing for Outpatient Treatment (ADOPT) Trial. J Clin Oncol, 2009: p. 
JCO.2008.19.6550. 
52. 
Cashen, A.F., et al., Preliminary Results of a Multicenter Phase II Trial of 5-Day 
Decitabine as Front-Line Therapy for Elderly Patients with Acute Myeloid 
Leukemia (AML). ASH Annual Meeting Abstracts, 2008. 112(11): p. 560-. 
53. 
Jabbour, E., et al., Evolution of decitabine development: accomplishments, 
ongoing investigations, and future strategies. Cancer, 2008. 112(11): p. 2341-51. 
54. 
Kantarjian, H., et al., Results of a randomized study of 3 schedules of low-dose 
decitabine in higher-risk myelodysplastic syndrome and chronic myelomonocytic 
leukemia. Blood, 2007. 109(1): p. 52-7. 
55. 
Blum, W.G., Klisovic, R., Liu, S., Kefauver, C., Grever, M. R., Schaaf, L., Chan, 
K., Byrd, J. C., Villalona-Calero, M., Marcucci,  G. Efficacy of a novel schedule 
of decitabine in previously untreated AML, age 60 or older. in ASCO Annual 
Meeting. 2009. 
56. 
van Groeningen, C.J., et al., Phase I and pharmacokinetic study of 5-aza-2'-
deoxycytidine (NSC 127716) in cancer patients. Cancer Res, 1986. 46(9): p. 
4831-6. 
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leukemia. Leuk Res, 1981. 5(6): p. 453-62. 
58. 
Lin, K.T., R.L. Momparler, and G.E. Rivard, Sample preparation for the 
determination of 5-aza-2'-deoxycytidine in plasma by high-performance liquid 
chromatography. J Chromatogr, 1985. 345(1): p. 162-7. 
59. 
Momparler, R.L., G.E. Rivard, and M. Gyger, Clinical trial on 5-aza-2'-
deoxycytidine in patients with acute leukemia. Pharmacol Ther, 1985. 30(3): p. 
277-86. 
60. 
Raska, K., Jurovcik, M., Sormova., Z., On the metabolism of 5-azacytidine and 5-
aza-2'-deoxycytidine in mice. Coll Czech Chem Commun, 1965. 30: p. 3001-
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61. 
Momparler, R.L., et al., Pilot phase I-II study on 5-aza-2'-deoxycytidine 
(Decitabine) in patients with metastatic lung cancer. Anticancer Drugs, 1997. 
8(4): p. 358-68. 


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62. 
Aparicio, A., et al., Phase I trial of continuous infusion 5-aza-2'-deoxycytidine. 
Cancer Chemother Pharmacol, 2003. 51(3): p. 231-9. 
63. 
Bryan, J., et al., Pharmacokinetic evaluation of decitabine for the treatment of 
leukemia. Expert Opin Drug Metab Toxicol, 2011. 7(5): p. 661-72. 
64. 
Scandura, J.M., et al., Phase 1 study of epigenetic priming with decitabine prior 
to standard induction chemotherapy for patients with AML. Blood, 2011. 118(6): 
p. 1472-80. 
65. 
Westfall, P.H. and S.A. Young, Resampling-based multiple testing: examples and 
methods for p-value adjustment. 1993, New York: John Wiley & Sons, Inc. 
66. 
Samlowski, W.E., et al., Evaluation of a 7-day continuous intravenous infusion of 
decitabine: inhibition of promoter-specific and global genomic DNA methylation. 
J Clin Oncol, 2005. 23(17): p. 3897-905. 
67. 
Blum, W., et al., Phase I study of decitabine alone or in combination with 
valproic acid in acute myeloid leukemia. J Clin Oncol, 2007. 25(25): p. 3884-91. 
68. 
Yang, A.S., et al., DNA methylation changes after 5-aza-2'-deoxycytidine therapy 
in patients with leukemia. Cancer Res, 2006. 66(10): p. 5495-503. 
69.       Cheson, B.D., et al., Clinical application and proposal for modification of the 
International Working Group (IWG) response criteria in myelodysplasia. Blood, 
2006. 108(2): p. 419-25. 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
 
NCT# NCT01687400 
 
 
Document Version Date:  04/21/2016 
 
IRB Approval Date:  06/14/2016 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 5 Version date: 04/21/16  
Page 1 of 51 
 
 
Genomic Predictors of Decitabine Response in AML/MDS 
 
Washington University School of Medicine, Division of Oncology 
660 South Euclid Avenue, Box 8056, St. Louis, MO 63110 
 
Protocol #: 201210102 
Protocol Version Date: 04/21/2016 
 
Coordinating Center: Washington University School of Medicine 
 
Principal Investigator 
John Welch, M.D./Ph.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-2626, Fax Number: (314) 362-9333 
Email: jwelch@wustl.edu 
 
Sub-Investigator 
Timothy Ley, M.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-8832, Fax Number: (314) 362-9333 
Email: timley@wustl.edu 
 
Pathology 
Eric Duncavage, M.D. 
Department of Anatomic and Molecular Pathology 
Telephone: (314) 362-8832 
Email: eduncavage@wustl.edu  
 
Statistician 
Feng Gao, Ph.D. 
Division of Biostatistics 
Telephone: (314) 362-3682 
E-mail: feng@wustl.edu 
 
IND# 116548 //Clinical Trials.gov # NCT01687400 
 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to obtain 
informed consent, may not be disclosed to another party unless law or regulations require such disclosure.  Persons 
to whom the information is disclosed must be informed that the information is confidential and may not be further 
disclosed by them. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 5 Version date: 04/21/16  
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Genomic Predictors of Decitabine Response in AML/MDS  
Principal Investigator Signature Page 
 
 
Principal Investigator: 
 
John Welch, M.D., Ph.D. 
 
 
 
 
 
Signature of Investigator 
 
Date 
 
 
 
Printed Name of Investigator 
 
 
 
By my signature, I agree to personally supervise the 
conduct of this study and to ensure its conduct in 
compliance with the protocol, informed consent, 
IRB/HRPO procedures, the Declaration of Helsinki, ICH 
Good Clinical Practices guidelines, and the applicable 
parts of the United States Code of Federal Regulations or 
local regulations governing the conduct of clinical 
studies. 
 
 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 5 Version date: 04/21/16  
Page 3 of 51 
 
Study Schema: 
 
 
Patients with progressive disease after two cycles (defined as a clear increase in bone marrow blast count, typically a 
> 50% increase in bone marrow blasts over baseline) should be removed from study. 
Patients with stable disease may continue on study and should undergo a diagnostic bone marrow biopsy at the end 
of even numbered cycles. 
Once AML patients achieve morphologic complete remission with incomplete blood count recovery (CRi) or MDS 
patients achieve a marrow complete response (Marrow CR) as defined in Section 9.0, decitabine 
administration may be decreased to 20 mg/m2/day for ten consecutive days every 6 weeks, to limit 
myelosuppressive toxicity and improve count recovery. After 2 cycles at 6 week intervals, the dose may be 
reduce to 20 mg/m2/day for five consecutive days in 6 week cycles, at the discretion of the treating physician. 
After two cycles of five consecutive days of treatment, if the patient meets the definition of CR with complete 
cytogenetic response as defined in Section 9.0, administration may further be decreased to three consecutive 
days per cycle at the discretion of the treating physician. 
Sample collection for correlative studies: 
Bone marrow biopsies should be performed at baseline, at Cycle 1 Day 10 ± 1, and at Day 28 ± 4 of Cycles 
1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be performed at the discretion of the 
treating physician to assess for relapse or progression.  
 
 
20 
mg/m
2/day  
Cycle 1 
20 
mg/m
2/day 
Cycle 2+ 
Day 1 
Day 28 
BM Bx 
PB 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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Genomic Predictors of Decitabine Response in AML/MDS 
Table of Contents 
Section 
 
Page 
1.0 
INTRODUCTION 
6 
1.1 
Rationale 
6 
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
8 
1.2.1 
Pharmacokinetics and Metabolism of Decitabine 
10 
1.2.2 
Experience with extended dosing schedules 
11 
1.3 
Correlative Studies 
12 
2.0 
OBJECTIVES 
14 
2.1 
Primary Objective 
14 
2.2 
Secondary Objectives 
14 
3.0 
PATIENT SELECTION 
15 
3.1 
Inclusion Criteria 
15 
3.2 
Exclusion Criteria 
15 
3.3 
Inclusion of Women and Minorities 
16 
4.0 
REGISTRATION PROCEDURES 
17 
4.1 
Confirmation of Patient Eligibility 
17 
4.2 
Patient Registration in the Siteman Cancer Center OnCore Database 
17 
4.3 
Assignment of UPN 
17 
5.0 
INVESTIGATIONAL PLAN 
19 
5.1 
Summary of Study Design 
19 
5.2 
Study Procedures 
20 
5.2.1 
Pre-study Procedures 
20 
5.2.2 
Baseline Evaluation 
20 
5.2.3 
Day 1 of Each Cycle 
20 
5.2.4 
Cycle 1 Days 1-10 
20 
5.2.5 
Cycle 1 Day 10 ± 1 
21 
5.2.6 
Weekly 
21 
5.2.7 
Every Other Week 
21 
5.2.8 
Cycles 1, 2, 4 and 6 Day 28 ± 4 days 
21 
5.2.9 
End-of-Study Procedures 
21 
5.2.10 
Post-study Follow-up 
22 
5.3 
Duration of Therapy 
22 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
22 
5.4.1 
Chemotherapy 
22 
5.4.2 
Growth Factors 
23 
5.4.3 
Transfusions 
23 
5.4.4 
Prophylactic Antimicrobial Agents 
23 
5.4.5 
Radiotherapy 
23 
6.0 
DOSE MODIFICATIONS 
24 
6.1 
Delay for Cytopenia or Infection 
24 
6.2 
Delay for Organ Dysfunction 
24 
6.3 
Modification in Dose or Schedule for Treatment Response 
24 
7.0 
PHARMACEUTICAL INFORMATION 
25 


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7.1 
Decitabine 
25 
7.1.1 
Study Drug Preparation 
25 
7.1.2 
Supplier 
25 
8.0 
CORRELATIVE STUDIES SAMPLE COLLECTIONS 
26 
9.0 
EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
27 
9.1 
Response Criteria - AML 
27 
9.2 
Response Criteria - MDS 
28 
9.2.1 
Hematologic Improvement (HI) 
28 
9.3.1 Cytogenetic Response 
29 
9.3 
Guidelines for Evaluation of Disease 
29 
9.4 
Other Secondary Efficacy Measures 
29 
9.4.1 
Treatment Failure, Overall Survival and Event-free Survival 
29 
9.4.2 
Duration of Remission and Relapse-free Survival for Patients Having Complete 
Remission 
30 
9.4.3 
Safety 
30 
10.0 REGULATORY AND REPORTING REQUIREMENTS 
31 
10.1 
Definitions 
31 
10.1.1 
Adverse Events (AEs) 
31 
10.1.2 
Serious Adverse Event (SAE) 
31 
10.1.3 
Unexpected Adverse Experience 
32 
10.1.4 
Life-Threatening Adverse Experience 
32 
10.1.5 
Unanticipated Problems 
32 
10.1.6 
Noncompliance 
32 
10.1.7 
Serious Noncompliance 
33 
10.1.8 
Protocol Exceptions 
33 
10.2 
Reporting to the Human Research Protection Office (HRPO) at Washington University:33 
10.3 
Reporting to the Quality Assurance and Safety Monitoring Committee (QASMC) at 
Washington University 
33 
10.4 
Reporting Requirements for Secondary Sites 
34 
10.5 
Reporting to Secondary Sites 
34 
10.6 
Reporting to the FDA 
34 
10.7 
Timeframe for Tracking Reportable Events: 
35 
11.0 DATA SAFETY MONITORING PLAN 
36 
12.0 AUDITING 
38 
13.0 MULTICENTER REGULATORY REQUIREMENTS 
39 
14.0 STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
40 
14.1 
Analysis Populations 
40 
14.2 
Statistical Analysis 
40 
14.2.1 
Descriptive Analyses 
40 
14.2.2 
Primary Endpoint Analysis 
40 
14.2.3 
Secondary Endpoint Analyses 
41 
14.2.4 
Safety Analysis 
42 
14.2.5 
Description of Planned Subgroup Analyses 
43 
14.3 
Sample Size 
43 
15.0 DATA SUBMISSION SCHEDULE 
44 
16.0 REFERENCES 
47 
  


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1.0 
INTRODUCTION 
 
1.1 
Rationale   
 
Personalized therapy for cancer requires that 1) patient-specific factors (e.g. patient-
specific mutations, expression profiles, or pharmacogenomics) influence treatment 
algorithms, and 2) once therapy is initiated, in vivo responsiveness is used to guide further 
therapy. The modern era of genomics has provided significant insights into cancer 
pathogenesis by describing patterns of germline and somatic variants, as well as defining 
disease-specific patterns of gene expression. However, the application and integration of 
this data with therapeutics remains a difficult task. 
 
Decitabine is a commonly used single-agent therapy for patients with acute myeloid 
leukemia (AML) and/or myelodysplastic syndrome (MDS). Historically, it has yielded 
clinical complete responses in approximately 25% of patients across diverse clinical trials, 
it is well tolerated, and it can be given as in the outpatient setting. However, the majority 
of patients do not respond to decitabine, the molecular etiology of decitabine sensitivity is 
unknown, and there are no algorithms to predict patient responsiveness a priori or even 
within one month of starting therapy.  
 
AML and MDS are two closely related hematological disorders characterized by 
ineffective hematopoiesis and malignant expansion of clonal myeloid cells.  Both AML 
and MDS frequently occur in elderly patients and some cases are secondary and/or related 
to long-term bone marrow damage after radiation or chemotherapy. Current therapy for 
AML consists of cytotoxic chemotherapy followed by consolidation chemotherapy or stem 
cell transplantation. Using these approaches, current cure rates may be as high as 60% for 
patients younger than 60 with good risk cytogenetics (e.g. t(15;17), t(8;21), inv(16)).[4]  
Outcomes in patients ≥ 60 years old and with relapsed disease remain poor with 2 year 
survival of only ~25%.[5] Historically, the available treatment for MDS has been 
symptomatic and has not been shown to be effective in producing sustained improvement 
in hematopoiesis or in delaying leukemic evolution[6, 7].  
 
Current AML therapy is poorly tolerated in elderly and pre-treated patients due to 
hematologic and non-hematologic toxicities; following such therapy, patients typically 
require 3-4 weeks of hospitalization for treatment of neutropenic fevers and for regular 
transfusions. Even among the relatively healthy patients enrolled in clinical trials, induction 
mortality in the elderly can be as high as 20-40% following standard induction therapy,[8-
11] and moderate to severe non-hematologic toxicity occurs in at least an additional 30% 
of patients.[9, 12-14] Consequently, many older AML patients are not offered traditional 
intensive chemotherapy, but receive supportive care alone.[15]  More effective and less 
toxic therapies are needed to provide durable remissions in a significant proportion of 
elderly AML in populations. 
 
Decitabine has been studied in MDS patients and in elderly and/or relapsed AML patients. 
It has been found to be reasonably well tolerated, can be given in the outpatient setting, 
and has overall response rates of 20-45%.[2, 3, 16-20] Our own experience has been of 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 5 Version date: 04/21/16  
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CR/PR rates of 25%, [2] and this is typical of many other groups for both MDS and AML 
patients (reviewed in [21]  Decitabine is an S-phase depended drug.  [22-24] However, 
decitabine is usually administered in one hour daily infusions during 3 to 5 consecutive 
days (too short to expose decitabine to most AML blasts during at least 2 cycles of 
replication, which divide every 2 to 7 days).[25-27]  Recent experience with increasing the 
number of infusions per cycle from 5 consecutive days to 10 consecutive days has 
improved overall response rates to 67%.[3] Response to decitabine can be slow, sometimes 
requiring 3 to 5 cycles before maximum response is observed.[2] As a consequence, many 
patients are exposed to prolonged therapy, gaining little benefit, before determining 
resistance. There are currently no biomarkers that predict response to decitabine. 
 
Decitabine is a cytosine analogue that cannot be methylated. Incorporation of decitabine 
into DNA prevents DNA methylation at cytosine residues and derepresses epigenetically 
silenced gene expression. Therefore, the reduction in total genomic DNA methylcytosine 
content acts as a marker of effective decitabine dosing. Indeed, two groups have recently 
correlated effective methylcytosine reduction and gene expression derepression to 
response (Figure 1.1).[3, 28] 
 
 
 
   
Figure 1.1. From Shen et al JCO (2010) 28:611 and Blum et al JCO (2007) 25: 3884. 
 
We and others have recently used whole genome sequencing to identify recurrent, somatic 
mutations in AML and MDS patients in four genes that are involved in methylcytosine 
regulation: DNMT3A, IDH1, IDH2, and TET2.[29-37] Collectively, nearly half of all 
AML/MDS patients have a mutation in one of these four genes.  DNMT3A mutations are 
associated with an adverse outcome in AML patients treated with cytotoxic 
chemotherapy.[35, 38] However, it is not yet clear whether the presence of mutations in 
one or more of these genes is associated with response or resistance to decitabine. 
 
In this study, we will treat patients with the current state-of-the-art decitabine schedule.[3] 
We will perform exome sequencing on all patients and correlate response with mutation 
status, rate of mutation clearance, expression profile, plasma decitabine levels, and DNA 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 5 Version date: 04/21/16  
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methylcytosine content reduction. This approach will comprehensively assess genomic and 
pharmacologic parameters that could clinically predict patient response to decitabine.  
 
In this study, we will combine analysis of patients with AML and MDS. Recent genomic 
analysis has shown that AML and MDS have shared mutations in many of the same genes, 
shared patterns of mutation acquisition, and shared patterns of mutation evolution.[39, 40] 
Furthermore, MDS, like AML, presents with clonal hematopoiesis, even when the 
morphologic blast count is zero.[36]Because this study will identify and track the rate of 
clearance of patient specific mutations, and because decitabine has been shown to have 
efficacy in both AML and MDS patients, [2, 3, 16-19, 41-45] combining analysis of AML 
and MDS patients is reasonable.  Asymptomatic MDS patients are excluded because these 
patients are typically followed with watchful-waiting rather than with chemotherapy. 
  
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
 
Methylation of cytosine in the CpG dinucleotide by DNA methyltransferase leads to 
transcriptional silencing of genes during normal development and has emerged as a 
significant mechanism for the loss of tumor suppressor gene expression in human cancer, 
including AML and MDS.[46, 47] Like azacitidine, decitabine (5-aza-2’-deoxycitidine) is 
incorporated into DNA during S phase and forms covalent adducts with DNA 
methyltransferase. The irreversible inhibition of this enzyme results in loss of methylation 
and is associated with differentiation.[48]  Decitabine is an approximately 5-fold more 
potent inhibitor of DNA methyltransferase than azacitidine, which might explain why 
decitabine can induce differentiation of myeloid progenitors in vitro at lower doses than 
azacitidine. In addition to their differentiation inducing activity at low doses, both agents 
have a direct cytotoxic effect at higher doses (1-2 µM).[49]  
 
Decitabine has been investigated in hematologic malignancies using a range of doses and 
schedules.  It is currently FDA approved for the treatment of adults with MDS at a dose of 
15 mg/m2 every 8 hours for 3 days, based on a Phase III study that demonstrated improved 
outcomes in the treatment arm compared to patients treated with supportive care alone.[15]  
However, the overall response rate in this trial was modest (17%). Of note, 18% of the 
patients in this trial had 20-30% bone marrow blasts, meeting the current WHO criteria for 
AML.  Subsequent phase I and II studies in AML, using a variety of low-dose decitabine 
regimens, have reported overall response rates of 22-49%.[2, 3, 16-19, 41-45] Comparisons 
of multiple low dose schedules have noted improved efficacy in the MDS population using 
a dose of 20 mg/m2 on Days 1-5 of a 28-day cycle.[50, 51] Because of these results, we 
have recently explored this dosing schedule in the treatment of elderly patients with 
AML.[52] In this study, we observed an overall response rate of 24%, and an additional 
44% of patients achieved stable disease during a median of 5 monthly cycles.[48] In this 
study, we noted that decitabine does not act quickly and the median time to response was 
3 months. These results are similar to the recently published experience in Germany 
treating 227 patients with 15 mg/m2 over 3 hours TID for 3 days every 6 weeks, which 
resulted in a CR/PR rate of 26%.[16]  
 


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A recent study using a prolonged administration schedule (20 mg/m2 IV over 1 hour on 
consecutive days 1-10) showed improved response (64% CR/PR) and improved time to 
respond (50% of patients had bone marrow blasts < 5% after 1 cycle of therapy).[3]  These 
results are an improvement over previous experience by other groups using decitabine to 
treat elderly AML patients or patients with relapsed AML, and this experience is 
summarized below: 
Trials of decitabine in AML 
Status 
Other Therapy 
DAC Dose 
(mg x days) 
Evaluable 
CR 
de novo 
daunomycin1 
90 mg/m2 x 5 
6 
100% 
Salvage 
None4 
0.75-80 mg/kg 
16 
  
None5 
37-81 mg/kg 
6 
33% 
None7.  Ara-C 
Refractory 
Various 
4 
0 
AMSA7 
250-500 mg/m2 x 6 
10 
80% 
idarubicin or 
AMSA3 
250 mg/m2 x 6 
56 
39% 
Elderly 
None6 
270-360 mg/m2 x 3 
10 
30% 
None2 
45 mg/m2 x 3 
4 
50% 
None8 
20 mg/m2day 1-5,8-12 
50 
23% 
None9 
20 mg/m2 x 5 
55 
25% 
 
None10 
50 mg/m2 CIVI x 3 
29 
48% 
 
None11 
20 mg/m2 x 10 
53 
64% 
 
ATRA cycle 212 
15 mg/m2 TID x 3 
227 
26% 
1 Schwartsmann G.  Leukemia 1997; 11(Suppl 1):S28-31. 
2 Lubbert M. ASH 2002 Abstract #4601. 
3 Willemze R.  Leukemia.  1997; 11(Suppl 1):S24-27 
4 Rivard GE.  Leukemia Research 1981; 5:453-462. 
5 Momparler RL.  Pharmac Therapy 1985; 30:277-286.  
6 Petti MC.  Leukemia.  1993; 7(Suppl 1):36-41.  
7 Richel DJ.  Br. J. Cancer 1991; 64:144-148 
8 Issa, JP. Blood 2004. 103: 1635. 
9 Cashen AF. ASH Annual Meeting. 2008 abstract 560. 
10 Wijermans PW. Leukemia 1997. 11: Suppl 1, 19-23. 
11 Blum, W. PNAS 2010; 107:7473. 
12 Lubbert. Haematologica 2011. ePub 11/04/2011. 
 
In general, decitabine is well tolerated. Unlike conventional cytotoxic chemotherapy, 
decitabine can be administered in the outpatient setting. However, AML and MDS are 
aggressive hematologic malignancies. Cytopenias commonly result from AML/MDS 
inhibition of normal hematopoiesis, and often do not resolve until a complete response is 
achieved. Thus, neutropenic sepsis and thrombocytopenic bleeding remain significant 
problems regardless of therapy, especially during the first 4-8 weeks. In our experience 
treating elderly AML patients, neutropenia occurred in 24%, sepsis in 9%, and bacteremia 
in 7%.  Similar results have been observed in MDS patients.[20, 53, 54] As a result, death 
is not uncommon in the first 8 weeks of therapy, occurring in 15-17% of patients, and most 
commonly resulting from infection and cytopenias related to disease and therapy.[16, 52, 
55] While unsettling, these numbers are better than many historical patients treated with 
cytotoxic chemotherapy, and suggest a need for continued improvements in therapy.[9, 12]  


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1.2.1 Pharmacokinetics and Metabolism of Decitabine 
 
In human subjects, after IV administration, decitabine displayed a distribution phase 
with a half-life of 7 minutes and a terminal phase elimination half-life of 10-35 
minutes as measured by bioassay.[56, 57]   The short plasma half-life is due to rapid 
inactivation of decitabine by deamination by liver cytidine deaminase.[23] Total 
body clearance (Clp) in humans was high with a mean value of 126 mL/min/kg.[58]  
 
Among five adult patients with advanced solid tumors administered seven courses 
of 100 mg/m2 of decitabine by 1-hour infusions,[56] maximum plasma 
concentration ranged from 1 to 4 µM. Disappearance from plasma was biphasic 
with a rapid initial decline to less than 0.1 µM during the first hour post infusion 
and a slower decline over the next two hours to 6 nM, the level of detectability. 
Plasma half-life (meanSE) was estimated to be approximately 71 minutes in the 
initial phase (t½α) and 355 minutes in the second phase (t½β). The area under the 
plasma concentration-time curve was 40888 ng.hour/mL for these patients, with a 
peak plasma concentration of 2.010.44 µM (0.440.1 µg/mL), while mean volume 
of distribution (Vdss) and CLp was 4.591.42 L/kg and 12621 mL/min/kg, 
respectively. The time course of DAC concentrations was similar in 1 of 2 patients 
administered 75 mg/m2, while DAC was usually undetectable in the plasma of 
patients administered 25 to 60 mg/m2. 
 
Using a longer infusion time (up to 40 hours), Rivard et al.[57] reported a plasma 
concentration of 0.1 to 0.4 g/mL. With infusion times of 40-60 hours, at an infusion 
rate of 1 mg/kg/h, plasma concentrations of 0.43-0.76 µg/mL were achieved.[59]  The 
steady-state plasma concentration at an infusion rate of 1 mg/kg/h was estimated to 
be 0.2-0.5 µg/mL. The half-life after discontinuing the infusion was 12-20 min.[57]  
 
Urinary excretion of unchanged decitabine was low, ranging from less than 0.01% to 
0.9% of the total dose, and there was no relationship between excretion and dose or 
plasma drug levels. High clearance values and a total urinary excretion of <1% of the 
administered dose suggest that decitabine is eliminated rapidly and largely by 
metabolic processes, including extrahepatic metabolism.[60] 
 
The steady state plasma concentrations (Css) ranged from 0.31 to 0.39 µg/mL (1.27 
to 1.60 µM) during the infusion of 100 mg/m² decitabine over a 6-hour period.[61] 
The range of concentrations during a 600 mg/m2 infusion over an 8-hour period was 
0.41-16 g/mL(1.68 to 4.76 µM).[60] These plasma concentrations are in the same 
range as the in vitro concentrations that activated the expression of tumor 
suppressor gene p16 in three human non-small-cell lung cancer (NSCLC) cell 
lines.[61]  
 
Steady state decitabine levels of 0.1 to 0.2 µM were achieved in solid tumor patients 
dosed with 30 mg/m²/day and 40 mg/m²/day by continuous intravenous infusion 
over 72 hours.[62]  


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Summary of published steady state serum levels are below and are in proportion to 
infusion rate (R2 = 0.98, p < 0.0001):[63] 
 
Rate 
Serum 
Concentration 
mg/m2/hr 
ng/ml 
nM 
5 
55 
241 
7.5 
80 
351 
15 
180 
789 
21 
182.4 
800 
22.5 
350 
1535 
42 
456 
2000 
84 
1140 
5000 
 
In our experience treating primary patient blasts in vitro, concentrations of 100 – 
500 nM are required maximize methylcytosine reduction without causing cellular 
toxicity. Because decitabine incorporation into DNA is S-phase dependent, 
exposure of cells during cell division is necessary for efficacy. 
  
 
 
 
1.2.2 Experience with extended dosing schedules 
 
Blum et al recently published their experience treating elderly AML patients with 
a 10-day course of decitabine 20 mg/m2/day as a 1-hour infusion.[3] This prolonged 
exposure course was designed to increase the number of AML blast divisions 
exposed to decitabine per cycle. Toxicities were very similar to a similar regiment 
given as a 3 and 5 day course,[2, 16] but the overall response (CR/PR) was much 
improved (64% vs 25%). In all three studies, the toxicities were primarily myeloid 
suppression and the resultant infectious and bleeding complications associated with 
leukemic and pharmacologic hematopoietic suppression. Furthermore, Blum et al 
noted that the percent methylcytosine reduction was improved (22% vs 11%) as 


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was the time to blast clearance (50% of patients who ultimately achieved a CR had 
≤ 5% bone marrow blasts after 1 cycle vs 0% of patients after 2 cycles). Finally, 
like Shen et al, they observed a correlation between the extent of methylcytosine 
reduction and response.[28]  This study suggests that improvement in response 
rates and time to response can be achieved by prolonged administration of 
decitabine. Further, that DNA methylcytosine reduction can be achieved with 
prolonged exposure to decitabine and that this might be a patient-specific biological 
marker of adequate dosing. 
  
Scandura et al. recently published a study of infusional decitabine prior to 
cytarabine + daunorubicin induction therapy for AML.[64] They escalated the 
duration of infusional therapy and reached their planned goal of 20 mg/m2/day x 7 
days of continuous therapy given prior to cytarabine and daunorubicin. They noted 
similar toxicities (primarily hematopoietic and the associated neutropenic 
infections), but that these were not different from their experience giving cytarabine 
+ daunorubicin alone. 
 
1.3 
Correlative Studies 
 
All correlative studies will be collected under HRPO# 201011766 (“Tissue Acquisition for 
Analysis of Genetic Progression Factors in Hematologic Diseases”) that all patients will 
be concurrently enrolled in. The goals of the correlative studies include: 
 
1. To correlate patient-specific mutations with response to decitabine. 
We will perform exome sequencing of each patient’s pre-study bone marrow sample 
and skin sample. We will determine AML/MDS-associated variants found in the bone 
marrow, but not skin sample. We will correlate the presence of mutations with response 
(especially mutations in DNMT3A, IDH1, IDH2, and TET2). This study is powered to 
identify mutations with >80% sensitivity to predict decitabine response if used as a 
clinical assay. 
 
2. To correlate the velocity and depth of response with response to decitabine. 
We will assess tumor burden by allele frequency of patient-specific mutations in bone 
marrow samples collected pre study, Cycle 1 Day 10±1, Cycle 1 Day 28±4, and Cycle 
2 Day 28±4. We will assess mutations in at least 24 recurrently mutated genes 
(including, but not limited to: FLT3, DNMT3A, NPM1, TET2, WT1, IDH1, IDH2, 
RUNX1, NRAS, TP53, U2AF1, KRAS, PHF6, MSTP9, PTPN11, KIT, SMC3, PRUNE2, 
ETV6, CEBPA, SMC1A, ASXL1, STAG2, RAD21) as well as all patient-specific 
mutations discovered during exome sequencing. The leukemic allele frequency will be 
measured using the Illumnia MySeq or HiSeq platform using DNA capture by gene 
specific biotinylated-oligonucleotides. Patient bone marrow samples will be assessed 
pre-study, Cycle 1 Day 10 ± 1, Cycle 1 Day 28 ± 4, and Cycle 2 Day 28 ± 4 and 
compared with patient specific skin samples (normal control). The velocity of response 
will be calculated as the natural log rate of patient-specific mutation allele frequency 
clearance. Depth of response will be defined as the lowest patient-specific mutation 


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allele frequency in a bone marrow sample. The velocity and depth of tumor response 
will be correlated to: 
 Achievement of clinical CR, 
 Time to best response, 
 Mutational status, 
 Time to disease progression. 
Because all patient-specific exome-discovered mutations will be assessed a second 
time in the primary bone marrow sample as well as the  Cycle 1 Day 10 ± 1, Cycle 1 
Day 28 ± 4, and Cycle 2 Day 28 ± 4 bone marrow samples, this study will also serve 
to validate all exome-discovered mutations. This is important, because exome 
sequencing is associated with a 20-50% false-discovery rate depending on type I error 
tolerated (mutation-calling algorithms are typically set for greater sensitivity than 
specificity).  Thus, many mutations discovered during exome sequencing are incorrect.  
This second analysis of these mutations will server to exclude these false positive calls. 
 
3. To correlate the bone marrow expression profile from pre-study and Cycle 1 Day 10 ± 
1 with response to decitabine. 
We will correlate expression signatures, and changes in expression signatures, with: 
 Achievement of clinical CR, 
 Mutation status. 
 
4. To correlate the extent of bone marrow DNA hypomethylation on Cycle 1 Day 10 ± 1 
with response to decitabine. 
We will assess the percent reduction in bone marrow methylcytosine content using LC-
MS. Patients will be evaluated pre-study and Cycle 1 Day 10 ± 1. The change in DNA 
methylcytosine content will be correlated to: 
 Achievement of clinical CR, 
 Change in expression signature. 
This will determine whether inadequate blast exposure to decitabine correlates with 
drug resistance. 
 


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2.0 
OBJECTIVES 
 
2.1 
Primary Objective 
 
To determine whether patient-specific mutations correlate with clinical overall response to 
decitabine in patients with AML or MDS. 
 
2.2 
Secondary Objectives 
 
- 
To determine whether 10 consecutive days per cycle of decitabine improves outcomes 
compared with historical controls treated with 5 consecutive days per cycle of 
decitabine. 
 
- 
To determine whether other biomarkers correlate with clinical overall response to 
decitabine in patients with AML or MDS. These will include:  
 
1. The velocity and depth of patient-specific mutation clearance; 
2. The bone marrow expression profile and change in expression profile during 
decitabine treatment; 
3. The decrease in bone marrow methylcytosine content in response to decitabine.  
 
 


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3.0 
PATIENT SELECTION 
 
3.1 
Inclusion Criteria 
 
One of the following: 
 Patient must have non-M3 AML and be > 60 years of age OR 
 non-M3 AML with relapsed disease OR 
 Symptomatic MDS with one of the following: 
□ Symptomatic anemia with either hemoglobin < 10.0 g/dL or requiring RBC 
transfusion 
□ Thrombocytopenia with a history of two or more platelet counts < 50,000/mcL 
or a significant hemorrhage requiring platelet transfusions 
□ Neutropenia with two or more ANC < 1,000/mcL 
 
All of the following: 
1. Patient must have an ECOG performance status ≤ 2. 
2. Patient must have >10% disease burden measured by cytomorphology, flow 
cytometry, or cytogenetics. 
3. Patient must have peripheral white blood cell count < 50,000/mcl. 
4. Patient must have adequate organ function, defined as: 
a. 
Total bilirubin < 1.5 x ULN 
b. 
AST/ALT < 2.5 x ULN 
c. 
Serum creatinine < 2.0 x ULN  
5. Patient must have undergone ≤ 2 cycles of prior hypomethylating agent (decitabine 
or azacitidine). 
6. Patient must be enrolled in HRPO# 201011766 (“Tissue Acquisition for Analysis 
of Genetic Progression Factors in Hematologic Diseases”). 
7. Patient must be > 18 years of age. 
8. Patient must be able to understand and willing to sign an IRB-approved written 
informed consent document. 
 
3.2 
Exclusion Criteria 
 
1. Patient must not be pregnant or nursing. 
2. The patient must not have acute promyelocytic leukemia or t(15;17) observed by 
FISH. 
3. Patient must not have known CNS leukemia.  
4. Patient must not have a history of positive HIV serology.  
5. Patient must not have a history of positive Hepatitis C serology. 
6. Patient must not have undergone a prior allogeneic stem cell transplant. 
7. Patient must not have any uncontrolled intercurrent illness including, but not 
limited to, ongoing or active infection, ongoing or active GVHD, congestive heart 
failure of NYHA class 3 or 4, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situation that would limit compliance with study 
requirements. 
8. Patient must not have had radiation therapy within 14 days of enrollment. 


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9. Patient must not have received any chemotherapy within 21 days of enrollment, 
and any acute treatment-related toxicities must have returned to baseline. Patients 
may be receiving hydrea at time of enrollment.    
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible for this trial. 


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4.0 
REGISTRATION PROCEDURES 
 
Patients must not start any protocol intervention prior to registration through the Siteman 
Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility by Washington University 
2. Registration of patient in the Siteman Cancer Center database 
3. Assignment of unique patient number (UPN) 
 
Once the patient has been entered in the Siteman Cancer Center OnCore database, the WUSM 
coordinator will forward verification of enrollment and the UPN via email. 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility collecting the information listed below and scanning and 
emailing it to the research coordinator listed in the Siteman Cancer Center Clinical Trials 
Core Protocol Procedures for Secondary Sites packet at least one business day prior to 
registering patient: 
1. Your name and contact information (telephone number, fax number, and email 
address) 
2. Your site PI’s name, the registering MD’s name, and your institution name 
3. Patient’s race, sex, and DOB 
4. Three letters (or two letters and a dash) for the patient’s initials 
5. Currently approved protocol version date 
6. Copy of signed consent form (patient name may be blacked out) 
7. Planned date of enrollment 
8. Completed eligibility checklist, signed and dated by a member of the study team 
9. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center OnCore Database 
 
Registrations may be submitted Monday through Friday between 8am and 5pm CT. Urgent 
late afternoon or early morning enrollments should be planned in advance and coordinated 
with the Washington University research coordinator.  Registration will be confirmed by 
the research coordinator or his/her delegate by email within one business day. Verification 
of eligibility and registration should be kept in the patient chart. 
 
All patients at all sites must be registered through the Siteman Cancer Center OnCore 
database at Washington University. 
 
4.3 
Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  Patients 


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will also be identified by first, middle, and last initials.  If the patient has no middle initial, 
a dash will be used on the case report forms (CRFs).  All data will be recorded with this 
identification number on the appropriate CRFs. 
 


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5.0 
INVESTIGATIONAL PLAN 
 
5.1 
Summary of Study Design 
 
This is an open-label, single-arm, Phase II, biomarker study of patients with AML or MDS 
who are treated with 10 consecutive days of decitabine. FAB-M3 AML is excluded from 
this trial, because other well-tolerated options exist for the treatment of FAB-M3 acute 
promyelocytic leukemia. AML patients who are < 60 years old and have not received 
cytotoxic induction chemotherapy are also excluded, because cytotoxic therapy offers a 
curative possibility for these patients.  MDS patients without cytopenias are also excluded 
because these patients can be followed with watchful-waiting. 
 
All patients will undergo bone marrow biopsies pre-study, on Cycle 1 Day 10 ± 1, and on 
Day 28 ± 4 of Cycles 1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. 
Patients will provide a skin biopsy sample pre-study. See Section 8.0 for details on 
correlative studies.  
 
Patients will receive decitabine 20 mg/m2/day as a 1-hour infusion on consecutive Days 1-
10 in 28-day cycles.  The dose will be calculated based on actual patient weight at the 
beginning of each cycle. 
 
Once AML patients achieve morphologic complete remission with incomplete blood count 
recovery (CRi) or MDS patients achieve a marrow complete response (Marrow CR) as 
defined in Section 9.0, decitabine administration may be decreased to 20 mg/m2/day for 
ten consecutive days every 6 weeks, to limit myelosuppressive toxicity and improve count 
recovery. After 2 cycles at 6 week intervals, the dose may be reduce to 20 mg/m2/day for 
five consecutive days in 6 week cycles, at the discretion of the treating physician.  After 
two cycles of five consecutive days of treatment, if the patient meets the definition of CR 
with complete cytogenetic response as defined in Section 9.0, administration may further 
be decreased to three consecutive days per cycle at the discretion of the treating physician. 
 
It will be a logistical goal of this trial consistently to return preliminary exome results to 
the treating physician by the end of Cycle 1 and validated results by the end of Cycle 2. 
However, this logistical goal is not a primary or secondary aim of this study. Given the 
limited data regarding mutation presence and drug response, we do not anticipate that the 
results will directly influence therapeutic decisions made by the treating physician. 
However, the development of comprehensive genomic pipelines and integration of this 
data into a clinical trial setting will be required before personalized medicine can be 
realized. 
 
 
 


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5.2 
Study Procedures 
 
5.2.1 Pre-study Procedures 
 
Pre-study procedures must take place no more than 14 days prior to the first dose 
of decitabine except where noted below. 
 
1. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments performed 
on bone marrow will include correlative studies (collected under HRPO# 
201011766), morphologic assessment, cytogenetics, and FISH.  
2. Bone marrow or peripheral blood for flow cytometry 
3. Skin biopsy sample (collected under HRPO# 201011766). There is no required 
time frame for this sample; it may have been collected months or even years 
prior to the first dose of decitabine.  
 
5.2.2 Baseline Evaluation 
 
Baseline evaluations must take place no more than 7 days prior to the first dose of 
decitabine.  
 
1. Medical history 
2. Physical exam including height, weight, and ECOG performance score. 
3. Record transfusion requirements for 8 weeks before first dose of study drug 
4. CBC with differential and platelets 
5. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
6. Urine βHCG pregnancy test if appropriate 
7. Clinical pathology should be carefully reviewed to rule-out acute 
promyelocytic leukemia (M3-AML), and discussion between the pathologist 
and treating physician should take place if warranted by morphology or 
immunophenotype. 
 
5.2.3 Day 1 of Each Cycle 
 
1. Physical exam, including weight  
2. CBC with differential and platelets 
3. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
 
5.2.4 Cycle 1 Days 1-10 
 
Decitabine administration, 20 mg/m2 per day given as a 1-hour continuous 
intravenous infusion.  The dose will be calculated based on actual patient weight at 


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the beginning of each cycle.  Patients may be premedicated with lorazepam, 
prochlorperazine, palonosetron, or ondansetron at the treating physician’s 
discretion. 
 
The clinical research coordinator should monitor for final FISH results to confirm 
that PML/RARA screening is negative. 
 
5.2.5 Cycle 1 Day 10 ± 1 
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH. 
 
5.2.6 Weekly 
 
CBC with differential and platelets. After the first cycle, this may be changed to 
every other week at the discretion of the treating physician. 
 
5.2.7 Every Other Week 
 
Serum chemistries, including bicarbonate, calcium, glucose, total protein, albumin, 
BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, ALT, alkaline 
phosphatase, LDH. After the first cycle, this may be changed to every month at the 
discretion of the treating physician. 
 
5.2.8 Cycles 1, 2, 4 and 6 Day 28 ± 4 days  
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include: correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH.  
 
Patients who achieve with clinical response (CRi or better) and are receiving cycles 
every 6 weeks, will undergo bone marrow biopsies on day 42 ± 4.  
 
5.2.9 End-of-Study Procedures  
 
1. Physical exam 
2. CBC with differential and platelets.  
3. Serum chemistries including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, and LDH.  
4. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments performed 
on bone marrow will include morphologic assessment, cytogenetics, and FISH.  


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5.2.10 Post-study Follow-up 
 
Patients are to be followed according to standard patient care procedures for 30 
days following the date of the last decitabine dose to monitor for toxicity, survival, 
and response. Thereafter, the investigator or designee will record information on 
subsequent therapy, disease progression, and/or death every 6 months for 2 years 
after last dose of study drug or until the patient is lost to follow-up or dies. 
 
5.3 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to the 
patient’s health and/or the patient no longer wishes to continue protocol therapy, the 
protocol therapy should be discontinued and the reason(s) for discontinuation documented 
in the case report forms. 
 
In the absence of treatment delays due to adverse events, treatment may continue 
indefinitely or until one of the following criteria applies: 
 
 Documented and confirmed disease progression 
 Death 
 Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
 General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
 Suspected pregnancy 
 Serious non-compliance with the study protocol 
 Lost to follow-up 
 Patient withdraws consent 
 Investigator removes the patient from study 
 The Siteman Cancer Center decides to close the study 
 
Patients who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar. 
 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
 
5.4.1 Chemotherapy 
 
Patients cannot receive any chemotherapy within 21 days of study enrollment and 
must have recovered from any treatment-related toxicities. However, patients may 
be receiving hydrea at enrollment and may continue on hydrea through Cycle 2 of 
decitabine. An indication for hydrea following 2 cycles of decitabine is evidence 
that the patient is not responding and decitabine should be discontinued. Other than 
decitabine, patients cannot receive chemotherapy during this trial.  
 


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5.4.2 Growth Factors 
 
Erythroid growth factors should not be routinely administered to patients during the 
study. 
 
Thrombopoietin receptor agonists should not be routinely administered to patients 
during the study. 
 
Filgrastim (G-CSF) or sargramostim (GM-CSF) may be administered to patients 
with septic shock or febrile neutropenia at the discretion of the treating physician. 
 
5.4.3 Transfusions 
 
 Packed red blood cells (RBCs) transfusion:   
RBC transfusions will be administered at the discretion of the treating 
physician.  
 
 Platelet transfusion:   
Platelet transfusions will be administered at the discretion of the treating 
physician  
 
5.4.4 Prophylactic Antimicrobial Agents  
 
Antimicrobial agents will be administered at the discretion of the treating physician. 
Common antimicrobial prophylaxis during cycles 1 and 2 include acyclovir, 
ciprofloxacin, and fluconazole. 
 
5.4.5 Radiotherapy 
 
Patients cannot receive radiotherapy within 14 days prior to study enrollment or 
while on study. 
 


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6.0 
DOSE MODIFICATIONS 
 
A criterion for discontinuation of the study is a delay of > 8 weeks between any two cycles.  
 
6.1 
Delay for Cytopenia or Infection 
 
As cytopenias are a defining feature of AML and MDS, their presence alone does not 
generally warrant dose modifications or delays. Patients will receive four cycles of therapy 
without adjustment for any cytopenia. Following Cycle 4, dose modifications or delays for 
cytopenias will be made at the discretion of the investigator.  
 
Patients with febrile neutropenia (temperature ≥ 38.5° with ANC < 1,000/l) or systemic 
infections should have treatment held until clinical resolution of the infection.  
 
6.2 
Delay for Organ Dysfunction 
 
If renal dysfunction (creatinine ≥ 3 X institutional upper limit of normal) or hepatic 
dysfunction (bilirubin ≥ 2.5 mg/dl or ALT/AST ≥ 3 X institutional upper limit of normal) 
occurs, therapy may not resume until the abnormalities have resolved to below the limits 
stated above. If renal or hepatic dysfunction occurs during a treatment course, the next dose 
of decitabine should be held, and serum chemistries should be checked twice per week.  
Decitabine can be resumed at full dose when creatinine, bilirubin, ALT and AST are below 
the limits listed above.  
 
For grade 3-4 non-hematologic toxicities related to decitabine, decitabine treatment should 
be delayed until recovery to baseline or to grade ≤ 1. Decitabine may be reintroduced with 
a 50% dose reduction, or at full dose at the discretion of the investigator. 
 
6.3 
Modification in Dose or Schedule for Treatment Response 
 
Once AML patients achieve morphologic complete remission with incomplete blood count 
recovery (CRi) or MDS patients achieve a marrow complete response (Marrow CR) as 
defined in Section 9.0, decitabine administration may be decreased to 20 mg/m2/day for 
ten consecutive days, and the cycles may be administered every 6 weeks to limit 
myelosuppressive toxicity, at the discretion of the treating physician. After two cycles at 
six week intervals, the dose may be reduced to 20 mg/m2/day for five consecutive days of 
treatment in six week intervals. If the patient meets the definition of CR with complete 
cytogenetic response as defined in Section 9.0, administration may further be decreased to 
three  consecutive days per cycle at the discretion of the treating physician. 
 


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7.0 
PHARMACEUTICAL INFORMATION 
 
7.1 
Decitabine 
 
Decitabine is an antimetabolite that can replace cytosine in DNA but, unlike cytosine, it 
cannot be methylated. The exact mechanism of decitabine activity has not been determined 
and may involve multiple pathways. Decitabine is thought primarily to act by correcting 
DNA methylation, a major mechanism for gene expression. In some cancer cells, 
hypermethylation blocks the activity of tumor suppressor genes, which regulate cell 
division and differentiation to prevent malignant transformation. When suppressor gene 
activity is blocked, cell division becomes unregulated, leading to the formation of 
neoplastic cells. Decitabine regulates DNA methylation (specifically targets the 
methyltransferase, DNMT1), effectively demethylating and reactivating different tumor 
suppressor genes. Because hypermethylation occurs early in the malignant transformation 
of cells, decitabine may have activity in chemoprevention. Other possible mechanisms for 
decitabine activity include cytotoxicity due to incorporation into DNA similar to other 
antimetabolites, methylation-independent induction of gene expression, and degradation of 
DNA methyltransferase 1. 
 
7.1.1 Study Drug Preparation  
 
OSHA Guidelines for handling cytotoxic drugs outlined in the American Journal 
of Hospital Pharmacy must be followed. 
 
Decitabine (5-aza-2’-deoxycytidine) is supplied as a lyophilized powder for 
injection, 50 mg in 20-ml vials. When reconstituted with 10 ml of sterile water for 
injection, each ml will contain 5 mg of decitabine and 6.8 mg of KH2PO4. The 
reconstituted solution should be diluted to 0.1-1.0 mg/mL with pre-chilled 0.9% 
sodium chloride intravenous infusion, which has been cooled to 2-8oC. 
Reconstitution of the powder results in a rapidly decomposing solution. Unless used 
within 15 minutes of reconstitution, the diluted solution must be prepared using 
cold (2—8 degrees C) infusion fluids and stored at 2—8 degrees C (36—46 degrees 
F) for a maximum of 7 hours. 
Decitabine will be administered per dosing schedule every 28 ± 4 days.  
 
Decitabine may be administered either as an inpatient or an outpatient.  
 
7.1.2 Supplier 
 
Decitabine is available commercially. 
 
 
  
 
  
 


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8.0 
CORRELATIVE STUDIES SAMPLE COLLECTIONS 
 
Bone marrow samples will be collected pre-study, on Cycle 1 Day 10 ± 1, and on Day 28 ± 4 of 
Cycle 1, 2, 4, and 6 for correlative studies. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. The bone 
marrow samples will be collected under HRPO# 201011766 (“Tissue Acquisition for Analysis of 
Genetic Progression Factors in Hematologic Diseases”) that all patients will be concurrently 
enrolled in.  
 
Skin biopsy sample will be collected as part of the patient’s participation in HRPO# 201011766 
(“Tissue Acquisition for Analysis of Genetic Progression Factors in Hematologic Malignancies”). 
There is no required time frame for this sample, and it may have been collected months or even 
years prior to the first dose of decitabine.  
 
 
 


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9.0 
EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
 
A secondary endpoint of this study is to estimate morphologic complete remission rate. 
  
9.1 
Response Criteria - AML 
 
AML Patients will be assessed for response according to the IWG criteria:1  
 
Morphologic complete remission (CR) – Defined as morphologic leukemia-free state, 
defined as less than 5% blasts in aspirate with marrow spicules and a count of ≥200 
nucleated cells and no blasts with Auer rods or any persistence of extramedullary disease. 
When erythroid precursors constitute less than 50% of bone marrow nucleated cells, the 
percentage of blasts is based on all nucleated cells; when there are 50% or more erythroid 
cells, the percentage blasts should be based on the nonerythroid cells. The presence of mild 
megaloblastoid changes may be permitted if they are thought to be consistent with 
treatment effect. However, persistence of pretreatment abnormalities (eg, pseudo-Pelger-
Hüet cells, ringed sideroblasts, dysplastic megakaryocytes) are not consistent with CR.  In 
addition, ANC >1000/μl and platelet count of ≥100,000/μl. Patient must be independent of 
transfusions. No duration requirement for this designation. 
 
Cytogenetic complete remission (CCR) – Defined as morphologic complete remission plus 
a reversion to a normal karyotype. For this study, reversion of a normal karyotype is 
defined as no clonal abnormalities detected in a minimum of 20 mitotic cells. 
 
Morphologic complete remission with incomplete blood count recovery (CRi) – Defined as 
morphologic complete recovery with the exception of neutropenia <1000/μl or 
thrombocytopenia <100,000/μl.  
 
Partial remission (PR) – Defined as meeting the above criteria for complete remission, 
with the following exceptions: decrease of ≥50% in the percentage of blast to 5-25% in the 
bone marrow aspirate. A value of 5% blasts in bone marrow with Auer rods present may 
also be considered a partial remission.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, CRi, PR, PD, or 
recurrence/morphologic relapse.   
 
Progressive disease (PD) – Defined as a clear increase in bone marrow blast count, 
typically a greater than 50% increase compared with baseline, or new extramedullary 
disease. Patients with a > 50% increase in peripheral blast count with an increase of the 
total peripheral white blood cell count to > 10,000/l should undergo evaluation by bone 
marrow biopsy to access for progressive disease.  
 
Recurrence/morphologic relapse - Defined as relapse following complete remission is 
defined as reappearance of blasts in the blood or the finding of ≥5% blasts in the bone 
marrow, not attributable to any other cause. New dysplastic changes is considered relapse. 


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If there are no blasts in the peripheral blood and 5-20% blasts in the bone marrow, bone 
marrow biopsy should be repeated in > 1 week to confirm relapse. 
 
9.2 
Response Criteria - MDS 
 
MDS Patients will be assessed for response according to the IWG criteria:69 
 
Complete remission (CR) – Defined as < 5% myeloblasts with normal maturation of all 
cell lines in the bone marrow and peripheral blood values of Hgb > 11g/dL, Platelets > 100 
x 109/L, Neutrophils > 1.0 x 109/L, and 0% blasts. Persistent dysplasia does not exclude 
CR but will be noted.  
 
Marrow Complete Response (Marrow CR) – Defined as < 5% myeloblasts in the bone 
marrow and a decrease by > 50% from pretreatment values, but not meeting the definition 
of CR above.  
 
Partial Remission (PR) – Defined as meeting the definition of CR above with a decrease 
of myeloeblasts in the bone marrow by > 50% from pretreatment values, but absolute 
myeloblasts still >5%.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, Marrow CR, PR, SD, 
PD, or recurrence/morphologic relapse.   
 
Progressive Disease//Relapse (PD) – Defined as ≥ 50% increase in blasts to > 5% blasts 
(for patients with less than 5% blasts at baseline only), ≥ 50% increase to > 10% blasts (for 
patients with 5-10% blasts at baseline only), ≥ 50% increase to > 20% blasts (for patients 
with l0-20% blasts at baseline only), ≥ 50% increase to > 30% blasts (for patients with 20-
30% blasts at baseline only) or any of the following: At least 50% decrement from 
maximum remission/response in granulocytes or platelets, reduction in Hgb by ≥ 2 g/dL, 
or New or worsened transfusion dependence not related to study drug toxicity. Or for 
patients with a CR, Marrow CR, or PR as defined above and subsequently development of 
one of the following: Return to pretreatment bone marrow blast percentage, decrement of 
≥ 50% from maximum remission/response levels in granulocytes or platelets, or reduction 
in Hgb concentration by ≥ 1.5 g/dL or transfusion dependence. 
 
9.2.1 Hematologic Improvement (HI) 
 
 
 
Progressive disease as defined above nullifies hematologic improvement.  
 
Erythroid response requires all of the following (only required if pretreatment HgB 
<11 g/dL): 
 Hgb increase by ≥ 1.5 g/dL 
 Relevant reduction of units of RBC transfusions by an absolute number of at 
least 4 RBC transfusions/8 wk compared with the pretreatment transfusion 
number in the previous 8 wk. Only RBC transfusions given for a Hgb of ≤ 9.0 
g/dL pretreatment will count in the RBC transfusion response evaluation 


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Platelet response requires one of the following (only required if pretreatment 
platelets < 100 x 109/L):  
 Absolute increase of ≥ 30 x 109/L (for patients starting with > 20 x 109/L 
platelets) 
 Increase from < 20 x 109/L to > 20 x 109/L and absolute increase > 100% (for 
patients starting with < 20 x 109/L) 
 
Neutrophil response requires the following (only required if pretreatment ANC 
<1.0 x 109/L):  
 At least 100% increase and an absolute increase > 0.5 x 109/L 
 
9.3.1 Cytogenetic Response  
 
Complete Cytogenetic Response – Defined as reversion to a normal karyotype. For 
this study, reversion of a normal karyotype is defined as no clonal abnormalities 
detected in a minimum of 20 mitotic cells. Progressive disease as defined above 
nullifies cytogenetic response.  
 
9.3 
Guidelines for Evaluation of Disease 
 
Bone marrow biopsy should be performed on Day 28 ± 4 of cycles 1, 2, 4 and 6 for clinical 
disease assessment. Following Cycle 6, bone marrow biopsies should be performed at the 
discretion of the treating physician to assess for relapse or progression. 
 
9.4 
Other Secondary Efficacy Measures 
 
9.4.1 Treatment Failure, Overall Survival and Event-free Survival 
 
Treatment failure is defined as failure to achieve a complete or partial remission 
following 4 cycles of therapy.  
 
Overall survival is defined as the date of first dose of study drug to the date of death 
from any cause.  Patients still alive at the time of the final analysis will be right-
censored. 
 
Event-free survival is defined as the interval from the date of first dose of study 
drug to date of treatment failure, recurrence, death due to any cause, or loss to 
follow up. 
 
 


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9.4.2 Duration of Remission and Relapse-free Survival for Patients Having 
Complete Remission 
 
Duration of complete remission is defined as the interval from the date that 
complete remission is documented to the date of recurrence, death due to any cause, 
or loss to follow-up.  
 
Relapse-free survival is defined only for those patients achieving a complete 
remission. It is defined as the interval from the date of first documentation of a 
leukemia-free state to the date of recurrence, death due to any cause, or loss to 
follow up.   
  
9.4.3 Safety 
 
Safety evaluation will include assessments of adverse experiences, medical history, 
physical examinations, vital signs, and laboratory assessments at baseline and 
throughout the study period. Local laboratories may be used for this study.  
 


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10.0 
REGULATORY AND REPORTING REQUIREMENTS 
 
The entities providing oversight of safety and compliance with the protocol require reporting as 
outlined below. 
 
The Washington University Human Research Protection Office (HRPO) requires that all events 
meeting the definition of unanticipated problem or serious noncompliance be reported as outlined 
in Section 10.2. 
 
The FDA requires that all serious and unexpected adverse events be reported as outlined in Section 
10.6.  In addition, any fatal or life-threatening adverse experiences where there is a reasonable 
possibility of relationship to study intervention must be reported. 
 
10.1 
Definitions 
 
10.1.1 Adverse Events (AEs) 
 
Definition:  any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading:  the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for 
all toxicity reporting.  A copy of the CTCAE version 4.0 can be downloaded from 
the CTEP web site:  http://ctep.cancer.gov/reporting/ctc.html.  Grade 1-2 events 
will be recorded for the first fifty enrolled patients. Subsequently, only events 
graded as 3 or greater will be recorded. Fifty patients should be sufficient to 
estimate the true incidence of any adverse event to within ± 14% with 95% 
confidence intervals. 
 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the 
terms listed that should be used are those provided by the Department of Health 
and Human Services’ Office for Human Research Protections (OHRP).  A copy of 
this 
guidance 
can 
be 
found 
on 
OHRP’s 
website: 
(http://www.hhs.gov/ohrp/policy/AdvEvntGuid.htm). 
 
10.1.2 Serious Adverse Event (SAE) 
 
Definition:  any adverse drug experience occurring at any dose that results in any 
of the following outcomes: 
o Death 
o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial disruption 
of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 


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o Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed above 
 
All unexpected SAEs must be reported to the FDA. 
 
10.1.3 Unexpected Adverse Experience 
 
Definition: any adverse drug experience, the specificity or severity of which is not 
consistent with the current investigator brochure (or risk information, if an IB is not 
required or available). 
 
Events that are both serious AND unexpected must be reported to the FDA.  
 
10.1.4 Life-Threatening Adverse Experience  
 
Definition: any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that, had it occurred in a more severe form, might have caused 
death. 
 
Life-threatening adverse experiences must be reported to the FDA. 
 
10.1.5 Unanticipated Problems 
 
Definition: 
 
 unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the 
IRB-approved research protocol and informed consent document; and (b) 
the characteristics of the subject population being studied; 
 related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the 
incident, experience, or outcome may have been caused by the procedures 
involved in the research); and 
 suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was 
previously known or recognized. 
 
10.1.6 Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that 
govern human subjects research or failure to follow the determinations of the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate 
choice to ignore regulations, institutional policies, or determinations of the IRB. 
 


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10.1.7 Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial 
harm to subjects or others, or that materially compromises the rights or welfare of 
participants. 
 
10.1.8 Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under the 
research team’s control. Exceptions apply only to a single participant or a singular 
situation. 
 
Local IRB Pre-approval of all protocol exceptions must be obtained prior to the 
event.  For secondary sites, the Washington University PI will issue approval of the 
exception, but it must also be submitted to the local IRB with documentation of 
approval forwarded to Washington University.  Washington University IRB 
approval is not required for protocol exceptions occurring at secondary sites. 
 
10.2 
Reporting to the Human Research Protection Office (HRPO) at Washington 
University: 
 
The PI is required to promptly notify the IRB of the following events: 
 
 Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct of 
the study. 
 Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 
 Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence of the 
event or notification to the PI of the event.  The death of a research participant that qualifies 
as a reportable event should be reported within 1 working day of the occurrence of the 
event or notification to the PI of the event. 
 
10.3 
Reporting to the Quality Assurance and Safety Monitoring Committee 
(QASMC) at Washington University 
 
The PI is required to notify the QASMC of any unanticipated problem occurring at WU or 
any BJH or SLCH institution that has been reported to and acknowledged by HRPO as 
reportable.  (Unanticipated problems reported to HRPO and withdrawn during the review 
process need not be reported to QASMC.) 
 
QASMC must be notified within 10 days of receipt of IRB acknowledgment via email to 
a QASMC auditor. 


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10.4 
Reporting Requirements for Secondary Sites 
 
The research team at each secondary site is required to promptly notify the Washington 
University PI and research coordinator of all reportable events (as described in Section 7.6) 
within 1 working day of the occurrence of the event or notification of the secondary site’s 
PI of the event.  This notification may take place via email if there is not yet enough 
information for a formal written report (using either an FDA MedWatch form if required 
or an institutional SAE reporting form if not).  A formal written report must be sent to the 
Washington University PI and research coordinator within 10 working days of the 
occurrence of the event or notification of the secondary site’s PI of the event.  The death 
of a research participant that qualifies as a reportable event should be reported within 1 
working day of the occurrence of the event or notification of the secondary site’s PI of the 
event. 
 
The research team at a secondary site is responsible for following its site’s guidelines for 
reporting applicable events to its site’s IRB according to its own institutional guidelines.  
The research team at Washington University is responsible for reporting all applicable 
events to the FDA.  
 
10.5 
Reporting to Secondary Sites 
 
The Washington University PI (or designee) will notify the research team at each secondary 
site of all reportable events that have occurred at other sites within 10 working days of the 
occurrence of the event or notification of the PI of the event.  This includes events that take 
place both at Washington University and at other secondary sites, if applicable. 
 
10.6 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER 
FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the Washington University principal investigator to report any 
unanticipated problem to the FDA as follows: 
 
 Report any unexpected fatal or life-threatening adverse experiences (Section 
10.1.4) associated with use of the drug by telephone or fax no later than 7 calendar 
days after initial receipt of the information.   
 Report any serious, unexpected adverse experiences (Section 10.1.2), as well as 
results from animal studies that suggest significant clinical risk within 15 calendar 
days after initial receipt of this information.   
 
All MedWatch forms will be sent by the investigator or investigator’s team to the FDA at 
the following address or by fax: 
 
Food and Drug Administration 


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Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
Secondary sites must submit a completed MedWatch form to the Washington University 
PI and research coordinator within 4 calendar days (for fatal or life-threatening adverse 
experiences) or 11 calendar days (for serious, unexpected adverse experiences).  The 
Washington University PI will be responsible for submitting all MedWatch forms from 
secondary sites to the FDA within the timeframes specified above. 
 
10.7 
Timeframe for Tracking Reportable Events:  
 
Adverse events will be tracked for 30 days following the last day of study treatment, or 
until the start of a new chemotherapy/treatment, whichever comes first. 


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11.0 
DATA SAFETY MONITORING PLAN 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, the 
Data and Safety Monitoring Committee (DSMC) will meet to review toxicity data at least every 6 
months following the activation of the first secondary site.  The report will be prepared by the 
statistician and will be submitted to the Quality Assurance and Safety Monitoring Committee 
(QASMC).  This report will include: 
 
 HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
 Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of study 
 History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol exceptions, 
error, or breach of confidentiality including start/stop dates and reason 
 Study-wide target accrual and study-wide actual accrual including numbers from 
participating sites 
 Protocol activation date at each participating site 
 Average rate of accrual observed in year 1, year 2, and subsequent years at each 
participating site 
 Expected accrual end date and accrual by site 
 Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
 Measures of efficacy 
 Early stopping rules with supporting data and list the number of participants who have 
met the early stopping rules 
 Summary of toxicities at all participating sites 
 Abstract submissions/publications 
 Summary of any recent literature that may affect the safety or ethics of the study  
 
Until such a time as the first secondary site activates this protocol, a semi-annual DSM report to 
be prepared by the study team will be submitted to the QASM Committee beginning 6 months 
after study activation at Washington University. 
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC according 
to institutional guidelines. 
 
A DSMC will consist of no fewer than 3 members including 2 clinical investigators and a 
biostatistician.  Like investigators, DSMC members are subject to the Washington University 
School of Medicine policies regarding standards of conduct. Individuals invited to serve on the 
DSMC will disclose any potential conflicts of interest to the trial principal investigator and/or 
appropriate university officials, in accordance with institution policies. Potential conflicts that 
develop during a trial or a member’s tenure on a DSMC must also be disclosed.  


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Refer to the Washington University Quality Assurance and Data Safety Monitoring Committee 
Policies and Procedures for full details on the responsibilities of the DSMC at 
http://www.siteman.wustl.edu/uploadedFiles/Research_Programs/Clinical_Research_Resources/
Protocol_Review_and_Monitoring_Committee/QASMCQualityAssurcance.pdf  
 
 


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12.0 
AUDITING 
 
Since Washington University is the coordinating center, each site will be audited annually by 
Siteman Cancer Center personnel (QASMC) unless the outside institution has an auditing 
mechanism in place and can provide a report. The outside sites will be asked to send copies of all 
audit materials, including source documentation.  The audit notification will be sent to the 
Washington University Research Patient Coordinator, who will obtain the audit materials from the 
participating institution. 
 
Notification of an upcoming audit will be sent to the research team one month ahead of the audit. 
Once accrual numbers are confirmed, and approximately 30 days prior to the audit, a list of the 
cases selected for review (up to 10 for each site) will be sent to the research team. However, if 
during the audit the need arises to review cases not initially selected, the research team will be 
asked to provide the additional charts within two working days. 
 
Additional details regarding the Auditing Policies and Procedures can be found at 
https://siteman.wustl.edu/wp-content/uploads/2015/QASMC-Policies-and-Procedures-
03.31.2015.pdf  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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13.0 
MULTICENTER REGULATORY REQUIREMENTS 
 
Washington University requires that each participating site sends its informed consent document 
to be reviewed and approved by the Washington University Regulatory Coordinator (or designee) 
prior to IRB/IEC submission.    
 
Site activation is defined as when the secondary site has received official written documentation 
from the coordinating center that the site has been approved to begin enrollment.  At a minimum, 
each participating institution must have the following documents on file at Washington University 
prior to study activation: 
 
 Documentation of IRB approval of the study in the form of a letter or other official 
document from the participating institution’s IRB.  This documentation must show 
which version of the protocol was approved by the IRB. 
 Documentation of IRB approval of an informed consent form. The consent must 
include a statement that data will be shared with Washington University, including the 
Quality Assurance and Safety Monitoring Committee (QASMC), the DSMC (if 
applicable), and the Washington University study team. 
 Documentation of FWA, signed FDA Form 1572 (if applicable), and the CVs of all 
participating investigators. 
 Protocol signature page signed and dated by the investigator at each participating site. 
 
The coordinating center Principal Investigator (or designee) is responsible for disseminating to the 
participating sites all study updates, amendments, reportable adverse events, etc.  Protocol/consent 
modifications and IB updates will be forwarded electronically to the secondary sites within 4 
weeks of obtaining Washington University IRB approval.  Activated secondary sites are expected 
to submit protocol/consent/IB modifications to their local IRBs within 4 weeks of receipt unless 
otherwise noted.  Upon the secondary sites obtaining local IRB approval, documentation of such 
shall be sent to the Washington University study team within 2 weeks of receipt of approval. 
 
Documentation of participating sites’ IRB approval of annual continuing reviews, protocol 
amendments or revisions, all SAE reports, and all protocol violations/deviations/exceptions must 
be kept on file at Washington University. 
 
The investigator or a designee from each institution must participate in a regular conference call 
to update and inform regarding the progress of the trial. 
 
 


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14.0 
STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
 
14.1 
Analysis Populations 
 
Intent-to-Treat - The intent-to-treat (ITT) population will be defined as all patients who 
receive at least one dose of decitabine.  
 
Efficacy-Evaluable - The efficacy-evaluable (EE) population will be defined as all patients 
who receive at least one cycle of treatment.   
 
Safety - The safety population will be defined as all patients with at least one dose of 
decitabine. 
 
14.2 
Statistical Analysis 
 
14.2.1 Descriptive Analyses 
 
Patient Disposition: The number of patients discontinued, the reasons for 
discontinuation, and the number of cycles administered will be summarized. 
 
Demographics and Baseline Characteristics: Descriptive summary statistics will 
be provided for demographic and important baseline characteristics.  For 
continuous variables, the number of patients, mean, standard deviation, median, 
minimum and maximum will be provided.  For categorical variables, the number 
and percent of patients in each demographic/characteristic category will be 
summarized. 
 
14.2.2 Primary Endpoint Analysis 
 
The primary end-point of this phase II study is the correlation biomarkers with 
overall response in AML/MDS patients to decitabine. 
 
We will correlate the presence of patient specific mutations (especially mutations 
in DNMT3A, IDH1, IDH2, and TET2) with overall response rates. Mutation rates 
in these genes and in other patient-specific genes will be correlated with overall 
response and their respective 95% confidence intervals will be provided. 
 
Statistical considerations. The relationship between AML/MDS-associated 
mutations and the responsiveness to decitabine will be described by contingency 
tables and assessed using Chi-square test or Fisher’s exact test as appropriate. The 
differentially expressed genes between responders and non-responders will be 
determined using the significance analysis of microarray (SAM). Multivariate 
logistic regression will also be fit to adjust the potential confounding effects of other 
demographic and clinical characteristics. The resultant critical P values will be 
corrected for multiple testing.  Since the Bonferroni correction is known to be too 
conservative, a permutation analysis (e.g., 10,000 random assignments for 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 5 Version date: 04/21/16  
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responsive status) will be used to derive an empirical critical P value.  The new 
critical p-value is the number of permutation tests which yield a lower p-value than 
the original test divided by the total number of permutation tests.[61]   
 
A power analysis was performed using Chi-square test for the association between 
mutation and response to decitabine. The frequency of DNMT3A, IDH1/2, and 
TET2 mutations in AML and MDS patients are each between 15% and 30% in 
multiple cohorts.[29-37] Table 3 shows the minimal detectable differences of 
response rates between mutant and wild-type cases, assuming an expected overall 
45% CR/CRi rate and 80% power. For an average of 20% frequency of a mutated 
gene, for example, 100 patients will provide 80% power to detect 30% difference 
at a 2-sided 0.1 alpha error. The purpose of this study is to determine whether any 
of these mutations result in a 
clinically 
significant 
association 
that 
would 
warrant pre-treatment testing. 
We believe that a reasonable 
cut off would be if at least 
30% of clinical response 
could be attributed to the 
presence 
of 
a 
specific 
mutation. 
However, 
we 
anticipate that more power 
could be achieved as we 
assess patient groups with 
combined characteristics (e.g. 
DNMT3A mutation or loss of 
DNMT3A 
expression). 
In 
addition, we expect that the 
majority of the enrolled 125 
patients can be used in the analysis (since all of our molecular information will be 
acquired within 30 days of enrollment, we anticipate a drop-out rate of 20% or less). 
 
14.2.3 Secondary Endpoint Analyses 
 
The overall response rate (CR/CRi/PR) and complete response rate (CR/CRi) will 
be compared with historical controls[2], and the 95% confidence intervals will be 
provided. 
 
The rate of patient specific mutation clearance after 10, 28, and 56 days of therapy 
will be determined using an Illumina based platform. We will compare the rate of 
mutation clearance between the patients who achieve a CR/CRi after 4 cycles and 
those who do not. We anticipate that we will identify between 6 and 25 exonic 
SNVs per case (the range obtained with >200 AML samples sequenced to date with 
whole genome or exome sequencing), and that the expected 95% confidence 
interval of the mean variant allele frequency will be ±2.56% and ±1.25%, 
Table 3. Detectable mutation associated response rates (80% power, α = 0.05) 
 
Total 
N 
% of patients 
with a given 
mutation  
Detectable 
response 
difference 
Sensitivity of mutation to 
predict response at minimum 
detectable response difference* 
100 
15%  
20%  
30% 
40% 
50% 
0.40 
0.35 
0.30 
0.27 
0.26 
80% 
75% 
67% 
61% 
58% 
* Assuming the overall response  rate (CR/Cri) of 45%   


Genomic Predictors of Decitabine Response in AML/MDS 
 
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respectively, if we assume an anticipated standard deviation of 3.2% within the 
same clone. The change of tumor burden over time and its association with clinical 
response will  be assessed using 2-way ANOVA for repeated measurement data, 
followed by the ad-hoc multiple comparisons for between-group differences (e.g., 
responders vs. non-responders) at given time points as well as the between-time 
differences within each group. The resultant critical P values will also be corrected 
using permutation analysis for multiple testing. 
 
The percent decrease in bone marrow methylcytosine will be calculated on day 
10±1. We will compare this reduction with previously published data and between 
the patients who achieve CR/CRi and those who do not. The correlation of peak 
plasma levels of decitabine with response will be assessed using 2-sample t-test or 
Mann-Whitney non-parametric rank-sum test as appropriate. The change of total 
bone marrow DNA methylcytosine from baseline and its association with both 
steady-state plasma drug levels and response will be assessed using 2-way ANOVA 
for repeated measurement data, followed by the ad-hoc multiple comparisons for 
between-group differences (e.g., responders vs. non-responders) and between-time 
differences. Similar analyses will be performed for the correlation of expression 
profiles and SNP analysis with pharmacokinetic data.   
 
Statistical power justification.  A power analysis was performed using two-sample 
t-test for between-group difference and paired t-test for the change over time. For 
tumor clearance, this will provide > 99% power to detect a 10% difference in the 
means at two separate time points for clones with 6 or more variants, and > 98% 
power to detect a 15% difference if there are 3 or more variants per clone with α = 
0.05 (power will decrease to 91% and 82%, respectively, in cases where the 
standard deviation is 4.7%, the maximum we have observed thus far). For 
pharmacologic correlations, we anticipate 85% power to determine a 25 ng/ml 
difference between the average Cmax of 45 evaluable responders and 55 evaluable 
non-responders with an anticipated average Cmax of 70 ng/ml and a standard 
deviation of 40 ng/ml within the entire cohort, and with α = 0.05.[22] We believe a 
less significant difference would not be sufficient to warrant application in clinical 
decision making (i.e. whether to continue therapy for more than 1 cycle). We 
anticipate 99% power to determine a difference of 20% in the reduction of bone 
marrow methylcytosine between 45 evaluable responders and 55 evaluable non-
responders with an expected standard deviation of 15% and α = 0.05.[28, 66-68] 
The difference in methylcytosine reduction previously observed was 30% between 
patients achieving CR/PR and those with only hematologic improvement.[28]  
 
Time to relapse and time to death will also be listed for each patient. 
 
14.2.4 Safety Analysis 
 
Extent of Exposure – Duration (number of cycles/infusions) of decitabine doses 
(cumulative and intensity) will be summarized. 
 


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Amendment 5 Version date: 04/21/16  
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Adverse Events - All adverse events will be coded using NCI CTCAE v. 4.0.  The 
incidence of treatment-emergent adverse events (number and percent of patients 
reporting the adverse event at least once during the study) will be summarized.  In 
addition, adverse events will be summarized by investigator attribution of 
relationship to study medication and by grade.  Similar summaries will be provided 
by treatment cycle. 
 
Clinical Laboratory Evaluation – Applicable laboratory parameters will be graded 
according to NCI CTCAE version 4.0. The incidence of maximum grade (number 
and percent of patients experiencing the maximum grade during the study) will be 
summarized for each laboratory parameter.  Similar summaries will be provided by 
treatment cycle.  Graphical displays of select hematological parameters will also be 
provided. 
 
Vital Signs, Physical Findings, and Other Safety Observations – Vital signs, 
findings from physical examinations, ECOG scores and weights will be presented 
in listing format. 
 
14.2.5 Description of Planned Subgroup Analyses 
 
The following subgroups (if sufficient numbers of patients) will be explored with 
respect to the primary and overall survival endpoints. 
 Age (60-69 years old vs ≥ 70 years old) 
 Cytogenetics 
 Patients with AML-associated mutations in DNMT3A, TET2, IDH1, or 
IDH2 vs those who do not have somatic variants in these genes. 
 
14.3 
Sample Size 
 
We anticipate initially enrolling 25 patients at Washington University. Once we establish 
a routine exome analysis with validation and calculation of the rate of patient-specific 
mutation clearance, we will enroll an additional 100 patients from at least 2 additional 
centers.  
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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15.0 
DATA SUBMISSION SCHEDULE 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to registration 
Patient Registration Form 
Disease Diagnosis Form 
Inclusion Criteria Worksheet 
Exclusion Criteria Worksheet 
 
Prior to starting treatment 
Treatment Record Form 
To be maintained throughout a patient’s treatment 
cycle.  The complete record is due at completion of 
treatment. 
Bone Marrow Sample Collection 
Form 
To be maintained throughout the course of the study 
at designated bone marrow sample collections time 
points (See Appendix 2). 
Pathology: Bone Marrow Biopsy 
To be completed upon the return of pathology 
report for each bone marrow biopsy. 
Study Calendar Form 
To be completed with the date of each time point 
upon occurrence. 
Labs Form 
To be completed upon the return of laboratory 
reports for each study time point (See Appendix 2) 
Dose Modifications 
To be maintained throughout the course of the study 
if dose modifications are performed. 
Therapy Response Form 
To be filled out upon remission (prior to, and/or on 
therapy) 
End of Study Form 
 
Due at the completion of the EOS visit, at the time 
of death, and/or at the time that the patient is lost to 
follow-up 
Follow Up Form 
 30 days post date of last decitabine dose. 
 Every 6 months for 2 years after the last dose of 
study drug. 
Record of Adverse Events 
At the time of any AE 
 
Any queries generated by Washington University must be responded to within 28 days of receipt 
by the participating site.  The Washington University research team will conduct a regular review 
of data status at all secondary sites, with appropriate corrective action to be requested as needed. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 5 Version date: 04/21/16  
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Appendix 1 
ECOG Performance Scale 
Activity Status 
Description 
0 
Asymptomatic, fully active, and able to carry on all predisease 
performance without restrictions. 
 
1 
Symptomatic, fully ambulatory but restricted in physically strenuous 
activity and able to carry out performance of a light or sedentary 
nature, eg, light housework, office work. 
 
2 
Symptomatic, ambulatory and capable of all self-care but unable to 
carry out any work activities.  Up and about more than 50% of 
waking hours: in bed less than 50% of day. 
 
3 
Symptomatic, capable of only limited self-care, confined to bed or 
chair more than 50% of waking hours, but not bedridden. 
 
4 
Completely disabled.  Cannot carry on any self-care.   Totally 
bedridden. 
 
5 
Dead 
 
 


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Appendix 2 
Study Flow Chart 
Study Flow Chart 
Test 
Pre-
study1 
Base-
line2 
All 
Cycles 
Day 1 
All Cycles 
Days 1-103 
Cycle 1 Day 
4 ± 1 
 
Cycle 1 
Day 10 ± 1 
and 28 ± 4 
Every 
week 
Every 
2 
weeks 
Even 
cycles 
Day 28±4 
End of 
Study 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Medical History 
 
X 
 
 
 
 
 
 
 
 
Physical Exam 
 
Xa 
Xb 
 
 
 
 
 
 
X 
Transfusion 
Requirementsc 
 
Xc 
Ongoing 
Bone Marrow 
Aspirate and Biopsy 
Xd 
 
 
 
 
Xd 
 
 
Xd 
Xd 
Flow Cytometrye 
X 
 
 
 
 
 
 
 
 
 
Pregnancy test 
 
X 
 
 
 
 
 
 
 
 
CBC with differential 
and platelets 
 
X 
X 
 
 
 
Xf 
 
 
X 
Serum Chemistriesg 
 
X 
X 
 
 
 
 
Xf 
 
X 
Decitabine 
administrationh 
 
 
 
X 
 
 
 
 
 
 
Adverse Events  
 
 
Ongoing 
Post-study follow-up 
 
 
 
 
 
 
 
 
 
Xi 
1 To be performed within 14 days of the first dose of decitabine.  
2 To be performed within 7 days of the first dose of decitabine.  
3 Decitabine may be reduced to Days 1-5 or Days 1-3. See Section 6.3 for details.  
a To include height, weight, and ECOG. 
b To include weight. 
c Care should be given to record the frequency of transfusions from 8 weeks prior to first dose of study drug through 
the duration of the study. 
d Bone marrow biopsy and aspirate should be performed at baseline, at Cycle 1 Day 10 ± 1, and at  Day 28 ± 4 of Cycles 
1, 2, 4 and 6 for clinical disease assessment. Patients who achieve with clinical response (CRi or better) and are receiving 
cycles every 6 weeks, will undergo bone marrow biopsies on day 42 ± 4. Following Cycle 6, bone marrow biopsies 
should be performed at the discretion of the treating physician to assess for relapse or progression. Standard bone marrow 
procurement procedures will be followed for collection of the tissue. To include: correlative studies (collected under 
HRPO# 201011766), morphologic assessment, cytogenetics and FISH at all time points. A skin biopsy sample (collected 
under HRPO# 201011766) is required at pre-study, but may have been collected months or even years prior to first dose 
of decitabine.  
e On bone marrow or peripheral blood.  
f After Cycle 1, these labs may be obtained every other week (CBC) or once a month (Chemistries) at the discretion of 
the treating physician. 
g Serum chemistries include bicarbonate, calcium, glucose, total protein, albumin, BUN, potassium, chloride, sodium, 
creatinine, total bilirubin, AST, ALT, alkaline phosphatase, and LDH. 
h Decitabine treatment will be 20 mg/m2 over a 1-hour IV infusion on consecutive Days 1-10 every 28-day cycle.   The 
dose will be calculated based on actual patient weight at the beginning of each cycle. Patients may be premedicated 
with lorazepam, prochlorperazine, palonosetron, or ondansetron at the treating physician’s discretion. 
i Patients will be followed for  30 days following the date of the last decitabine dose to monitor for toxicity, survival 
and response. Thereafter, the investigator or designee will record information on subsequent therapy, disease 
progression, and death, every 6 months for 2 years after first dose of study drug or until the patient is lost to follow-up.


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Aparicio, A., et al., Phase I trial of continuous infusion 5-aza-2'-deoxycytidine. 
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63. 
Bryan, J., et al., Pharmacokinetic evaluation of decitabine for the treatment of 
leukemia. Expert Opin Drug Metab Toxicol, 2011. 7(5): p. 661-72. 
64. 
Scandura, J.M., et al., Phase 1 study of epigenetic priming with decitabine prior 
to standard induction chemotherapy for patients with AML. Blood, 2011. 118(6): 
p. 1472-80. 
65. 
Westfall, P.H. and S.A. Young, Resampling-based multiple testing: examples and 
methods for p-value adjustment. 1993, New York: John Wiley & Sons, Inc. 
66. 
Samlowski, W.E., et al., Evaluation of a 7-day continuous intravenous infusion of 
decitabine: inhibition of promoter-specific and global genomic DNA methylation. 
J Clin Oncol, 2005. 23(17): p. 3897-905. 
67. 
Blum, W., et al., Phase I study of decitabine alone or in combination with 
valproic acid in acute myeloid leukemia. J Clin Oncol, 2007. 25(25): p. 3884-91. 
68. 
Yang, A.S., et al., DNA methylation changes after 5-aza-2'-deoxycytidine therapy 
in patients with leukemia. Cancer Res, 2006. 66(10): p. 5495-503. 
69.       Cheson, B.D., et al., Clinical application and proposal for modification of the 
International Working Group (IWG) response criteria in myelodysplasia. Blood, 
2006. 108(2): p. 419-25. 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
 
NCT# NCT01687400 
 
 
Document Version Date:  04/16/2015 
 
IRB Approval Date:  05/05/2015 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 4 Version date: 04/16/15  
Page 1 of 51 
 
 
Genomic Predictors of Decitabine Response in AML/MDS 
 
Washington University School of Medicine 
Division of Oncology 
660 South Euclid Avenue, Box 8056 
St. Louis, MO 63110 
 
Protocol #: 201210102 
Protocol Version Date: 04/16/15 
 
Coordinating Center: Washington University School of Medicine 
 
Principal Investigator 
John Welch, M.D./Ph.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-2626, Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Sub-Investigator 
Timothy Ley, M.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-8832, Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Pathology 
Jeffery M. Klco, M.D., Ph.D. 
Department of Anatomic and Molecular Pathology 
Telephone: (314) 362-8832 
Email: klcoj@wustl.edu 
 
Statistician 
Feng Gao, Ph.D. 
Division of Biostatistics 
Telephone: (314) 362-3682 
E-mail: feng@wubios.wustl.edu 
 
IND# 116548 
Clinical Trials.gov # NCT01687400 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to obtain 
informed consent, may not be disclosed to another party unless law or regulations require such disclosure.  Persons 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 4 Version date: 04/16/15  
Page 2 of 51 
to whom the information is disclosed must be informed that the information is confidential and may not be further 
disclosed by them. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 4 Version date: 04/16/15  
Page 3 of 51 
Genomic Predictors of Decitabine Response in AML/MDS  
Principal Investigator Signature Page 
 
 
Principal Investigator: 
 
John Welch, M.D., Ph.D. 
 
 
 
 
 
Signature of Investigator 
 
Date 
 
 
 
Printed Name of Investigator 
 
 
 
By my signature, I agree to personally supervise the 
conduct of this study and to ensure its conduct in 
compliance with the protocol, informed consent, 
IRB/HRPO procedures, the Declaration of Helsinki, ICH 
Good Clinical Practices guidelines, and the applicable 
parts of the United States Code of Federal Regulations or 
local regulations governing the conduct of clinical 
studies. 
 
 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 4 Version date: 04/16/15  
Page 4 of 51 
 
Study Schema: 
 
 
Patients with progressive disease after two cycles (defined as a clear increase in bone marrow blast count, typically a 
> 50% increase in bone marrow blasts over baseline) should be removed from study. 
Patients with stable disease may continue on study and should undergo a diagnostic bone marrow biopsy at the end 
of even numbered cycles. 
Once AML patients achieve morphologic complete remission with incomplete blood count recovery (CRi) or MDS 
patients achieve a marrow complete response (Marrow CR) as defined in Section 9.0, decitabine 
administration may be decreased to 20 mg/m2/day for ten consecutive days every 6 weeks, to limit 
myelosuppressive toxicity and improve count recovery. After 2 cycles at 6 week intervals, the dose may be 
reduce to 20 mg/m2/day for five consecutive days in 6 week cycles, at the discretion of the treating physcian. 
After two cycles of five consecutive days of treatment, if the patient meets the definition of CR with complete 
cytogenetic response as defined in Section 9.0, administration may further be decreased to three consecutive 
days per cycle at the discretion of the treating physician. 
Sample collection for correlative studies: 
Bone marrow biopsies should be performed at baseline, at Cycle 1 Day 10 ± 1, and at Day 28 ± 4 of Cycles 
1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be performed at the discretion of the 
treating physician to assess for relapse or progression.  
Genomic Predictors of Decitabine Response in AML/MDS 
Table of Contents 
Section 
 
Page 
1.0 INTRODUCTION .................................................................................. 8 
1.1 
Rationale ............................................................................................ 8 
1.2 
Decitabine and Hypomethylating agents in AML/MDS ................. 10 
1.2.1 
Pharmacokinetics and Metabolism of Decitabine ................... 12 
1.2.2 
Experience with extended dosing schedules ........................... 13 
1.3 
Correlative Studies .......................................................................... 14 
20 
mg/m
2/day  
Cycle 1 
20 
mg/m
2/day 
Cycle 2+ 
Day 1 
Day 28 
BM Bx 
PB 


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2.0 OBJECTIVES ....................................................................................... 16 
2.1 
Primary Objective ............................................................................ 16 
2.2 
Secondary Objectives ...................................................................... 16 
3.0 PATIENT SELECTION ....................................................................... 17 
3.1 
Inclusion Criteria ............................................................................. 17 
3.2 
Exclusion Criteria ............................................................................ 17 
3.3 
Inclusion of Women and Minorities ................................................ 18 
4.0 REGISTRATION PROCEDURES ...................................................... 19 
4.1 
Confirmation of Patient Eligibility .................................................. 19 
4.2 
Patient Registration in the Siteman Cancer Center Database .......... 19 
4.3 
Assignment of UPN ......................................................................... 19 
5.0 INVESTIGATIONAL PLAN .............................................................. 21 
5.1 
Summary of Study Design............................................................... 21 
5.2 
Study Procedures ............................................................................. 22 
5.2.1 
Pre-study Procedures ............................................................... 22 
5.2.2 
Baseline Evaluation ................................................................. 22 
5.2.3 
Day 1 of Each Cycle ............................................................... 22 
5.2.4 
Cycle 1 Days 1-10 ................................................................... 22 
5.2.5 
Cycle 1 Day 10 ± 1 ................................................................. 23 
5.2.6 
Weekly .................................................................................... 23 
5.2.7 
Every Other Week ................................................................... 23 
5.2.8 
Cycles 1, 2, 4 and 6 Day 28 ± 4 days ...................................... 23 
5.2.9 
End-of-Study Procedures ........................................................ 23 
5.2.10 
Post-study Follow-up .............................................................. 23 
5.3 
Duration of Therapy ........................................................................ 24 
5.4 
Concomitant Therapy and Supportive Care Guidelines .................. 24 
5.4.1 
Chemotherapy ......................................................................... 24 
5.4.2 
Growth Factors ........................................................................ 24 
5.4.3 
Transfusions ............................................................................ 25 
5.4.4 
Prophylactic Antimicrobial Agents ......................................... 25 
5.4.5 
Radiotherapy ........................................................................... 25 
6.0 DOSE MODIFICATIONS ................................................................... 26 
6.1 
Delay for Cytopenia or Infection ..................................................... 26 
6.2 
Delay for Organ Dysfunction .......................................................... 26 
6.3 
Modification in Dose or Schedule for Treatment Response ........... 26 
7.0 PHARMACEUTICAL INFORMATION ............................................ 27 
7.1 
Decitabine ........................................................................................ 27 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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7.1.1 
Study Drug Preparation ........................................................... 27 
7.1.2 
Supplier ................................................................................... 27 
8.0 CORRELATIVE STUDIES SAMPLE COLLECTIONS .................... 28 
9.0 EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES29 
9.1 
Response Criteria - AML ................................................................ 29 
9.2 
Response Criteria - MDS................................................................. 30 
9.2.1 
Hematologic Improvement (HI) .............................................. 30 
9.3.1 Cytogenetic Response ....................................................................... 31 
9.3 
Guidelines for Evaluation of Disease .............................................. 31 
9.4 
Other Secondary Efficacy Measures ............................................... 31 
9.4.1 
Treatment Failure, Overall Survival and Event-free Survival 31 
9.4.2 
Duration of Remission and Relapse-free Survival for 
Patients Having Complete Remission ..................................... 31 
9.4.3 
Safety ...................................................................................... 32 
10.0 ADVERSE EVENT REPORTING ...................................................... 33 
10.1 Adverse Events (AEs) ..................................................................... 33 
10.2 Unanticipated Problems................................................................... 33 
10.3 Noncompliance ................................................................................ 33 
10.4 Serious Noncompliance ................................................................... 33 
10.5 Protocol Exceptions ......................................................................... 34 
10.6 Reporting to the Human Research Protection Office (HRPO) and 
the Quality Assurance and Safety Monitoring Committee 
(QASMC) at Washington University: ............................................. 34 
10.7 Reporting Requirements for Secondary Sites .................................. 34 
10.8 Reporting to Secondary Sites .......................................................... 35 
10.9 Reporting to the FDA ...................................................................... 35 
10.10 Timeframe for Reporting Required Events: .................................... 36 
11.0 DATA SAFETY MONITORING PLAN ............................................. 37 
12.0 AUDITING ........................................................................................... 38 
13.0 MULTICENTER REGULATORY REQUIREMENTS ...................... 39 
14.0 STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE ........ 40 
14.1 Analysis Populations ....................................................................... 40 
14.2 Statistical Analysis .......................................................................... 40 
14.2.1 
Descriptive Analyses ............................................................... 40 
14.2.2 
Primary Endpoint Analysis ..................................................... 40 
14.2.3 
Secondary Endpoint Analyses ................................................ 41 
14.2.4 
Safety Analysis ....................................................................... 42 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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14.2.5 
Description of Planned Subgroup Analyses ............................ 43 
14.3 Sample Size ..................................................................................... 43 
15.0 DATA SUBMISSION SCHEDULE .................................................... 44 
16.0 REFERENCES ..................................................................................... 47 
  


Genomic Predictors of Decitabine Response in AML/MDS 
 
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1.0 
INTRODUCTION 
 
1.1 
Rationale   
 
Personalized therapy for cancer requires that 1) patient-specific factors (e.g. patient-
specific mutations, expression profiles, or pharmacogenomics) influence treatment 
algorithms, and 2) once therapy is initiated, in vivo responsiveness is used to guide further 
therapy. The modern era of genomics has provided significant insights into cancer 
pathogenesis by describing patterns of germline and somatic variants, as well as defining 
disease-specific patterns of gene expression. However, the application and integration of 
this data with therapeutics remains a difficult task. 
 
Decitabine is a commonly used single-agent therapy for patients with acute myeloid 
leukemia (AML) and/or myelodysplastic syndrome (MDS). Historically, it has yielded 
clinical complete responses in approximately 25% of patients across diverse clinical trials, 
it is well tolerated, and it can be given as in the outpatient setting. However, the majority 
of patients do not respond to decitabine, the molecular etiology of decitabine sensitivity is 
unknown, and there are no algorithms to predict patient responsiveness a priori or even 
within one month of starting therapy.  
 
AML and MDS are two closely related hematological disorders characterized by 
ineffective hematopoiesis and malignant expansion of clonal myeloid cells.  Both AML 
and MDS frequently occur in elderly patients and some cases are secondary and/or related 
to long-term bone marrow damage after radiation or chemotherapy. Current therapy for 
AML consists of cytotoxic chemotherapy followed by consolidation chemotherapy or stem 
cell transplantation. Using these approaches, current cure rates may be as high as 60% for 
patients younger than 60 with good risk cytogenetics (e.g. t(15;17), t(8;21), inv(16)).[4]  
Outcomes in patients ≥ 60 years old and with relapsed disease remain poor with 2 year 
survival of only ~25%.[5] Historically, the available treatment for MDS has been 
symptomatic and has not been shown to be effective in producing sustained improvement 
in hematopoiesis or in delaying leukemic evolution[6, 7].  
 
Current AML therapy is poorly tolerated in elderly and pre-treated patients due to 
hematologic and non-hematologic toxicities; following such therapy, patients typically 
require 3-4 weeks of hospitalization for treatment of neutropenic fevers and for regular 
transfusions. Even among the relatively healthy patients enrolled in clinical trials, induction 
mortality in the elderly can be as high as 20-40% following standard induction therapy,[8-
11] and moderate to severe non-hematologic toxicity occurs in at least an additional 30% 
of patients.[9, 12-14] Consequently, many older AML patients are not offered traditional 
intensive chemotherapy, but receive supportive care alone.[15]  More effective and less 
toxic therapies are needed to provide durable remissions in a significant proportion of 
elderly AML in populations. 
 
Decitabine has been studied in MDS patients and in elderly and/or relapsed AML patients. 
It has been found to be reasonably well tolerated, can be given in the outpatient setting, 
and has overall response rates of 20-45%.[2, 3, 16-20] Our own experience has been of 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 4 Version date: 04/16/15  
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CR/PR rates of 25%, [2] and this is typical of many other groups for both MDS and AML 
patients (reviewed in [21]  Decitabine is an S-phase depended drug.  [22-24] However, 
decitabine is usually administered in one hour daily infusions during 3 to 5 consecutive 
days (too short to expose decitabine to most AML blasts during at least 2 cycles of 
replication, which divide every 2 to 7 days).[25-27]  Recent experience with increasing the 
number of infusions per cycle from 5 consecutive days to 10 consecutive days has 
improved overall response rates to 67%.[3] Response to decitabine can be slow, sometimes 
requiring 3 to 5 cycles before maximum response is observed.[2] As a consequence, many 
patients are exposed to prolonged therapy, gaining little benefit, before determining 
resistance. There are currently no biomarkers that predict response to decitabine. 
 
Decitabine is a cytosine analogue that cannot be methylated. Incorporation of decitabine 
into DNA prevents DNA methylation at cytosine residues and derepresses epigenetically 
silenced gene expression. Therefore, the reduction in total genomic DNA methylcytosine 
content acts as a marker of effective decitabine dosing. Indeed, two groups have recently 
correlated effective methylcytosine reduction and gene expression derepression to 
response (Figure 1.1).[3, 28] 
 
 
 
   
Figure 1.1. From Shen et al JCO (2010) 28:611 and Blum et al JCO (2007) 25: 3884. 
 
We and others have recently used whole genome sequencing to identify recurrent, somatic 
mutations in AML and MDS patients in four genes that are involved in methylcytosine 
regulation: DNMT3A, IDH1, IDH2, and TET2.[29-37] Collectively, nearly half of all 
AML/MDS patients have a mutation in one of these four genes.  DNMT3A mutations are 
associated with an adverse outcome in AML patients treated with cytotoxic 
chemotherapy.[35, 38] However, it is not yet clear whether the presence of mutations in 
one or more of these genes is associated with response or resistance to decitabine. 
 
In this study, we will treat patients with the current state-of-the-art decitabine schedule.[3] 
We will perform exome sequencing on all patients and correlate response with mutation 
status, rate of mutation clearance, expression profile, plasma decitabine levels, and DNA 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 4 Version date: 04/16/15  
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methylcytosine content reduction. This approach will comprehensively assess genomic and 
pharmacologic parameters that could clinically predict patient response to decitabine.  
 
In this study, we will combine analysis of patients with AML and MDS. Recent genomic 
analysis has shown that AML and MDS have shared mutations in many of the same genes, 
shared patterns of mutation acquisition, and shared patterns of mutation evolution.[39, 40] 
Furthermore, MDS, like AML, presents with clonal hematopoiesis, even when the 
morphologic blast count is zero.[36]Because this study will identify and track the rate of 
clearance of patient specific mutations, and because decitabine has been shown to have 
efficacy in both AML and MDS patients, [2, 3, 16-19, 41-45] combining analysis of AML 
and MDS patients is reasonable.  Asymptomatic MDS patients are excluded because these 
patients are typically followed with watchful-waiting rather than with chemotherapy. 
  
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
 
Methylation of cytosine in the CpG dinucleotide by DNA methyltransferase leads to 
transcriptional silencing of genes during normal development and has emerged as a 
significant mechanism for the loss of tumor suppressor gene expression in human cancer, 
including AML and MDS.[46, 47] Like azacitidine, decitabine (5-aza-2’-deoxycitidine) is 
incorporated into DNA during S phase and forms covalent adducts with DNA 
methyltransferase. The irreversible inhibition of this enzyme results in loss of methylation 
and is associated with differentiation.[48]  Decitabine is an approximately 5-fold more 
potent inhibitor of DNA methyltransferase than azacitidine, which might explain why 
decitabine can induce differentiation of myeloid progenitors in vitro at lower doses than 
azacitidine. In addition to their differentiation inducing activity at low doses, both agents 
have a direct cytotoxic effect at higher doses (1-2 µM).[49]  
 
Decitabine has been investigated in hematologic malignancies using a range of doses and 
schedules.  It is currently FDA approved for the treatment of adults with MDS at a dose of 
15 mg/m2 every 8 hours for 3 days, based on a Phase III study that demonstrated improved 
outcomes in the treatment arm compared to patients treated with supportive care alone.[15]  
However, the overall response rate in this trial was modest (17%). Of note, 18% of the 
patients in this trial had 20-30% bone marrow blasts, meeting the current WHO criteria for 
AML.  Subsequent phase I and II studies in AML, using a variety of low-dose decitabine 
regimens, have reported overall response rates of 22-49%.[2, 3, 16-19, 41-45] Comparisons 
of multiple low dose schedules have noted improved efficacy in the MDS population using 
a dose of 20 mg/m2 on Days 1-5 of a 28-day cycle.[50, 51] Because of these results, we 
have recently explored this dosing schedule in the treatment of elderly patients with 
AML.[52] In this study, we observed an overall response rate of 24%, and an additional 
44% of patients achieved stable disease during a median of 5 monthly cycles.[48] In this 
study, we noted that decitabine does not act quickly and the median time to response was 
3 months. These results are similar to the recently published experience in Germany 
treating 227 patients with 15 mg/m2 over 3 hours TID for 3 days every 6 weeks, which 
resulted in a CR/PR rate of 26%.[16]  
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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A recent study using a prolonged administration schedule (20 mg/m2 IV over 1 hour on 
consecutive days 1-10) showed improved response (64% CR/PR) and improved time to 
respond (50% of patients had bone marrow blasts < 5% after 1 cycle of therapy).[3]  These 
results are an improvement over previous experience by other groups using decitabine to 
treat elderly AML patients or patients with relapsed AML, and this experience is 
summarized below: 
Trials of decitabine in AML 
Status 
Other Therapy 
DAC Dose 
(mg x days) 
Evaluable 
CR 
de novo 
daunomycin1 
90 mg/m2 x 5 
6 
100% 
Salvage 
None4 
0.75-80 mg/kg 
16 
  
None5 
37-81 mg/kg 
6 
33% 
None7.  Ara-C 
Refractory 
Various 
4 
0 
AMSA7 
250-500 mg/m2 x 6 
10 
80% 
idarubicin or 
AMSA3 
250 mg/m2 x 6 
56 
39% 
Elderly 
None6 
270-360 mg/m2 x 3 
10 
30% 
None2 
45 mg/m2 x 3 
4 
50% 
None8 
20 mg/m2day 1-5,8-12 
50 
23% 
None9 
20 mg/m2 x 5 
55 
25% 
 
None10 
50 mg/m2 CIVI x 3 
29 
48% 
 
None11 
20 mg/m2 x 10 
53 
64% 
 
ATRA cycle 212 
15 mg/m2 TID x 3 
227 
26% 
1 Schwartsmann G.  Leukemia 1997; 11(Suppl 1):S28-31. 
2 Lubbert M. ASH 2002 Abstract #4601. 
3 Willemze R.  Leukemia.  1997; 11(Suppl 1):S24-27 
4 Rivard GE.  Leukemia Research 1981; 5:453-462. 
5 Momparler RL.  Pharmac Therapy 1985; 30:277-286.  
6 Petti MC.  Leukemia.  1993; 7(Suppl 1):36-41.  
7 Richel DJ.  Br. J. Cancer 1991; 64:144-148 
8 Issa, JP. Blood 2004. 103: 1635. 
9 Cashen AF. ASH Annual Meeting. 2008 abstract 560. 
10 Wijermans PW. Leukemia 1997. 11: Suppl 1, 19-23. 
11 Blum, W. PNAS 2010; 107:7473. 
12 Lubbert. Haematologica 2011. ePub 11/04/2011. 
 
In general, decitabine is well tolerated. Unlike conventional cytotoxic chemotherapy, 
decitabine can be administered in the outpatient setting. However, AML and MDS are 
aggressive hematologic malignancies. Cytopenias commonly result from AML/MDS 
inhibition of normal hematopoiesis, and often do not resolve until a complete response is 
achieved. Thus, neutropenic sepsis and thrombocytopenic bleeding remain significant 
problems regardless of therapy, especially during the first 4-8 weeks. In our experience 
treating elderly AML patients, neutropenia occurred in 24%, sepsis in 9%, and bacteremia 
in 7%.  Similar results have been observed in MDS patients.[20, 53, 54] As a result, death 
is not uncommon in the first 8 weeks of therapy, occurring in 15-17% of patients, and most 
commonly resulting from infection and cytopenias related to disease and therapy.[16, 52, 
55] While unsettling, these numbers are better than many historical patients treated with 
cytotoxic chemotherapy, and suggest a need for continued improvements in therapy.[9, 12]  


Genomic Predictors of Decitabine Response in AML/MDS 
 
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1.2.1 Pharmacokinetics and Metabolism of Decitabine 
 
In human subjects, after IV administration, decitabine displayed a distribution phase 
with a half-life of 7 minutes and a terminal phase elimination half-life of 10-35 
minutes as measured by bioassay.[56, 57]   The short plasma half-life is due to rapid 
inactivation of decitabine by deamination by liver cytidine deaminase.[23] Total 
body clearance (Clp) in humans was high with a mean value of 126 mL/min/kg.[58]  
 
Among five adult patients with advanced solid tumors administered seven courses 
of 100 mg/m2 of decitabine by 1-hour infusions,[56] maximum plasma 
concentration ranged from 1 to 4 µM. Disappearance from plasma was biphasic 
with a rapid initial decline to less than 0.1 µM during the first hour post infusion 
and a slower decline over the next two hours to 6 nM, the level of detectability. 
Plasma half-life (meanSE) was estimated to be approximately 71 minutes in the 
initial phase (t½α) and 355 minutes in the second phase (t½β). The area under the 
plasma concentration-time curve was 40888 ng.hour/mL for these patients, with a 
peak plasma concentration of 2.010.44 µM (0.440.1 µg/mL), while mean volume 
of distribution (Vdss) and CLp was 4.591.42 L/kg and 12621 mL/min/kg, 
respectively. The time course of DAC concentrations was similar in 1 of 2 patients 
administered 75 mg/m2, while DAC was usually undetectable in the plasma of 
patients administered 25 to 60 mg/m2. 
 
Using a longer infusion time (up to 40 hours), Rivard et al.[57] reported a plasma 
concentration of 0.1 to 0.4 g/mL. With infusion times of 40-60 hours, at an infusion 
rate of 1 mg/kg/h, plasma concentrations of 0.43-0.76 µg/mL were achieved.[59]  The 
steady-state plasma concentration at an infusion rate of 1 mg/kg/h was estimated to 
be 0.2-0.5 µg/mL. The half-life after discontinuing the infusion was 12-20 min.[57]  
 
Urinary excretion of unchanged decitabine was low, ranging from less than 0.01% to 
0.9% of the total dose, and there was no relationship between excretion and dose or 
plasma drug levels. High clearance values and a total urinary excretion of <1% of the 
administered dose suggest that decitabine is eliminated rapidly and largely by 
metabolic processes, including extrahepatic metabolism.[60] 
 
The steady state plasma concentrations (Css) ranged from 0.31 to 0.39 µg/mL (1.27 
to 1.60 µM) during the infusion of 100 mg/m² decitabine over a 6-hour period.[61] 
The range of concentrations during a 600 mg/m2 infusion over an 8-hour period was 
0.41-16 g/mL(1.68 to 4.76 µM).[60] These plasma concentrations are in the same 
range as the in vitro concentrations that activated the expression of tumor 
suppressor gene p16 in three human non-small-cell lung cancer (NSCLC) cell 
lines.[61]  
 
Steady state decitabine levels of 0.1 to 0.2 µM were achieved in solid tumor patients 
dosed with 30 mg/m²/day and 40 mg/m²/day by continuous intravenous infusion 
over 72 hours.[62]  


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 4 Version date: 04/16/15  
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Summary of published steady state serum levels are below and are in proportion to 
infusion rate (R2 = 0.98, p < 0.0001):[63] 
 
Rate 
Serum 
Concentration 
mg/m2/hr 
ng/ml 
nM 
5 
55 
241 
7.5 
80 
351 
15 
180 
789 
21 
182.4 
800 
22.5 
350 
1535 
42 
456 
2000 
84 
1140 
5000 
 
In our experience treating primary patient blasts in vitro, concentrations of 100 – 
500 nM are required maximize methylcytosine reduction without causing cellular 
toxicity. Because decitabine incorporation into DNA is S-phase dependent, 
exposure of cells during cell division is necessary for efficacy. 
  
 
 
 
1.2.2 Experience with extended dosing schedules 
 
Blum et al recently published their experience treating elderly AML patients with 
a 10-day course of decitabine 20 mg/m2/day as a 1-hour infusion.[3] This prolonged 
exposure course was designed to increase the number of AML blast divisions 
exposed to decitabine per cycle. Toxicities were very similar to a similar regiment 
given as a 3 and 5 day course,[2, 16] but the overall response (CR/PR) was much 
improved (64% vs 25%). In all three studies, the toxicities were primarily myeloid 
suppression and the resultant infectious and bleeding complications associated with 
leukemic and pharmacologic hematopoietic suppression. Furthermore, Blum et al 
noted that the percent methylcytosine reduction was improved (22% vs 11%) as 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 4 Version date: 04/16/15  
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was the time to blast clearance (50% of patients who ultimately achieved a CR had 
≤ 5% bone marrow blasts after 1 cycle vs 0% of patients after 2 cycles). Finally, 
like Shen et al, they observed a correlation between the extent of methylcytosine 
reduction and response.[28]  This study suggests that improvement in response 
rates and time to response can be achieved by prolonged administration of 
decitabine. Further, that DNA methylcytosine reduction can be achieved with 
prolonged exposure to decitabine and that this might be a patient-specific biological 
marker of adequate dosing. 
  
Scandura et al. recently published a study of infusional decitabine prior to 
cytarabine + daunorubicin induction therapy for AML.[64] They escalated the 
duration of infusional therapy and reached their planned goal of 20 mg/m2/day x 7 
days of continuous therapy given prior to cytarabine and daunorubicin. They noted 
similar toxicities (primarily hematopoietic and the associated neutropenic 
infections), but that these were not different from their experience giving cytarabine 
+ daunorubicin alone. 
 
1.3 
Correlative Studies 
 
All correlative studies will be collected under HRPO# 201011766 (“Tissue Acquisition for 
Analysis of Genetic Progression Factors in Hematologic Diseases”) that all patients will 
be concurrently enrolled in. The goals of the correlative studies include: 
 
1. To correlate patient-specific mutations with response to decitabine. 
We will perform exome sequencing of each patient’s pre-study bone marrow sample 
and skin sample. We will determine AML/MDS-associated variants found in the bone 
marrow, but not skin sample. We will correlate the presence of mutations with response 
(especially mutations in DNMT3A, IDH1, IDH2, and TET2). This study is powered to 
identify mutations with >80% sensitivity to predict decitabine response if used as a 
clinical assay. 
 
2. To correlate the velocity and depth of response with response to decitabine. 
We will assess tumor burden by allele frequency of patient-specific mutations in bone 
marrow samples collected pre study, Cycle 1 Day 10±1, Cycle 1 Day 28±4, and Cycle 
2 Day 28±4. We will assess mutations in at least 24 recurrently mutated genes 
(including, but not limited to: FLT3, DNMT3A, NPM1, TET2, WT1, IDH1, IDH2, 
RUNX1, NRAS, TP53, U2AF1, KRAS, PHF6, MSTP9, PTPN11, KIT, SMC3, PRUNE2, 
ETV6, CEBPA, SMC1A, ASXL1, STAG2, RAD21) as well as all patient-specific 
mutations discovered during exome sequencing. The leukemic allele frequency will be 
measured using the Illumnia MySeq or HiSeq platform using DNA capture by gene 
specific biotinylated-oligonucleotides. Patient bone marrow samples will be assessed 
pre-study, Cycle 1 Day 10 ± 1, Cycle 1 Day 28 ± 4, and Cycle 2 Day 28 ± 4 and 
compared with patient specific skin samples (normal control). The velocity of response 
will be calculated as the natural log rate of patient-specific mutation allele frequency 
clearance. Depth of response will be defined as the lowest patient-specific mutation 


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allele frequency in a bone marrow sample. The velocity and depth of tumor response 
will be correlated to: 
 Achievement of clinical CR, 
 Time to best response, 
 Mutational status, 
 Time to disease progression. 
Because all patient-specific exome-discovered mutations will be assessed a second 
time in the primary bone marrow sample as well as the  Cycle 1 Day 10 ± 1, Cycle 1 
Day 28 ± 4, and Cycle 2 Day 28 ± 4 bone marrow samples, this study will also serve 
to validate all exome-discovered mutations. This is important, because exome 
sequencing is associated with a 20-50% false-discovery rate depending on type I error 
tolerated (mutation-calling algorithms are typically set for greater sensitivity than 
specificity).  Thus, many mutations discovered during exome sequencing are incorrect.  
This second analysis of these mutations will server to exclude these false positive calls. 
 
3. To correlate the bone marrow expression profile from pre-study and Cycle 1 Day 10 ± 
1 with response to decitabine. 
We will correlate expression signatures, and changes in expression signatures, with: 
 Achievement of clinical CR, 
 Mutation status. 
 
 
4. To correlate the extent of bone marrow DNA hypomethylation on Cycle 1 Day 10 ± 1 
with response to decitabine. 
We will assess the percent reduction in bone marrow methylcytosine content using LC-
MS. Patients will be evaluated pre-study and Cycle 1 Day 10 ± 1. The change in DNA 
methylcytosine content will be correlated to: 
 Achievement of clinical CR, 
 Change in expression signature. 
This will determine whether inadequate blast exposure to decitabine correlates with 
drug resistance. 
 


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2.0 
OBJECTIVES 
 
2.1 
Primary Objective 
 
To determine whether patient-specific mutations correlate with clinical overall response to 
decitabine in patients with AML or MDS. 
 
2.2 
Secondary Objectives 
 
- 
To determine whether 10 consecutive days per cycle of decitabine improves outcomes 
compared with historical controls treated with 5 consecutive days per cycle of 
decitabine. 
 
- 
To determine whether other biomarkers correlate with clinical overall response to 
decitabine in patients with AML or MDS. These will include:  
 
1. The velocity and depth of patient-specific mutation clearance; 
2. The bone marrow expression profile and change in expression profile during 
decitabine treatment; 
3. The decrease in bone marrow methylcytosine content in response to decitabine.  
 
 


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3.0 
PATIENT SELECTION 
 
3.1 
Inclusion Criteria 
 
One of the following: 
 Patient must have non-M3 AML and be > 60 years of age OR 
 non-M3 AML with relapsed disease OR 
 Symptomatic MDS with one of the following: 
□ Symptomatic anemia with either hemoglobin < 10.0 g/dL or requiring RBC 
transfusion 
□ Thrombocytopenia with a history of two or more platelet counts < 50,000/mcL 
or a significant hemorrhage requiring platelet transfusions 
□ Neutropenia with two or more ANC < 1,000/mcL 
 
All of the following: 
1. Patient must have an ECOG performance status ≤ 2. 
2. Patient must have >10% disease burden measured by cytomorphology, flow 
cytometry, or cytogenetics. 
3. Patient must have peripheral white blood cell count < 50,000/mcl. 
4. Patient must have adequate organ function, defined as: 
a. 
Total bilirubin < 1.5 x ULN 
b. 
AST/ALT < 2.5 x ULN 
c. 
Serum creatinine < 2.0 x ULN  
5. Patient must have undergone ≤ 2 cycles of prior hypomethylating agent (decitabine 
or azacitidine). 
6. Patient must be enrolled in HRPO# 201011766 (“Tissue Acquisition for Analysis 
of Genetic Progression Factors in Hematologic Diseases”). 
7. Patient must be > 18 years of age. 
8. Patient must be able to understand and willing to sign an IRB-approved written 
informed consent document. 
 
3.2 
Exclusion Criteria 
 
1. Patient must not be pregnant or nursing. 
2. Patient must not have known CNS leukemia.  
3. Patient must not have a history of positive HIV serology.  
4. Patient must not have a history of positive Hepatitis C serology. 
5. Patient must not have undergone a prior allogeneic stem cell transplant. 
6. Patient must not have any uncontrolled intercurrent illness including, but not 
limited to, ongoing or active infection, ongoing or active GVHD, congestive heart 
failure of NYHA class 3 or 4, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situation that would limit compliance with study 
requirements. 
7. Patient must not have had radiation therapy within 14 days of enrollment. 
8. Patient must not have received any chemotherapy within 21 days of enrollment, 
and any acute treatment-related toxicities must have returned to baseline. Patients 


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may be receiving hydrea at time of enrollment.    
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible for this trial. 


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4.0 
REGISTRATION PROCEDURES 
 
Patients must not start any protocol intervention prior to registration through the Siteman 
Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility by Washington University 
2. Registration of patient in the Siteman Cancer Center database 
3. Assignment of unique patient number (UPN) 
 
Once the patient has been entered in the Siteman Cancer Center database, the WUSM coordinator 
will forward verification of enrollment and the UPN via email. 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility collecting the information listed below and scanning and 
emailing it to the research coordinator listed in the Siteman Cancer Center Clinical Trials 
Core Protocol Procedures for Secondary Sites packet at least one business day prior to 
registering patient: 
1. Your name and contact information (telephone number, fax number, and email 
address) 
2. Your site PI’s name, the registering MD’s name, and your institution name 
3. Patient’s race, sex, and DOB 
4. Three letters (or two letters and a dash) for the patient’s initials 
5. Currently approved protocol version date 
6. Copy of signed consent form (patient name may be blacked out) 
7. Planned date of enrollment 
8. Completed eligibility checklist, signed and dated by a member of the study team 
9. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center Database 
 
Registrations may be submitted Monday through Friday between 8am and 5pm CT. Urgent 
late afternoon or early morning enrollments should be planned in advance and coordinated 
with the Washington University research coordinator.  Registration will be confirmed by 
the research coordinator or his/her delegate by email within one business day. Verification 
of eligibility and registration should be kept in the patient chart. 
 
All patients at all sites must be registered through the Siteman Cancer Center database at 
Washington University. 
 
4.3 
Assignment of UPN 
 


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Each patient will be identified with a unique patient number (UPN) for this study.  Patients 
will also be identified by first, middle, and last initials.  If the patient has no middle initial, 
a dash will be used on the case report forms (CRFs).  All data will be recorded with this 
identification number on the appropriate CRFs. 
 


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5.0 
INVESTIGATIONAL PLAN 
 
5.1 
Summary of Study Design 
 
This is an open-label, single-arm, Phase II, biomarker study of patients with AML or MDS 
who are treated with 10 consecutive days of decitabine. FAB-M3 AML is excluded from 
this trial, because other well-tolerated options exist for the treatment of FAB-M3 acute 
promyelocytic leukemia. AML patients who are < 60 years old and have not received 
cytotoxic induction chemotherapy are also excluded, because cytotoxic therapy offers a 
curative possibility for these patients.  MDS patients without cytopenias are also excluded 
because these patients can be followed with watchful-waiting. 
 
All patients will undergo bone marrow biopsies pre-study, on Cycle 1 Day 10 ± 1, and on 
Day 28 ± 4 of Cycles 1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. 
Patients will provide a skin biopsy sample pre-study. See Section 8.0 for details on 
correlative studies.  
 
Patients will receive decitabine 20 mg/m2/day as a 1-hour infusion on consecutive Days 1-
10 in 28-day cycles.  The dose will be calculated based on actual patient weight at the 
beginning of each cycle. 
 
Once AML patients achieve morphologic complete remission with incomplete blood count 
recovery (CRi) or MDS patients achieve a marrow complete response (Marrow CR) as 
defined in Section 9.0, decitabine administration may be decreased to 20 mg/m2/day for 
ten consecutive days every 6 weeks, to limit myelosuppressive toxicity and improve count 
recovery. After 2 cycles at 6 week intervals, the dose may be reduce to 20 mg/m2/day for 
five consecutive days in 6 week cycles, at the discretion of the treating physician.  After 
two cycles of five consecutive days of treatment, if the patient meets the definition of CR 
with complete cytogenetic response as defined in Section 9.0, administration may further 
be decreased to three consecutive days per cycle at the discretion of the treating physician. 
 
It will be a logistical goal of this trial consistently to return preliminary exome results to 
the treating physician by the end of Cycle 1 and validated results by the end of Cycle 2. 
However, this logistical goal is not a primary or secondary aim of this study. Given the 
limited data regarding mutation presence and drug response, we do not anticipate that the 
results will directly influence therapeutic decisions made by the treating physician. 
However, the development of comprehensive genomic pipelines and integration of this 
data into a clinical trial setting will be required before personalized medicine can be 
realized. 
 
 
 


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5.2 
Study Procedures 
 
5.2.1 Pre-study Procedures 
 
Pre-study procedures must take place no more than 14 days prior to the first dose 
of decitabine except where noted below. 
 
1. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments performed 
on bone marrow will include correlative studies (collected under HRPO# 
201011766), morphologic assessment, cytogenetics, and FISH.  
2. Bone marrow or peripheral blood for flow cytometry 
3. Skin biopsy sample (collected under HRPO# 201011766). There is no required 
time frame for this sample; it may have been collected months or even years 
prior to the first dose of decitabine.  
 
5.2.2 Baseline Evaluation 
 
Baseline evaluations must take place no more than 7 days prior to the first dose of 
decitabine.  
 
1. Medical history 
2. Physical exam including height, weight, and ECOG performance score. 
3. Record transfusion requirements for 8 weeks before first dose of study drug 
4. CBC with differential and platelets 
5. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
6. Urine βHCG pregnancy test if appropriate 
 
5.2.3 Day 1 of Each Cycle 
 
1. Physical exam, including weight  
2. CBC with differential and platelets 
3. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
 
5.2.4 Cycle 1 Days 1-10 
 
Decitabine administration, 20 mg/m2 per day given as a 1-hour continuous 
intravenous infusion.  The dose will be calculated based on actual patient weight at 
the beginning of each cycle.  Patients may be premedicated with lorazepam, 
prochlorperazine, palonosetron, or ondansetron at the treating physician’s 
discretion. 
 


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. 
  
5.2.5 Cycle 1 Day 10 ± 1 
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH. 
 
5.2.6 Weekly 
 
CBC with differential and platelets. After the first cycle, this may be changed to 
every other week at the discretion of the treating physician. 
 
5.2.7 Every Other Week 
 
Serum chemistries, including bicarbonate, calcium, glucose, total protein, albumin, 
BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, ALT, alkaline 
phosphatase, LDH. After the first cycle, this may be changed to every month at the 
discretion of the treating physician. 
 
5.2.8 Cycles 1, 2, 4 and 6 Day 28 ± 4 days  
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include: correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH.  
 
Patients who achieve with clinical response (CRi or better) and are receiving cycles 
every 6 weeks, will undergo bone marrow biopsies on day 42 ± 4.  
 
5.2.9 End-of-Study Procedures  
 
1. Physical exam 
2. CBC with differential and platelets.  
3. Serum chemistries including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, and LDH.  
4. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments performed 
on bone marrow will include morphologic assessment, cytogenetics, and FISH.  
 
5.2.10 Post-study Follow-up 
 
Patients are to be followed according to standard patient care procedures for 30 
days following the date of the last decitabine dose to monitor for toxicity, survival, 


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and response. Thereafter, the investigator or designee will record information on 
subsequent therapy, disease progression, and/or death every 6 months for 2 years 
after last dose of study drug or until the patient is lost to follow-up or dies. 
 
5.3 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to the 
patient’s health and/or the patient no longer wishes to continue protocol therapy, the 
protocol therapy should be discontinued and the reason(s) for discontinuation documented 
in the case report forms. 
 
In the absence of treatment delays due to adverse events, treatment may continue 
indefinitely or until one of the following criteria applies: 
 
 Documented and confirmed disease progression 
 Death 
 Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
 General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
 Suspected pregnancy 
 Serious non-compliance with the study protocol 
 Lost to follow-up 
 Patient withdraws consent 
 Investigator removes the patient from study 
 The Siteman Cancer Center decides to close the study 
 
Patients who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar. 
 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
 
5.4.1 Chemotherapy 
 
Patients cannot receive any chemotherapy within 21 days of study enrollment and 
must have recovered from any treatment-related toxicities. However, patients may 
be receiving hydrea at enrollment and may continue on hydrea through Cycle 2 of 
decitabine. An indication for hydrea following 2 cycles of decitabine is evidence 
that the patient is not responding and decitabine should be discontinued. Other than 
decitabine, patients cannot receive chemotherapy during this trial.  
 
5.4.2 Growth Factors 
 
Erythroid growth factors should not be routinely administered to patients during the 
study. 
 


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Thrombopoietin receptor agonists should not be routinely administered to patients 
during the study. 
 
Filgrastim (G-CSF) or sargramostim (GM-CSF) may be administered to patients 
with septic shock or febrile neutropenia at the discretion of the treating physician. 
 
5.4.3 Transfusions 
 
 Packed red blood cells (RBCs) transfusion:   
RBC transfusions will be administered at the discretion of the treating 
physician.  
 
 Platelet transfusion:   
Platelet transfusions will be administered at the discretion of the treating 
physician  
5.4.4 Prophylactic Antimicrobial Agents  
 
Antimicrobial agents will be administered at the discretion of the treating physician. 
Common antimicrobial prophylaxis during cycles 1 and 2 include acyclovir, 
ciprofloxacin, and fluconazole. 
 
5.4.5 Radiotherapy 
 
Patients cannot receive radiotherapy within 14 days prior to study enrollment or 
while on study. 
 


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6.0 
DOSE MODIFICATIONS 
 
A criterion for discontinuation of the study is a delay of > 8 weeks between any two cycles.  
 
6.1 
Delay for Cytopenia or Infection 
 
As cytopenias are a defining feature of AML and MDS, their presence alone does not 
generally warrant dose modifications or delays. Patients will receive four cycles of therapy 
without adjustment for any cytopenia. Following Cycle 4, dose modifications or delays for 
cytopenias will be made at the discretion of the investigator.  
 
Patients with febrile neutropenia (temperature ≥ 38.5° with ANC < 1,000/l) or systemic 
infections should have treatment held until clinical resolution of the infection.  
 
6.2 
Delay for Organ Dysfunction 
 
If renal dysfunction (creatinine ≥ 3 X institutional upper limit of normal) or hepatic 
dysfunction (bilirubin ≥ 2.5 mg/dl or ALT/AST ≥ 3 X institutional upper limit of normal) 
occurs, therapy may not resume until the abnormalities have resolved to below the limits 
stated above. If renal or hepatic dysfunction occurs during a treatment course, the next dose 
of decitabine should be held, and serum chemistries should be checked twice per week.  
Decitabine can be resumed at full dose when creatinine, bilirubin, ALT and AST are below 
the limits listed above.  
 
For grade 3-4 non-hematologic toxicities related to decitabine, decitabine treatment should 
be delayed until recovery to baseline or to grade ≤ 1. Decitabine may be reintroduced with 
a 50% dose reduction, or at full dose at the discretion of the investigator. 
 
6.3 
Modification in Dose or Schedule for Treatment Response 
 
Once AML patients achieve morphologic complete remission with incomplete blood count 
recovery (CRi) or MDS patients achieve a marrow complete response (Marrow CR) as 
defined in Section 9.0, decitabine administration may be decreased to 20 mg/m2/day for 
ten consecutive days, and the cycles may be administered every 6 weeks to limit 
myelosuppressive toxicity, at the discretion of the treating physician. After two cycles at 
six week intervals, the dose may be reduced to 20 mg/m2/day for five consecutive days of 
treatment in six week intervals. If the patient meets the definition of CR with complete 
cytogenetic response as defined in Section 9.0, administration may further be decreased to 
three  consecutive days per cycle at the discretion of the treating physician. 
 


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7.0 
PHARMACEUTICAL INFORMATION 
 
7.1 
Decitabine 
 
Decitabine is an antimetabolite that can replace cytosine in DNA but, unlike cytosine, it 
cannot be methylated. The exact mechanism of decitabine activity has not been determined 
and may involve multiple pathways. Decitabine is thought primarily to act by correcting 
DNA methylation, a major mechanism for gene expression. In some cancer cells, 
hypermethylation blocks the activity of tumor suppressor genes, which regulate cell 
division and differentiation to prevent malignant transformation. When suppressor gene 
activity is blocked, cell division becomes unregulated, leading to the formation of 
neoplastic cells. Decitabine regulates DNA methylation (specifically targets the 
methyltransferase, DNMT1), effectively demethylating and reactivating different tumor 
suppressor genes. Because hypermethylation occurs early in the malignant transformation 
of cells, decitabine may have activity in chemoprevention. Other possible mechanisms for 
decitabine activity include cytotoxicity due to incorporation into DNA similar to other 
antimetabolites, methylation-independent induction of gene expression, and degradation of 
DNA methyltransferase 1. 
 
7.1.1 Study Drug Preparation  
 
OSHA Guidelines for handling cytotoxic drugs outlined in the American Journal 
of Hospital Pharmacy must be followed. 
 
Decitabine (5-aza-2’-deoxycytidine) is supplied as a lyophilized powder for 
injection, 50 mg in 20-ml vials. When reconstituted with 10 ml of sterile water for 
injection, each ml will contain 5 mg of decitabine and 6.8 mg of KH2PO4. The 
reconstituted solution should be diluted to 0.1-1.0 mg/mL with pre-chilled 0.9% 
sodium chloride intravenous infusion, which has been cooled to 2-8oC. 
Reconstitution of the powder results in a rapidly decomposing solution. Unless used 
within 15 minutes of reconstitution, the diluted solution must be prepared using 
cold (2—8 degrees C) infusion fluids and stored at 2—8 degrees C (36—46 degrees 
F) for a maximum of 7 hours. 
Decitabine will be administered per dosing schedule every 28 ± 4 days.  
 
Decitabine may be administered either as an inpatient or an outpatient.  
 
7.1.2 Supplier 
 
Decitabine is available commercially. 
 
 
  
 
  
 


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8.0 
CORRELATIVE STUDIES SAMPLE COLLECTIONS 
 
Bone marrow samples will be collected pre-study, on Cycle 1 Day 10 ± 1, and on Day 28 ± 4 of 
Cycle 1, 2, 4, and 6 for correlative studies. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. The bone 
marrow samples will be collected under HRPO# 201011766 (“Tissue Acquisition for Analysis of 
Genetic Progression Factors in Hematologic Diseases”) that all patients will be concurrently 
enrolled in.  
 
Skin biopsy sample will be collected as part of the patient’s participation in HRPO# 201011766 
(“Tissue Acquisition for Analysis of Genetic Progression Factors in Hematologic Malignancies”). 
There is no required time frame for this sample, and it may have been collected months or even 
years prior to the first dose of decitabine.  
 
 
 


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9.0 
EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
 
A secondary endpoint of this study is to estimate morphologic complete remission rate. 
  
9.1 
Response Criteria - AML 
 
AML Patients will be assessed for response according to the IWG criteria:1  
 
Morphologic complete remission (CR) – Defined as morphologic leukemia-free state, 
defined as less than 5% blasts in aspirate with marrow spicules and a count of ≥200 
nucleated cells and no blasts with Auer rods or any persistence of extramedullary disease. 
When erythroid precursors constitute less than 50% of bone marrow nucleated cells, the 
percentage of blasts is based on all nucleated cells; when there are 50% or more erythroid 
cells, the percentage blasts should be based on the nonerythroid cells. The presence of mild 
megaloblastoid changes may be permitted if they are thought to be consistent with 
treatment effect. However, persistence of pretreatment abnormalities (eg, pseudo-Pelger-
Hüet cells, ringed sideroblasts, dysplastic megakaryocytes) are not consistent with CR.  In 
addition, ANC >1000/μl and platelet count of ≥100,000/μl. Patient must be independent of 
transfusions. No duration requirement for this designation. 
 
Cytogenetic complete remission (CCR) – Defined as morphologic complete remission plus 
a reversion to a normal karyotype. For this study, reversion of a normal karyotype is 
defined as no clonal abnormalities detected in a minimum of 20 mitotic cells. 
 
Morphologic complete remission with incomplete blood count recovery (CRi) – Defined as 
morphologic complete recovery with the exception of neutropenia <1000/μl or 
thrombocytopenia <100,000/μl.  
 
Partial remission (PR) – Defined as meeting the above criteria for complete remission, 
with the following exceptions: decrease of ≥50% in the percentage of blast to 5-25% in the 
bone marrow aspirate. A value of 5% blasts in bone marrow with Auer rods present may 
also be considered a partial remission.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, CRi, PR, PD, or 
recurrence/morphologic relapse.   
 
Progressive disease (PD) – Defined as a clear increase in bone marrow blast count, 
typically a greater than 50% increase compared with baseline, or new extramedullary 
disease. Patients with a > 50% increase in peripheral blast count with an increase of the 
total peripheral white blood cell count to > 10,000/l should undergo evaluation by bone 
marrow biopsy to access for progressive disease.  
 
Recurrence/morphologic relapse - Defined as relapse following complete remission is 
defined as reappearance of blasts in the blood or the finding of ≥5% blasts in the bone 
marrow, not attributable to any other cause. New dysplastic changes is considered relapse. 


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If there are no blasts in the peripheral blood and 5-20% blasts in the bone marrow, bone 
marrow biopsy should be repeated in > 1 week to confirm relapse. 
 
9.2 
Response Criteria - MDS 
 
MDS Patients will be assessed for response according to the IWG criteria:69 
 
Complete remission (CR) – Defined as < 5% myeloblasts with normal maturation of all 
cell lines in the bone marrow and peripheral blood values of Hgb > 11g/dL, Platelets > 100 
x 109/L, Neutrophils > 1.0 x 109/L, and 0% blasts. Persistent dysplasia does not exclude 
CR but will be noted.  
 
Marrow Complete Response (Marrow CR) – Defined as < 5% myeloblasts in the bone 
marrow and a decrease by > 50% from pretreatment values, but not meeting the definition 
of CR above.  
 
Partial Remission (PR) – Defined as meeting the definition of CR above with a decrease 
of myeloeblasts in the bone marrow by > 50% from pretreatment values, but absolute 
myeloblasts still >5%.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, Marrow CR, PR, SD, 
PD, or recurrence/morphologic relapse.   
 
Progressive Disease//Relapse (PD) – Defined as ≥ 50% increase in blasts to > 5% blasts 
(for patients with less than 5% blasts at baseline only), ≥ 50% increase to > 10% blasts (for 
patients with 5-10% blasts at baseline only), ≥ 50% increase to > 20% blasts (for patients 
with l0-20% blasts at baseline only), ≥ 50% increase to > 30% blasts (for patients with 20-
30% blasts at baseline only) or any of the following: At least 50% decrement from 
maximum remission/response in granulocytes or platelets, reduction in Hgb by ≥ 2 g/dL, 
or New or worsened transfusion dependence not related to study drug toxicity. Or for 
patients with a CR, Marrow CR, or PR as defined above and subsequently development of 
one of the following: Return to pretreatment bone marrow blast percentage, decrement of 
≥ 50% from maximum remission/response levels in granulocytes or platelets, or reduction 
in Hgb concentration by ≥ 1.5 g/dL or transfusion dependence. 
 
9.2.1 Hematologic Improvement (HI) 
 
 
 
Progressive disease as defined above nullifies hematologic improvement.  
 
Erythroid response requires all of the following (only required if pretreatment HgB 
<11 g/dL): 
 Hgb increase by ≥ 1.5 g/dL 
 Relevant reduction of units of RBC transfusions by an absolute number of at 
least 4 RBC transfusions/8 wk compared with the pretreatment transfusion 
number in the previous 8 wk. Only RBC transfusions given for a Hgb of ≤ 9.0 
g/dL pretreatment will count in the RBC transfusion response evaluation 


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Platelet response requires one of the following (only required if pretreatment 
platelets < 100 x 109/L):  
 Absolute increase of ≥ 30 x 109/L (for patients starting with > 20 x 109/L 
platelets) 
 Increase from < 20 x 109/L to > 20 x 109/L and absolute increase > 100% (for 
patients starting with < 20 x 109/L) 
 
Neutrophil response requires the following (only required if pretreatment ANC 
<1.0 x 109/L):  
 At least 100% increase and an absolute increase > 0.5 x 109/L 
 
9.3.1 Cytogenetic Response  
 
Complete Cytogenetic Response – Defined as reversion to a normal karyotype. For 
this study, reversion of a normal karyotype is defined as no clonal abnormalities 
detected in a minimum of 20 mitotic cells. Progressive disease as defined above 
nullifies cytogenetic response.  
 
9.3 
Guidelines for Evaluation of Disease 
 
Bone marrow biopsy should be performed on Day 28 ± 4 of cycles 1, 2, 4 and 6 for clinical 
disease assessment. Following Cycle 6, bone marrow biopsies should be performed at the 
discretion of the treating physician to assess for relapse or progression. 
 
9.4 
Other Secondary Efficacy Measures 
 
9.4.1 Treatment Failure, Overall Survival and Event-free Survival 
 
Treatment failure is defined as failure to achieve a complete or partial remission 
following 4 cycles of therapy.  
 
Overall survival is defined as the date of first dose of study drug to the date of death 
from any cause.  Patients still alive at the time of the final analysis will be right-
censored. 
 
Event-free survival is defined as the interval from the date of first dose of study 
drug to date of treatment failure, recurrence, death due to any cause, or loss to 
follow up. 
 
9.4.2 Duration of Remission and Relapse-free Survival for Patients Having 
Complete Remission 
 
Duration of complete remission is defined as the interval from the date that 
complete remission is documented to the date of recurrence, death due to any cause, 


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or loss to follow-up.  
 
Relapse-free survival is defined only for those patients achieving a complete 
remission. It is defined as the interval from the date of first documentation of a 
leukemia-free state to the date of recurrence, death due to any cause, or loss to 
follow up.   
  
9.4.3 Safety 
 
Safety evaluation will include assessments of adverse experiences, medical history, 
physical examinations, vital signs, and laboratory assessments at baseline and 
throughout the study period. Local laboratories may be used for this study.  
 


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10.0 
ADVERSE EVENT REPORTING  
 
10.1 
Adverse Events (AEs) 
 
Definition:  any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading:  the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for all 
toxicity reporting.  A copy of the CTCAE version 4.0 can be downloaded from the CTEP 
web site:  http://ctep.cancer.gov/reporting/ctc.html.  Grade 1-2 events will be recorded for 
the first fifty enrolled patients. Subsequently, only events graded as 3 or greater will be 
recorded. Fifty patients should be sufficient to estimate the true incidence of any adverse 
event to within ± 14% with 95% confidence intervals. 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the terms 
listed that should be used are those provided by the Department of Health and Human 
Services’ Office for Human Research Protections (OHRP).  A copy of this guidance can 
be found on OHRP’s website: (http://www.hhs.gov/ohrp/policy/AdvEvntGuid.htm). 
 
10.2 
Unanticipated Problems 
 
Definition: 
 
 unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied; 
 related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the incident, 
experience, or outcome may have been caused by the procedures involved in the 
research); and 
 suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
 
10.3 
Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that govern 
human 
subjects 
research 
or 
failure 
to 
follow 
the 
determinations 
of 
the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate choice to 
ignore regulations, institutional policies, or determinations of the IRB. 
 
10.4 
Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial harm 
to subjects or others, or that materially compromises the rights or welfare of participants. 


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10.5 
Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under the research 
team’s control. Exceptions apply only to a single participant or a singular situation. 
 
Local IRB Pre-approval of all protocol exceptions must be obtained prior to the event.  For 
secondary sites, the Washington University PI will issue approval of the exception, but it 
must also be submitted to the local IRB with documentation of approval forwarded to 
Washington University.  HRPO approval is not required for protocol exceptions occurring 
at secondary sites. 
 
10.6 
Reporting to the Human Research Protection Office (HRPO) and the Quality 
Assurance and Safety Monitoring Committee (QASMC) at Washington 
University: 
 
The PI is required to promptly notify the IRB of the following events: 
 
 Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct of 
the study. 
 Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 
 Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence of the 
event or notification to the PI of the event.  The death of a research participant that qualifies 
as a reportable event should be reported within 1 working day of the occurrence of the 
event or notification to the PI of the event. 
 
10.7 
Reporting Requirements for Secondary Sites 
 
The research team at each secondary site is required to promptly notify the Washington 
University PI and research coordinator of all reportable events (as described in Section 7.6) 
within 1 working day of the occurrence of the event or notification of the secondary site’s 
PI of the event.  This notification may take place via email if there is not yet enough 
information for a formal written report (using either an FDA MedWatch form if required 
or an institutional SAE reporting form if not).  A formal written report must be sent to the 
Washington University PI and research coordinator within 10 working days of the 
occurrence of the event or notification of the secondary site’s PI of the event.  The death 
of a research participant that qualifies as a reportable event should be reported within 1 
working day of the occurrence of the event or notification of the secondary site’s PI of the 
event. 
 


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The research team at a secondary site is responsible for following its site’s guidelines for 
reporting applicable events to its site’s IRB according to its own institutional guidelines. 
 
10.8 
Reporting to Secondary Sites 
 
The Washington University PI (or designee) will notify the research team at each secondary 
site of all reportable events that have occurred at other sites (as described in Section 7.6) 
within 10 working days of the occurrence of the event or notification of the PI of the event.  
This includes events that take place both at Washington University and at other secondary 
sites, if applicable. 
 
10.9 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER 
FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the investigator to report any unanticipated problem to the FDA as 
follows: 
 
 Report any unexpected fatal or life-threatening adverse experiences associated with 
use of the drug (i.e., there is a reasonable possibility that the experience may have 
been caused by the drug) by telephone or fax no later than 7 calendar days after 
initial receipt of the information.  A life-threatening adverse experience is defined 
as any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that, had it occurred in a more severe form, might have caused 
death. 
 Report any serious, unexpected adverse experiences, as well as results from animal 
studies that suggest significant clinical risk within 15 calendar days after initial 
receipt of this information.  A serious adverse drug experience is defined as any 
adverse drug experience occurring at any dose that results in any of the following 
outcomes: 
o Death 
o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial disruption 
of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 
o Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed above 
An unexpected adverse drug experience is defined as any adverse drug experience, 
the specificity or severity of which is not consistent with the current investigator 
brochure (or risk information, if an IB is not required or available). 


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All MedWatch forms will be sent by the investigator or investigator’s team to the FDA at 
the following address or by fax: 
 
Food and Drug Administration 
Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
Secondary sites must submit a completed MedWatch form to the Washington University 
PI and research coordinator within 4 calendar days (for fatal or life-threatening adverse 
experiences) or 11 calendar days (for serious, unexpected adverse experiences).  The 
Washington University PI will be responsible for submitting all MedWatch forms from 
secondary sites to the FDA within the timeframes specified above. 
 
10.10 Timeframe for Tracking Reportable Events:  
 
Reportable adverse events will be tracked for 30 days following the last day of study 
treatment, or until the start of a new chemotherapy/treatment, whichever comes first. 


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11.0 
DATA SAFETY MONITORING PLAN 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, the 
Principal Investigator will provide a Data and Safety Monitoring (DSM) report to the Washington 
University Quality Assurance and Safety Monitoring Committee (QASMC) semi-annually 
beginning six months following activation of the first secondary site.  
 
The Principal Investigator will review all patient data at least every six months, and provide a 
semi-annual report to the QASMC. This report will include: 
 HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
 Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of study 
 History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol exceptions, 
error, or breach of confidentiality including start/stop dates and reason 
 Study-wide target accrual and study-wide actual accrual including numbers from 
participating sites 
 Protocol activation date at each participating site 
 Average rate of accrual observed in year 1, year 2, and subsequent years at each 
participating site 
 Expected accrual end date and accrual by site 
 Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
 Measures of efficacy 
 Early stopping rules with supporting data and list the number of participants who have 
met the early stopping rules 
 Summary of toxicities at all participating sites 
 Abstract submissions/publications 
 Summary of any recent literature that may affect the safety or ethics of the study  
 
Until such a time as the first secondary site activates this protocol, a semi-annual DSM report to 
be prepared by the study team will be submitted to the QASM Committee beginning 6 months 
after study activation at Washington University. 
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC according 
to institutional guidelines. 
 


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12.0 
AUDITING 
 
Since Washington University is the coordinating center, each site will be audited annually by 
Siteman Cancer Center personnel (QASMC) unless the outside institution has an auditing 
mechanism in place and can provide a report. The outside sites will be asked to send copies of all 
audit materials, including source documentation.  The audit notification will be sent to the 
Washington University Research Patient Coordinator, who will obtain the audit materials from the 
participating institution. 
 
Notification of an upcoming audit will be sent to the research team one month ahead of the audit. 
Once accrual numbers are confirmed, and approximately 30 days prior to the audit, a list of the 
cases selected for review (up to 10 for each site) will be sent to the research team. However, if 
during the audit the need arises to review cases not initially selected, the research team will be 
asked to provide the additional charts within two working days. 
 
Additional details regarding the Auditing Policies and procedures can be found at 
http://www.siteman.wustl.edu/uploadedFiles/Research_Programs/Clinical_Research_Resources/
Protocol_Review_and_Monitoring_Committee/QASMCQualityAssurcance.pdf  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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13.0 
MULTICENTER REGULATORY REQUIREMENTS 
 
Washington University requires that each participating site sends its informed consent document 
to be reviewed and approved by the Washington University Regulatory Coordinator (or designee) 
prior to IRB/IEC submission.    
 
Each participating institution must have the following documents on file at Washington University 
prior to first subject enrollment: 
 Documentation of IRB approval of the study in the form of a letter or other official 
document from the participating institution’s IRB.  This documentation must show 
which version of the protocol was approved by the IRB. 
 Documentation of IRB approval of an informed consent form. The consent must 
include a statement that data will be shared with Washington University, including the 
Quality Assurance and Safety Monitoring Committee (QASMC), the DSMC (if 
applicable), and the Washington University study team. 
 Documentation of FWA, signed FDA Form 1572, and signed and dated CVs of all 
participating investigators. 
 Documentation of training in protection of human subjects by all participating 
investigators. 
 Protocol signature page signed and dated by the investigator at each participating site. 
 
The Principal Investigator is responsible for disseminating to the participating sites all study 
updates, amendments, reportable adverse events, etc.  There will be one current version of the 
protocol document at any given time and each participating institution will utilize that document.  
Protocol/consent modifications and IB updates will be forwarded electronically to the secondary 
sites within 2 weeks of obtaining Washington University IRB approval with acknowledgement of 
receipt requested.  Secondary sites are to submit protocol/consent/IB modifications to their local 
IRBs within 4 weeks of receipt, and confirmation of submission must be forwarded to the 
appropriate contact person on the Washington University study team at the time of submission.  
Upon the secondary sites obtaining local IRB approval, documentation of such shall be sent to the 
Washington University study team within 2 weeks of receipt of approval. 
 
Documentation of participating sites’ IRB approval of annual continuing reviews, protocol 
amendments or revisions, all SAE reports, and all protocol violations/deviations/exceptions must 
be kept on file at Washington University. 
 
The investigator or a designee from each institution must participate in a regular conference call 
to update and inform regarding the progress of the trial. 
 
 


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14.0 
STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
 
14.1 
Analysis Populations 
 
Intent-to-Treat - The intent-to-treat (ITT) population will be defined as all patients who 
receive at least one dose of decitabine.  
 
Efficacy-Evaluable - The efficacy-evaluable (EE) population will be defined as all patients 
who receive at least one cycle of treatment.   
 
Safety - The safety population will be defined as all patients with at least one dose of 
decitabine. 
 
14.2 
Statistical Analysis 
 
14.2.1 Descriptive Analyses 
 
Patient Disposition: The number of patients discontinued, the reasons for 
discontinuation, and the number of cycles administered will be summarized. 
 
Demographics and Baseline Characteristics: Descriptive summary statistics will 
be provided for demographic and important baseline characteristics.  For 
continuous variables, the number of patients, mean, standard deviation, median, 
minimum and maximum will be provided.  For categorical variables, the number 
and percent of patients in each demographic/characteristic category will be 
summarized. 
 
14.2.2 Primary Endpoint Analysis 
 
The primary end-point of this phase II study is the correlation biomarkers with 
overall response in AML/MDS patients to decitabine. 
 
We will correlate the presence of patient specific mutations (especially mutations 
in DNMT3A, IDH1, IDH2, and TET2) with overall response rates. Mutation rates 
in these genes and in other patient-specific genes will be correlated with overall 
response and their respective 95% confidence intervals will be provided. 
 
Statistical considerations. The relationship between AML/MDS-associated 
mutations and the responsiveness to decitabine will be described by contingency 
tables and assessed using Chi-square test or Fisher’s exact test as appropriate. The 
differentially expressed genes between responders and non-responders will be 
determined using the significance analysis of microarray (SAM). Multivariate 
logistic regression will also be fit to adjust the potential confounding effects of other 
demographic and clinical characteristics. The resultant critical P values will be 
corrected for multiple testing.  Since the Bonferroni correction is known to be too 
conservative, a permutation analysis (e.g., 10,000 random assignments for 


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responsive status) will be used to derive an empirical critical P value.  The new 
critical p-value is the number of permutation tests which yield a lower p-value than 
the original test divided by the total number of permutation tests.[61]   
 
A power analysis was performed using Chi-square test for the association between 
mutation and response to decitabine. The frequency of DNMT3A, IDH1/2, and 
TET2 mutations in AML and MDS patients are each between 15% and 30% in 
multiple cohorts.[29-37] Table 3 shows the minimal detectable differences of 
response rates between mutant and wild-type cases, assuming an expected overall 
45% CR/CRi rate and 80% power. For an average of 20% frequency of a mutated 
gene, for example, 100 patients will provide 80% power to detect 30% difference 
at a 2-sided 0.1 alpha error. The purpose of this study is to determine whether any 
of these mutations result in a 
clinically 
significant 
association 
that 
would 
warrant pre-treatment testing. 
We believe that a reasonable 
cut off would be if at least 
30% of clinical response 
could be attributed to the 
presence 
of 
a 
specific 
mutation. 
However, 
we 
anticipate that more power 
could be achieved as we 
assess patient groups with 
combined characteristics (e.g. 
DNMT3A mutation or loss of 
DNMT3A 
expression). 
In 
addition, we expect that the 
majority of the enrolled 125 
patients can be used in the analysis (since all of our molecular information will be 
acquired within 30 days of enrollment, we anticipate a drop-out rate of 20% or less). 
 
14.2.3 Secondary Endpoint Analyses 
 
The overall response rate (CR/CRi/PR) and complete response rate (CR/CRi) will 
be compared with historical controls[2], and the 95% confidence intervals will be 
provided. 
 
The rate of patient specific mutation clearance after 10, 28, and 56 days of therapy 
will be determined using an Illumina based platform. We will compare the rate of 
mutation clearance between the patients who achieve a CR/CRi after 4 cycles and 
those who do not. We anticipate that we will identify between 6 and 25 exonic 
SNVs per case (the range obtained with >200 AML samples sequenced to date with 
whole genome or exome sequencing), and that the expected 95% confidence 
interval of the mean variant allele frequency will be ±2.56% and ±1.25%, 
Table 3. Detectable mutation associated response rates (80% power, α = 0.05) 
 
Total 
N 
% of patients 
with a given 
mutation  
Detectable 
response 
difference 
Sensitivity of mutation to 
predict response at minimum 
detectable response difference* 
100 
15%  
20%  
30% 
40% 
50% 
0.40 
0.35 
0.30 
0.27 
0.26 
80% 
75% 
67% 
61% 
58% 
* Assuming the overall response  rate (CR/Cri) of 45%   


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respectively, if we assume an anticipated standard deviation of 3.2% within the 
same clone. The change of tumor burden over time and its association with clinical 
response will  be assessed using 2-way ANOVA for repeated measurement data, 
followed by the ad-hoc multiple comparisons for between-group differences (e.g., 
responders vs. non-responders) at given time points as well as the between-time 
differences within each group. The resultant critical P values will also be corrected 
using permutation analysis for multiple testing. 
 
 
The percent decrease in bone marrow methylcytosine will be calculated on day 
10±1. We will compare this reduction with previously published data and between 
the patients who achieve CR/CRi and those who do not. The correlation of peak 
plasma levels of decitabine with response will be assessed using 2-sample t-test or 
Mann-Whitney non-parametric rank-sum test as appropriate. The change of total 
bone marrow DNA methylcytosine from baseline and its association with both 
steady-state plasma drug levels and response will be assessed using 2-way ANOVA 
for repeated measurement data, followed by the ad-hoc multiple comparisons for 
between-group differences (e.g., responders vs. non-responders) and between-time 
differences. Similar analyses will be performed for the correlation of expression 
profiles and SNP analysis with pharmacokinetic data.   
 
Statistical power justification.  A power analysis was performed using two-sample 
t-test for between-group difference and paired t-test for the change over time. For 
tumor clearance, this will provide > 99% power to detect a 10% difference in the 
means at two separate time points for clones with 6 or more variants, and > 98% 
power to detect a 15% difference if there are 3 or more variants per clone with α = 
0.05 (power will decrease to 91% and 82%, respectively, in cases where the 
standard deviation is 4.7%, the maximum we have observed thus far). For 
pharmacologic correlations, we anticipate 85% power to determine a 25 ng/ml 
difference between the average Cmax of 45 evaluable responders and 55 evaluable 
non-responders with an anticipated average Cmax of 70 ng/ml and a standard 
deviation of 40 ng/ml within the entire cohort, and with α = 0.05.[22] We believe a 
less significant difference would not be sufficient to warrant application in clinical 
decision making (i.e. whether to continue therapy for more than 1 cycle). We 
anticipate 99% power to determine a difference of 20% in the reduction of bone 
marrow methylcytosine between 45 evaluable responders and 55 evaluable non-
responders with an expected standard deviation of 15% and α = 0.05.[28, 66-68] 
The difference in methylcytosine reduction previously observed was 30% between 
patients achieving CR/PR and those with only hematologic improvement.[28]  
 
Time to relapse and time to death will also be listed for each patient. 
 
14.2.4 Safety Analysis 
 
Extent of Exposure – Duration (number of cycles/infusions) of decitabine doses 
(cumulative and intensity) will be summarized. 


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Adverse Events - All adverse events will be coded using NCI CTCAE v. 4.0.  The 
incidence of treatment-emergent adverse events (number and percent of patients 
reporting the adverse event at least once during the study) will be summarized.  In 
addition, adverse events will be summarized by investigator attribution of 
relationship to study medication and by grade.  Similar summaries will be provided 
by treatment cycle. 
 
Clinical Laboratory Evaluation – Applicable laboratory parameters will be graded 
according to NCI CTCAE version 4.0. The incidence of maximum grade (number 
and percent of patients experiencing the maximum grade during the study) will be 
summarized for each laboratory parameter.  Similar summaries will be provided by 
treatment cycle.  Graphical displays of select hematological parameters will also be 
provided. 
 
Vital Signs, Physical Findings, and Other Safety Observations – Vital signs, 
findings from physical examinations, ECOG scores and weights will be presented 
in listing format. 
 
14.2.5 Description of Planned Subgroup Analyses 
 
The following subgroups (if sufficient numbers of patients) will be explored with 
respect to the primary and overall survival endpoints. 
 Age (60-69 years old vs ≥ 70 years old) 
 Cytogenetics 
 Patients with AML-associated mutations in DNMT3A, TET2, IDH1, or 
IDH2 vs those who do not have somatic variants in these genes. 
 
14.3 
Sample Size 
 
We anticipate initially enrolling 25 patients at Washington University. Once we establish 
a routine exome analysis with validation and calculation of the rate of patient-specific 
mutation clearance, we will enroll an additional 100 patients from at least 2 additional 
centers.  
 


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15.0 
DATA SUBMISSION SCHEDULE 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to registration 
Patient Registration Form 
Disease Diagnosis Form 
Inclusion Criteria Worksheet 
Exclusion Criteria Worksheet 
 
Prior to starting treatment 
Treatment Record Form 
To be maintained throughout a patient’s treatment 
cycle.  The complete record is due at completion of 
treatment. 
Bone Marrow Sample Collection 
Form 
To be maintained throughout the course of the 
study at designated bone marrow sample 
collections time points (See Appendix 2). 
Pathology: Bone Marrow Biopsy 
To be completed upon the return of pathology 
report for each bone marrow biopsy. 
Study Calendar Form 
To be completed with the date of each time point 
upon occurrence. 
Labs Form 
To be completed upon the return of laboratory 
reports for each study time point (See Appendix 2) 
Dose Modifications 
To be maintained throughout the course of the 
study if dose modifications are performed. 
Therapy Response Form 
To be filled out upon remission (prior to, and/or on 
therapy) 
End of Study Form 
 
Due at the completion of the EOS visit, at the time 
of death, and/or at the time that the patient is lost to 
follow-up 
Follow Up Form 
 30 days post date of last decitabine dose. 
 Every 6 months for 2 years after the last dose of 
study drug. 
Record of Adverse Events 
At the time of any AE 
 
Any queries generated by Washington University must be responded to within 28 days of receipt 
by the participating site.  The Washington University research team will conduct a regular review 
of data status at all secondary sites, with appropriate corrective action to be requested as needed. 
 


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Appendix 1 
ECOG Performance Scale 
Activity Status 
Description 
0 
Asymptomatic, fully active, and able to carry on all predisease 
performance without restrictions. 
 
1 
Symptomatic, fully ambulatory but restricted in physically strenuous 
activity and able to carry out performance of a light or sedentary 
nature, eg, light housework, office work. 
 
2 
Symptomatic, ambulatory and capable of all self-care but unable to 
carry out any work activities.  Up and about more than 50% of 
waking hours: in bed less than 50% of day. 
 
3 
Symptomatic, capable of only limited self-care, confined to bed or 
chair more than 50% of waking hours, but not bedridden. 
 
4 
Completely disabled.  Cannot carry on any self-care.   Totally 
bedridden. 
 
5 
Dead 
 
 


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Appendix 2 
Study Flow Chart 
Study Flow Chart 
Test 
Pre-
study1 
Base-
line2 
All 
Cycles 
Day 1 
All Cycles 
Days 1-103 
Cycle 1 Day 
4 ± 1 
 
Cycle 1 
Day 10 ± 1 
and 28 ± 4 
Every 
week 
Every 
2 
weeks 
Even 
cycles 
Day 28±4 
End of 
Study 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Medical History 
 
X 
 
 
 
 
 
 
 
 
Physical Exam 
 
Xa 
Xb 
 
 
 
 
 
 
X 
Transfusion 
Requirementsc 
 
Xc 
Ongoing 
Bone Marrow 
Aspirate and Biopsy 
Xd 
 
 
 
 
Xd 
 
 
Xd 
Xd 
Flow Cytometrye 
X 
 
 
 
 
 
 
 
 
 
Pregnancy test 
 
X 
 
 
 
 
 
 
 
 
CBC with differential 
and platelets 
 
X 
X 
 
 
 
Xf 
 
 
X 
Serum Chemistriesg 
 
X 
X 
 
 
 
 
Xf 
 
X 
Decitabine 
administrationh 
 
 
 
X 
 
 
 
 
 
 
Adverse Events  
 
 
Ongoing 
Post-study follow-up 
 
 
 
 
 
 
 
 
 
Xi 
1 To be performed within 14 days of the first dose of decitabine.  
2 To be performed within 7 days of the first dose of decitabine.  
3 Decitabine may be reduced to Days 1-5 or Days 1-3. See Section 6.3 for details.  
a To include height, weight, and ECOG. 
b To include weight. 
c Care should be given to record the frequency of transfusions from 8 weeks prior to first dose of study drug through 
the duration of the study. 
d Bone marrow biopsy and aspirate should be performed at baseline, at Cycle 1 Day 10 ± 1, and at  Day 28 ± 4 of Cycles 
1, 2, 4 and 6 for clinical disease assessment. Patients who achieve with clinical response (CRi or better) and are receiving 
cycles every 6 weeks, will undergo bone marrow biopsies on day 42 ± 4. Following Cycle 6, bone marrow biopsies 
should be performed at the discretion of the treating physician to assess for relapse or progression. Standard bone marrow 
procurement procedures will be followed for collection of the tissue. To include: correlative studies (collected under 
HRPO# 201011766), morphologic assessment, cytogenetics and FISH at all time points. A skin biopsy sample (collected 
under HRPO# 201011766) is required at pre-study, but may have been collected months or even years prior to first dose 
of decitabine.  
e On bone marrow or peripheral blood.  
f After Cycle 1, these labs may be obtained every other week (CBC) or once a month (Chemistries) at the discretion of 
the treating physician. 
g Serum chemistries include bicarbonate, calcium, glucose, total protein, albumin, BUN, potassium, chloride, sodium, 
creatinine, total bilirubin, AST, ALT, alkaline phosphatase, and LDH. 
h Decitabine treatment will be 20 mg/m2 over a 1-hour IV infusion on consecutive Days 1-10 every 28-day cycle.   The 
dose will be calculated based on actual patient weight at the beginning of each cycle. Patients may be premedicated 
with lorazepam, prochlorperazine, palonosetron, or ondansetron at the treating physician’s discretion. 
i Patients will be followed for  30 days following the date of the last decitabine dose to monitor for toxicity, survival 
and response. Thereafter, the investigator or designee will record information on subsequent therapy, disease 
progression, and death, every 6 months for 2 years after first dose of study drug or until the patient is lost to follow-up.


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 4 Version date: 04/16/15  
Page 47 of 51 
16.0 
REFERENCES 
 
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Amendment 4 Version date: 04/16/15  
Page 48 of 51 
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32. 
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Mutations in Epigenetic Modifiers in AML. Hematol Oncol Clin North Am, 2011. 
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Myeloid Leukemia and Myelodysplastic Syndrome. PLoS One, 2011. 6(10): p. 
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41. 
Kantarjian, H.M., et al., Survival advantage with decitabine versus intensive 
chemotherapy in patients with higher risk myelodysplastic syndrome: comparison 
with historical experience. Cancer, 2007. 109(6): p. 1133-7. 
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Lubbert, M., et al., Continued Low-Dose Decitabine (DAC) Is an Active First-
Line Treatment in All Cytogenetic Subgroups of Older AML Patients: Results of 
the FR00331 Multicenter Phase II Study. ASH Annual Meeting Abstracts, 2007. 
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Valproic Acid in Acute Myeloid Leukemia. J Clin Oncol, 2007. 25(25): p. 3884-
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Issa, J.-P.J., et al., Phase 1 study of low-dose prolonged exposure schedules of the 
hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in hematopoietic 
malignancies. Blood, 2004. 103(5): p. 1635-1640. 
45. 
Garcia-Manero, G., et al., Phase 1/2 study of the combination of 5-aza-2'-
deoxycytidine with valproic acid in patients with leukemia. Blood, 2006. 108(10): 
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Issa, J.P., S.B. Baylin, and J.G. Herman, DNA methylation changes in 
hematologic malignancies: biologic and clinical implications. Leukemia, 1997. 
11 Suppl 1: p. S7-11. 
47. 
Ganetzky, R.D., et al., Differences Between Normal and Leukemic Stem Cell-
Specific Methylome Indicates Aberrantly Silenced Genes Involved in the 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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Pathogenesis of Malignant Evolution. ASH Annual Meeting Abstracts, 2008. 
112(11): p. 599-. 
48. 
Christman, J.K., 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA 
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Oncogene, 2002. 21(35): p. 5483-95. 
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Lubbert, M., DNA methylation inhibitors in the treatment of leukemias, 
myelodysplastic syndromes and hemoglobinopathies: clinical results and possible 
mechanisms of action. Curr Top Microbiol Immunol, 2000. 249: p. 135-64. 
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62. 
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Cancer Chemother Pharmacol, 2003. 51(3): p. 231-9. 
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Bryan, J., et al., Pharmacokinetic evaluation of decitabine for the treatment of 
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Samlowski, W.E., et al., Evaluation of a 7-day continuous intravenous infusion of 
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Genomic Predictors of Decitabine Response in AML/MDS 
 
 
NCT# NCT01687400 
 
 
Document Version Date:  11/13/2014 
 
IRB Approval Date:  11/17/2014 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 3 Version date: 11/13/14  
Page 1 of 50 
 
 
Genomic Predictors of Decitabine Response in AML/MDS 
 
Washington University School of Medicine 
Division of Oncology 
660 South Euclid Avenue, Box 8056 
St. Louis, MO 63110 
 
Protocol #: 201210102 
Protocol Version Date: 11/13/14 
 
Coordinating Center: Washington University School of Medicine 
 
Principal Investigator 
John Welch, M.D./Ph.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-2626, Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Sub-Investigator 
Timothy Ley, M.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-8832, Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Pathology 
Jeffery M. Klco, M.D., Ph.D. 
Department of Anatomic and Molecular Pathology 
Telephone: (314) 362-8832 
Email: klcoj@wustl.edu 
 
Statistician 
Feng Gao, Ph.D. 
Division of Biostatistics 
Telephone: (314) 362-3682 
E-mail: feng@wubios.wustl.edu 
 
IND# 116548 
Clinical Trials.gov # NCT01687400 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to obtain 
informed consent, may not be disclosed to another party unless law or regulations require such disclosure.  Persons 
to whom the information is disclosed must be informed that the information is confidential and may not be further 
disclosed by them. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 3 Version date: 11/13/14  
Page 2 of 50 
Genomic Predictors of Decitabine Response in AML/MDS  
Principal Investigator Signature Page 
 
 
Principal Investigator: 
 
John Welch, M.D., Ph.D. 
 
 
 
 
 
Signature of Investigator 
 
Date 
 
 
 
Printed Name of Investigator 
 
 
 
By my signature, I agree to personally supervise the 
conduct of this study and to ensure its conduct in 
compliance with the protocol, informed consent, 
IRB/HRPO procedures, the Declaration of Helsinki, ICH 
Good Clinical Practices guidelines, and the applicable 
parts of the United States Code of Federal Regulations or 
local regulations governing the conduct of clinical 
studies. 
 
 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 3 Version date: 11/13/14  
Page 3 of 50 
 
Study Schema: 
 
 
Patients with progressive disease after two cycles (defined as a clear increase in bone marrow blast count, typically a 
> 50% increase in bone marrow blasts over baseline) should be removed from study. 
Patients with stable disease may continue on study and should undergo a diagnostic bone marrow biopsy at the end 
of even numbered cycles. 
Once AML patients achieve morphologic complete remission with incomplete blood count recovery (CRi) or MDS 
patients achieve a marrow complete response (Marrow CR) as defined in Section 9.0, decitabine 
administration may be decreased to 20 mg/m2/day for ten consecutive days every 6 weeks, to limit 
myelosuppressive toxicity and improve count recovery. After 2 cycles at 6 week intervals, the dose may be 
reduce to 20 mg/m2/day for five consecutive days in 6 week cycles, at the discretion of the treating physcian. 
After two cycles of five consecutive days of treatment, if the patient meets the definition of CR with complete 
cytogenetic response as defined in Section 9.0, administration may further be decreased to three consecutive 
days per cycle at the discretion of the treating physician. 
Sample collection for correlative studies: 
Bone marrow biopsies should be performed at baseline, at Cycle 1 Day 10 ± 1, and at Day 28 ± 4 of Cycles 
1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be performed at the discretion of the 
treating physician to assess for relapse or progression.  
Peripheral blood samples should be collected on Day 4± 1 of Cycle 1. Collection should be obtained 45 ± 
15 minutes after decitabine infusion is initiated.
20 
mg/m
2/day  
Cycle 1 
20 
mg/m
2/day 
Cycle 2+ 
Day 1 
Day 28 
BM Bx 
PB 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 3 Version date: 11/13/14  
Page 4 of 50 
Genomic Predictors of Decitabine Response in AML/MDS 
Table of Contents 
Section 
 
Page 
1.0 INTRODUCTION .......................................................................................................7 
1.1 
Rationale .................................................................................................................7 
1.2 
Decitabine and Hypomethylating agents in AML/MDS ........................................9 
1.2.1 
Pharmacokinetics and Metabolism of Decitabine ........................................11 
1.2.2 
Experience with extended dosing schedules ................................................12 
1.3 
Correlative Studies ...............................................................................................13 
2.0 OBJECTIVES ............................................................................................................15 
2.1 
Primary Objective .................................................................................................15 
2.2 
Secondary Objectives ...........................................................................................15 
3.0 PATIENT SELECTION ............................................................................................16 
3.1 
Inclusion Criteria ..................................................................................................16 
3.2 
Exclusion Criteria .................................................................................................16 
3.3 
Inclusion of Women and Minorities .....................................................................17 
4.0 REGISTRATION PROCEDURES ...........................................................................18 
4.1 
Confirmation of Patient Eligibility .......................................................................18 
4.2 
Patient Registration in the Siteman Cancer Center Database ...............................18 
4.3 
Assignment of UPN ..............................................................................................18 
5.0 INVESTIGATIONAL PLAN ...................................................................................20 
5.1 
Summary of Study Design....................................................................................20 
5.2 
Study Procedures ..................................................................................................21 
5.2.1 
Pre-study Procedures ....................................................................................21 
5.2.2 
Baseline Evaluation ......................................................................................21 
5.2.3 
Day 1 of Each Cycle ....................................................................................21 
5.2.4 
Cycle 1 Days 1-10 ........................................................................................21 
5.2.5 
Cycle 1 Day 4 ± 1 ........................................................................................22 
5.2.6 
Cycle 1 Day 10 ± 1 ......................................................................................22 
5.2.7 
Weekly .........................................................................................................22 
5.2.8 
Every Other Week ........................................................................................22 
5.2.9 
Cycles 1, 2, 4 and 6Day 28 ± 4 days ............................................................22 
5.2.10 
End-of-Study Procedures .............................................................................22 
5.2.11 
Post-study Follow-up ...................................................................................23 
5.3 
Duration of Therapy .............................................................................................23 


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Amendment 3 Version date: 11/13/14  
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5.4 
Concomitant Therapy and Supportive Care Guidelines .......................................23 
5.4.1 
Chemotherapy ..............................................................................................23 
5.4.2 
Growth Factors .............................................................................................24 
5.4.3 
Transfusions .................................................................................................24 
5.4.4 
Prophylactic Antimicrobial Agents ..............................................................24 
5.4.5 
Radiotherapy ................................................................................................24 
6.0 DOSE MODIFICATIONS ........................................................................................25 
6.1 
Delay for Cytopenia or Infection ..........................................................................25 
6.2 
Delay for Organ Dysfunction ...............................................................................25 
6.3 
Modification in Dose or Schedule for Treatment Response ................................25 
7.0 PHARMACEUTICAL INFORMATION .................................................................26 
7.1 
Decitabine .............................................................................................................26 
7.1.1 
Study Drug Preparation ................................................................................26 
7.1.2 
Supplier ........................................................................................................26 
7.1.3 
Toxicities ...................................................... Error! Bookmark not defined. 
8.0 CORRELATIVE STUDIES SAMPLE COLLECTIONS .........................................27 
9.0 EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES....................28 
9.1 
Response Criteria - AML .....................................................................................28 
9.2 
Response Criteria - MDS......................................................................................29 
9.2.1 
Hematologic Improvement (HI) ...................................................................29 
9.3.1 Cytogenetic Response ............................................................................................30 
9.3 
Guidelines for Evaluation of Disease ...................................................................30 
9.4 
Other Secondary Efficacy Measures ....................................................................30 
9.4.1 
Treatment Failure, Overall Survival and Event-free Survival .....................30 
9.4.2 
Duration of Remission and Relapse-free Survival for 
Patients Having Complete Remission ..........................................................30 
9.4.3 
Safety ...........................................................................................................31 
10.0 ADVERSE EVENT REPORTING ...........................................................................32 
10.1 Adverse Events (AEs) ..........................................................................................32 
10.2 Unanticipated Problems........................................................................................32 
10.3 Noncompliance .....................................................................................................32 
10.4 Serious Noncompliance ........................................................................................32 
10.5 Protocol Exceptions ..............................................................................................33 
10.6 Reporting to the Human Research Protection Office (HRPO) and 
the Quality Assurance and Safety Monitoring Committee 
(QASMC) at Washington University: ..................................................................33 
10.7 Reporting Requirements for Secondary Sites .......................................................33 
10.8 Reporting to Secondary Sites ...............................................................................34 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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10.9 Reporting to the FDA ...........................................................................................34 
10.10 Timeframe for Reporting Required Events: .........................................................35 
11.0 DATA SAFETY MONITORING PLAN ..................................................................36 
12.0 AUDITING ................................................................................................................37 
13.0 MULTICENTER REGULATORY REQUIREMENTS ...........................................38 
14.0 STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE .............................39 
14.1 Analysis Populations ............................................................................................39 
14.2 Statistical Analysis ...............................................................................................39 
14.2.1 
Descriptive Analyses ....................................................................................39 
14.2.2 
Primary Endpoint Analysis ..........................................................................39 
14.2.3 
Secondary Endpoint Analyses .....................................................................40 
14.2.4 
Safety Analysis ............................................................................................41 
14.2.5 
Description of Planned Subgroup Analyses .................................................42 
14.3 Sample Size ..........................................................................................................42 
15.0 DATA SUBMISSION SCHEDULE .........................................................................43 
16.0 REFERENCES ..........................................................................................................46 
  


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1.0 
INTRODUCTION 
 
1.1 
Rationale   
 
Personalized therapy for cancer requires that 1) patient-specific factors (e.g. patient-
specific mutations, expression profiles, or pharmacogenomics) influence treatment 
algorithms, and 2) once therapy is initiated, in vivo responsiveness is used to guide further 
therapy. The modern era of genomics has provided significant insights into cancer 
pathogenesis by describing patterns of germline and somatic variants, as well as defining 
disease-specific patterns of gene expression. However, the application and integration of 
this data with therapeutics remains a difficult task. 
 
Decitabine is a commonly used single-agent therapy for patients with acute myeloid 
leukemia (AML) and/or myelodysplastic syndrome (MDS). Historically, it has yielded 
clinical complete responses in approximately 25% of patients across diverse clinical trials, 
it is well tolerated, and it can be given as in the outpatient setting. However, the majority 
of patients do not respond to decitabine, the molecular etiology of decitabine sensitivity is 
unknown, and there are no algorithms to predict patient responsiveness a priori or even 
within one month of starting therapy.  
 
AML and MDS are two closely related hematological disorders characterized by 
ineffective hematopoiesis and malignant expansion of clonal myeloid cells.  Both AML 
and MDS frequently occur in elderly patients and some cases are secondary and/or related 
to long-term bone marrow damage after radiation or chemotherapy. Current therapy for 
AML consists of cytotoxic chemotherapy followed by consolidation chemotherapy or stem 
cell transplantation. Using these approaches, current cure rates may be as high as 60% for 
patients younger than 60 with good risk cytogenetics (e.g. t(15;17), t(8;21), inv(16)).[4]  
Outcomes in patients ≥ 60 years old and with relapsed disease remain poor with 2 year 
survival of only ~25%.[5] Historically, the available treatment for MDS has been 
symptomatic and has not been shown to be effective in producing sustained improvement 
in hematopoiesis or in delaying leukemic evolution[6, 7].  
 
Current AML therapy is poorly tolerated in elderly and pre-treated patients due to 
hematologic and non-hematologic toxicities; following such therapy, patients typically 
require 3-4 weeks of hospitalization for treatment of neutropenic fevers and for regular 
transfusions. Even among the relatively healthy patients enrolled in clinical trials, induction 
mortality in the elderly can be as high as 20-40% following standard induction therapy,[8-
11] and moderate to severe non-hematologic toxicity occurs in at least an additional 30% 
of patients.[9, 12-14] Consequently, many older AML patients are not offered traditional 
intensive chemotherapy, but receive supportive care alone.[15]  More effective and less 
toxic therapies are needed to provide durable remissions in a significant proportion of 
elderly AML in populations. 
 
Decitabine has been studied in MDS patients and in elderly and/or relapsed AML patients. 
It has been found to be reasonably well tolerated, can be given in the outpatient setting, 
and has overall response rates of 20-45%.[2, 3, 16-20] Our own experience has been of 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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CR/PR rates of 25%, [2] and this is typical of many other groups for both MDS and AML 
patients (reviewed in [21]  Decitabine is an S-phase depended drug.  [22-24] However, 
decitabine is usually administered in one hour daily infusions during 3 to 5 consecutive 
days (too short to expose decitabine to most AML blasts during at least 2 cycles of 
replication, which divide every 2 to 7 days).[25-27]  Recent experience with increasing the 
number of infusions per cycle from 5 consecutive days to 10 consecutive days has 
improved overall response rates to 67%.[3] Response to decitabine can be slow, sometimes 
requiring 3 to 5 cycles before maximum response is observed.[2] As a consequence, many 
patients are exposed to prolonged therapy, gaining little benefit, before determining 
resistance. There are currently no biomarkers that predict response to decitabine. 
 
Decitabine is a cytosine analogue that cannot be methylated. Incorporation of decitabine 
into DNA prevents DNA methylation at cytosine residues and derepresses epigenetically 
silenced gene expression. Therefore, the reduction in total genomic DNA methylcytosine 
content acts as a marker of effective decitabine dosing. Indeed, two groups have recently 
correlated effective methylcytosine reduction and gene expression derepression to 
response (Figure 1.1).[3, 28] 
 
 
 
   
Figure 1.1. From Shen et al JCO (2010) 28:611 and Blum et al JCO (2007) 25: 3884. 
 
We and others have recently used whole genome sequencing to identify recurrent, somatic 
mutations in AML and MDS patients in four genes that are involved in methylcytosine 
regulation: DNMT3A, IDH1, IDH2, and TET2.[29-37] Collectively, nearly half of all 
AML/MDS patients have a mutation in one of these four genes.  DNMT3A mutations are 
associated with an adverse outcome in AML patients treated with cytotoxic 
chemotherapy.[35, 38] However, it is not yet clear whether the presence of mutations in 
one or more of these genes is associated with response or resistance to decitabine. 
 
In this study, we will treat patients with the current state-of-the-art decitabine schedule.[3] 
We will perform exome sequencing on all patients and correlate response with mutation 
status, rate of mutation clearance, expression profile, plasma decitabine levels, and DNA 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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methylcytosine content reduction. This approach will comprehensively assess genomic and 
pharmacologic parameters that could clinically predict patient response to decitabine.  
 
In this study, we will combine analysis of patients with AML and MDS. Recent genomic 
analysis has shown that AML and MDS have shared mutations in many of the same genes, 
shared patterns of mutation acquisition, and shared patterns of mutation evolution.[39, 40] 
Furthermore, MDS, like AML, presents with clonal hematopoiesis, even when the 
morphologic blast count is zero.[36]Because this study will identify and track the rate of 
clearance of patient specific mutations, and because decitabine has been shown to have 
efficacy in both AML and MDS patients, [2, 3, 16-19, 41-45] combining analysis of AML 
and MDS patients is reasonable.  Asymptomatic MDS patients are excluded because these 
patients are typically followed with watchful-waiting rather than with chemotherapy. 
  
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
 
Methylation of cytosine in the CpG dinucleotide by DNA methyltransferase leads to 
transcriptional silencing of genes during normal development and has emerged as a 
significant mechanism for the loss of tumor suppressor gene expression in human cancer, 
including AML and MDS.[46, 47] Like azacitidine, decitabine (5-aza-2’-deoxycitidine) is 
incorporated into DNA during S phase and forms covalent adducts with DNA 
methyltransferase. The irreversible inhibition of this enzyme results in loss of methylation 
and is associated with differentiation.[48]  Decitabine is an approximately 5-fold more 
potent inhibitor of DNA methyltransferase than azacitidine, which might explain why 
decitabine can induce differentiation of myeloid progenitors in vitro at lower doses than 
azacitidine. In addition to their differentiation inducing activity at low doses, both agents 
have a direct cytotoxic effect at higher doses (1-2 µM).[49]  
 
Decitabine has been investigated in hematologic malignancies using a range of doses and 
schedules.  It is currently FDA approved for the treatment of adults with MDS at a dose of 
15 mg/m2 every 8 hours for 3 days, based on a Phase III study that demonstrated improved 
outcomes in the treatment arm compared to patients treated with supportive care alone.[15]  
However, the overall response rate in this trial was modest (17%). Of note, 18% of the 
patients in this trial had 20-30% bone marrow blasts, meeting the current WHO criteria for 
AML.  Subsequent phase I and II studies in AML, using a variety of low-dose decitabine 
regimens, have reported overall response rates of 22-49%.[2, 3, 16-19, 41-45] Comparisons 
of multiple low dose schedules have noted improved efficacy in the MDS population using 
a dose of 20 mg/m2 on Days 1-5 of a 28-day cycle.[50, 51] Because of these results, we 
have recently explored this dosing schedule in the treatment of elderly patients with 
AML.[52] In this study, we observed an overall response rate of 24%, and an additional 
44% of patients achieved stable disease during a median of 5 monthly cycles.[48] In this 
study, we noted that decitabine does not act quickly and the median time to response was 
3 months. These results are similar to the recently published experience in Germany 
treating 227 patients with 15 mg/m2 over 3 hours TID for 3 days every 6 weeks, which 
resulted in a CR/PR rate of 26%.[16]  
 


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A recent study using a prolonged administration schedule (20 mg/m2 IV over 1 hour on 
consecutive days 1-10) showed improved response (64% CR/PR) and improved time to 
respond (50% of patients had bone marrow blasts < 5% after 1 cycle of therapy).[3]  These 
results are an improvement over previous experience by other groups using decitabine to 
treat elderly AML patients or patients with relapsed AML, and this experience is 
summarized below: 
Trials of decitabine in AML 
Status 
Other Therapy 
DAC Dose 
(mg x days) 
Evaluable 
CR 
de novo 
daunomycin1 
90 mg/m2 x 5 
6 
100% 
Salvage 
None4 
0.75-80 mg/kg 
16 
  
None5 
37-81 mg/kg 
6 
33% 
None7.  Ara-C 
Refractory 
Various 
4 
0 
AMSA7 
250-500 mg/m2 x 6 
10 
80% 
idarubicin or 
AMSA3 
250 mg/m2 x 6 
56 
39% 
Elderly 
None6 
270-360 mg/m2 x 3 
10 
30% 
None2 
45 mg/m2 x 3 
4 
50% 
None8 
20 mg/m2day 1-5,8-12 
50 
23% 
None9 
20 mg/m2 x 5 
55 
25% 
 
None10 
50 mg/m2 CIVI x 3 
29 
48% 
 
None11 
20 mg/m2 x 10 
53 
64% 
 
ATRA cycle 212 
15 mg/m2 TID x 3 
227 
26% 
1 Schwartsmann G.  Leukemia 1997; 11(Suppl 1):S28-31. 
2 Lubbert M. ASH 2002 Abstract #4601. 
3 Willemze R.  Leukemia.  1997; 11(Suppl 1):S24-27 
4 Rivard GE.  Leukemia Research 1981; 5:453-462. 
5 Momparler RL.  Pharmac Therapy 1985; 30:277-286.  
6 Petti MC.  Leukemia.  1993; 7(Suppl 1):36-41.  
7 Richel DJ.  Br. J. Cancer 1991; 64:144-148 
8 Issa, JP. Blood 2004. 103: 1635. 
9 Cashen AF. ASH Annual Meeting. 2008 abstract 560. 
10 Wijermans PW. Leukemia 1997. 11: Suppl 1, 19-23. 
11 Blum, W. PNAS 2010; 107:7473. 
12 Lubbert. Haematologica 2011. ePub 11/04/2011. 
 
In general, decitabine is well tolerated. Unlike conventional cytotoxic chemotherapy, 
decitabine can be administered in the outpatient setting. However, AML and MDS are 
aggressive hematologic malignancies. Cytopenias commonly result from AML/MDS 
inhibition of normal hematopoiesis, and often do not resolve until a complete response is 
achieved. Thus, neutropenic sepsis and thrombocytopenic bleeding remain significant 
problems regardless of therapy, especially during the first 4-8 weeks. In our experience 
treating elderly AML patients, neutropenia occurred in 24%, sepsis in 9%, and bacteremia 
in 7%.  Similar results have been observed in MDS patients.[20, 53, 54] As a result, death 
is not uncommon in the first 8 weeks of therapy, occurring in 15-17% of patients, and most 
commonly resulting from infection and cytopenias related to disease and therapy.[16, 52, 
55] While unsettling, these numbers are better than many historical patients treated with 
cytotoxic chemotherapy, and suggest a need for continued improvements in therapy.[9, 12]  


Genomic Predictors of Decitabine Response in AML/MDS 
 
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1.2.1 Pharmacokinetics and Metabolism of Decitabine 
 
In human subjects, after IV administration, decitabine displayed a distribution phase 
with a half-life of 7 minutes and a terminal phase elimination half-life of 10-35 
minutes as measured by bioassay.[56, 57]   The short plasma half-life is due to rapid 
inactivation of decitabine by deamination by liver cytidine deaminase.[23] Total 
body clearance (Clp) in humans was high with a mean value of 126 mL/min/kg.[58]  
 
Among five adult patients with advanced solid tumors administered seven courses 
of 100 mg/m2 of decitabine by 1-hour infusions,[56] maximum plasma 
concentration ranged from 1 to 4 µM. Disappearance from plasma was biphasic 
with a rapid initial decline to less than 0.1 µM during the first hour post infusion 
and a slower decline over the next two hours to 6 nM, the level of detectability. 
Plasma half-life (meanSE) was estimated to be approximately 71 minutes in the 
initial phase (t½α) and 355 minutes in the second phase (t½β). The area under the 
plasma concentration-time curve was 40888 ng.hour/mL for these patients, with a 
peak plasma concentration of 2.010.44 µM (0.440.1 µg/mL), while mean volume 
of distribution (Vdss) and CLp was 4.591.42 L/kg and 12621 mL/min/kg, 
respectively. The time course of DAC concentrations was similar in 1 of 2 patients 
administered 75 mg/m2, while DAC was usually undetectable in the plasma of 
patients administered 25 to 60 mg/m2. 
 
Using a longer infusion time (up to 40 hours), Rivard et al.[57] reported a plasma 
concentration of 0.1 to 0.4 g/mL. With infusion times of 40-60 hours, at an infusion 
rate of 1 mg/kg/h, plasma concentrations of 0.43-0.76 µg/mL were achieved.[59]  The 
steady-state plasma concentration at an infusion rate of 1 mg/kg/h was estimated to 
be 0.2-0.5 µg/mL. The half-life after discontinuing the infusion was 12-20 min.[57]  
 
Urinary excretion of unchanged decitabine was low, ranging from less than 0.01% to 
0.9% of the total dose, and there was no relationship between excretion and dose or 
plasma drug levels. High clearance values and a total urinary excretion of <1% of the 
administered dose suggest that decitabine is eliminated rapidly and largely by 
metabolic processes, including extrahepatic metabolism.[60] 
 
The steady state plasma concentrations (Css) ranged from 0.31 to 0.39 µg/mL (1.27 
to 1.60 µM) during the infusion of 100 mg/m² decitabine over a 6-hour period.[61] 
The range of concentrations during a 600 mg/m2 infusion over an 8-hour period was 
0.41-16 g/mL(1.68 to 4.76 µM).[60] These plasma concentrations are in the same 
range as the in vitro concentrations that activated the expression of tumor 
suppressor gene p16 in three human non-small-cell lung cancer (NSCLC) cell 
lines.[61]  
 
Steady state decitabine levels of 0.1 to 0.2 µM were achieved in solid tumor patients 
dosed with 30 mg/m²/day and 40 mg/m²/day by continuous intravenous infusion 
over 72 hours.[62]  


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Summary of published steady state serum levels are below and are in proportion to 
infusion rate (R2 = 0.98, p < 0.0001):[63] 
 
Rate 
Serum 
Concentration 
mg/m2/hr 
ng/ml 
nM 
5 
55 
241 
7.5 
80 
351 
15 
180 
789 
21 
182.4 
800 
22.5 
350 
1535 
42 
456 
2000 
84 
1140 
5000 
 
In our experience treating primary patient blasts in vitro, concentrations of 100 – 
500 nM are required maximize methylcytosine reduction without causing cellular 
toxicity. Because decitabine incorporation into DNA is S-phase dependent, 
exposure of cells during cell division is necessary for efficacy. 
  
 
 
 
1.2.2 Experience with extended dosing schedules 
 
Blum et al recently published their experience treating elderly AML patients with 
a 10-day course of decitabine 20 mg/m2/day as a 1-hour infusion.[3] This prolonged 
exposure course was designed to increase the number of AML blast divisions 
exposed to decitabine per cycle. Toxicities were very similar to a similar regiment 
given as a 3 and 5 day course,[2, 16] but the overall response (CR/PR) was much 
improved (64% vs 25%). In all three studies, the toxicities were primarily myeloid 
suppression and the resultant infectious and bleeding complications associated with 
leukemic and pharmacologic hematopoietic suppression. Furthermore, Blum et al 
noted that the percent methylcytosine reduction was improved (22% vs 11%) as 


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was the time to blast clearance (50% of patients who ultimately achieved a CR had 
≤ 5% bone marrow blasts after 1 cycle vs 0% of patients after 2 cycles). Finally, 
like Shen et al, they observed a correlation between the extent of methylcytosine 
reduction and response.[28]  This study suggests that improvement in response 
rates and time to response can be achieved by prolonged administration of 
decitabine. Further, that DNA methylcytosine reduction can be achieved with 
prolonged exposure to decitabine and that this might be a patient-specific biological 
marker of adequate dosing. 
  
Scandura et al. recently published a study of infusional decitabine prior to 
cytarabine + daunorubicin induction therapy for AML.[64] They escalated the 
duration of infusional therapy and reached their planned goal of 20 mg/m2/day x 7 
days of continuous therapy given prior to cytarabine and daunorubicin. They noted 
similar toxicities (primarily hematopoietic and the associated neutropenic 
infections), but that these were not different from their experience giving cytarabine 
+ daunorubicin alone. 
 
1.3 
Correlative Studies 
 
With exception of the peripheral blood sample collected on Cycle 1 Day 4, all correlative 
studies will be collected under HRPO# 201011766 (“Tissue Acquisition for Analysis of 
Genetic Progression Factors in Hematologic Diseases”) that all patients will be 
concurrently enrolled in. The goals of the correlative studies include: 
 
1. To correlate patient-specific mutations with response to decitabine. 
We will perform exome sequencing of each patient’s pre-study bone marrow sample 
and skin sample. We will determine AML/MDS-associated variants found in the bone 
marrow, but not skin sample. We will correlate the presence of mutations with response 
(especially mutations in DNMT3A, IDH1, IDH2, and TET2). This study is powered to 
identify mutations with >80% sensitivity to predict decitabine response if used as a 
clinical assay. 
 
2. To correlate the velocity and depth of response with response to decitabine. 
We will assess tumor burden by allele frequency of patient-specific mutations in bone 
marrow samples collected pre study, Cycle 1 Day 10±1, Cycle 1 Day 28±4, and Cycle 
2 Day 28±4. We will assess mutations in at least 24 recurrently mutated genes 
(including, but not limited to: FLT3, DNMT3A, NPM1, TET2, WT1, IDH1, IDH2, 
RUNX1, NRAS, TP53, U2AF1, KRAS, PHF6, MSTP9, PTPN11, KIT, SMC3, PRUNE2, 
ETV6, CEBPA, SMC1A, ASXL1, STAG2, RAD21) as well as all patient-specific 
mutations discovered during exome sequencing. The leukemic allele frequency will be 
measured using the Illumnia MySeq or HiSeq platform using DNA capture by gene 
specific biotinylated-oligonucleotides. Patient bone marrow samples will be assessed 
pre-study, Cycle 1 Day 10 ± 1, Cycle 1 Day 28 ± 4, and Cycle 2 Day 28 ± 4 and 
compared with patient specific skin samples (normal control). The velocity of response 
will be calculated as the natural log rate of patient-specific mutation allele frequency 
clearance. Depth of response will be defined as the lowest patient-specific mutation 


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allele frequency in a bone marrow sample. The velocity and depth of tumor response 
will be correlated to: 
 Achievement of clinical CR, 
 Time to best response, 
 Mutational status, 
 Time to disease progression. 
Because all patient-specific exome-discovered mutations will be assessed a second 
time in the primary bone marrow sample as well as the  Cycle 1 Day 10 ± 1, Cycle 1 
Day 28 ± 4, and Cycle 2 Day 28 ± 4 bone marrow samples, this study will also serve 
to validate all exome-discovered mutations. This is important, because exome 
sequencing is associated with a 20-50% false-discovery rate depending on type I error 
tolerated (mutation-calling algorithms are typically set for greater sensitivity than 
specificity).  Thus, many mutations discovered during exome sequencing are incorrect.  
This second analysis of these mutations will server to exclude these false positive calls. 
 
3. To correlate the bone marrow expression profile from pre-study and Cycle 1 Day 10 ± 
1 with response to decitabine. 
We will correlate expression signatures, and changes in expression signatures, with: 
 Achievement of clinical CR, 
 Mutation status. 
 
4. To correlate steady-state plasma decitabine concentration on Cycle 1 Day 4 ± 1 with: 
 
Achievement of CR. 
 
5. To correlate the extent of bone marrow DNA hypomethylation on Cycle 1 Day 10 ± 1 
with response to decitabine. 
We will assess the percent reduction in bone marrow methylcytosine content using LC-
MS. Patients will be evaluated pre-study and Cycle 1 Day 10 ± 1. The change in DNA 
methylcytosine content will be correlated to: 
 Achievement of clinical CR, 
 Change in expression signature. 
This will determine whether inadequate blast exposure to decitabine correlates with 
drug resistance. 
 


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2.0 
OBJECTIVES 
 
2.1 
Primary Objective 
 
To determine whether patient-specific mutations correlate with clinical overall response to 
decitabine in patients with AML or MDS. 
 
2.2 
Secondary Objectives 
 
- 
To determine whether 10 consecutive days per cycle of decitabine improves outcomes 
compared with historical controls treated with 5 consecutive days per cycle of 
decitabine. 
 
- 
To determine whether other biomarkers correlate with clinical overall response to 
decitabine in patients with AML or MDS. These will include:  
 
1. The velocity and depth of patient-specific mutation clearance; 
2. The bone marrow expression profile and change in expression profile during 
decitabine treatment; 
3. The steady-state plasma decitabine concentrations; 
4. The decrease in bone marrow methylcytosine content in response to decitabine.  
 
 


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3.0 
PATIENT SELECTION 
 
3.1 
Inclusion Criteria 
 
One of the following: 
 Patient must have non-M3 AML and be > 60 years of age OR 
 non-M3 AML with relapsed disease OR 
 Symptomatic MDS with one of the following: 
□ Symptomatic anemia with either hemoglobin < 10.0 g/dL or requiring RBC 
transfusion 
□ Thrombocytopenia with a history of two or more platelet counts < 50,000/mcL 
or a significant hemorrhage requiring platelet transfusions 
□ Neutropenia with two or more ANC < 1,000/mcL 
 
All of the following: 
1. Patient must have an ECOG performance status ≤ 2. 
2. Patient must have >10% disease burden measured by cytomorphology, flow 
cytometry, or cytogenetics. 
3. Patient must have peripheral white blood cell count < 50,000/mcl. 
4. Patient must have adequate organ function, defined as: 
a. 
Total bilirubin < 1.5 x ULN 
b. 
AST/ALT < 2.5 x ULN 
c. 
Serum creatinine < 2.0 x ULN  
5. Patient must have undergone ≤ 2 cycles of prior hypomethylating agent (decitabine 
or azacitidine). 
6. Patient must be enrolled in HRPO# 201011766 (“Tissue Acquisition for Analysis 
of Genetic Progression Factors in Hematologic Diseases”). 
7. Patient must be > 18 years of age. 
8. Patient must be able to understand and willing to sign an IRB-approved written 
informed consent document. 
 
3.2 
Exclusion Criteria 
 
1. Patient must not be pregnant or nursing. 
2. Patient must not have known CNS leukemia.  
3. Patient must not have a history of positive HIV serology.  
4. Patient must not have a history of positive Hepatitis C serology. 
5. Patient must not have undergone a prior allogeneic stem cell transplant. 
6. Patient must not have any uncontrolled intercurrent illness including, but not 
limited to, ongoing or active infection, ongoing or active GVHD, congestive heart 
failure of NYHA class 3 or 4, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situation that would limit compliance with study 
requirements. 
7. Patient must not have had radiation therapy within 14 days of enrollment. 
8. Patient must not have received any chemotherapy within 21 days of enrollment, 
and any acute treatment-related toxicities must have returned to baseline. Patients 


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may be receiving hydrea at time of enrollment.    
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible for this trial. 


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4.0 
REGISTRATION PROCEDURES 
 
Patients must not start any protocol intervention prior to registration through the Siteman 
Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility by Washington University 
2. Registration of patient in the Siteman Cancer Center database 
3. Assignment of unique patient number (UPN) 
 
Once the patient has been entered in the Siteman Cancer Center database, the WUSM coordinator 
will forward verification of enrollment and the UPN via email. 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility collecting the information listed below and scanning and 
emailing it to the research coordinator listed in the Siteman Cancer Center Clinical Trials 
Core Protocol Procedures for Secondary Sites packet at least one business day prior to 
registering patient: 
1. Your name and contact information (telephone number, fax number, and email 
address) 
2. Your site PI’s name, the registering MD’s name, and your institution name 
3. Patient’s race, sex, and DOB 
4. Three letters (or two letters and a dash) for the patient’s initials 
5. Currently approved protocol version date 
6. Copy of signed consent form (patient name may be blacked out) 
7. Planned date of enrollment 
8. Completed eligibility checklist, signed and dated by a member of the study team 
9. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center Database 
 
Registrations may be submitted Monday through Friday between 8am and 5pm CT. Urgent 
late afternoon or early morning enrollments should be planned in advance and coordinated 
with the Washington University research coordinator.  Registration will be confirmed by 
the research coordinator or his/her delegate by email within one business day. Verification 
of eligibility and registration should be kept in the patient chart. 
 
All patients at all sites must be registered through the Siteman Cancer Center database at 
Washington University. 
 
4.3 
Assignment of UPN 
 


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Each patient will be identified with a unique patient number (UPN) for this study.  Patients 
will also be identified by first, middle, and last initials.  If the patient has no middle initial, 
a dash will be used on the case report forms (CRFs).  All data will be recorded with this 
identification number on the appropriate CRFs. 
 


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5.0 
INVESTIGATIONAL PLAN 
 
5.1 
Summary of Study Design 
 
This is an open-label, single-arm, Phase II, biomarker study of patients with AML or MDS 
who are treated with 10 consecutive days of decitabine. FAB-M3 AML is excluded from 
this trial, because other well-tolerated options exist for the treatment of FAB-M3 acute 
promyelocytic leukemia. AML patients who are < 60 years old and have not received 
cytotoxic induction chemotherapy are also excluded, because cytotoxic therapy offers a 
curative possibility for these patients.  MDS patients without cytopenias are also excluded 
because these patients can be followed with watchful-waiting. 
 
All patients will undergo bone marrow biopsies pre-study, on Cycle 1 Day 10 ± 1, and on 
Day 28 ± 4 of Cycles 1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. 
Patients will provide a skin biopsy sample pre-study. Patients will also provide peripheral 
blood samples on Cycle 1 Day 4 ± 1 for pharmacokinetic studies. See Section 8.0 for details 
on correlative studies.  
 
Patients will receive decitabine 20 mg/m2/day as a 1-hour infusion on consecutive Days 1-
10 in 28-day cycles.  The dose will be calculated based on actual patient weight at the 
beginning of each cycle. 
 
Once AML patients achieve morphologic complete remission with incomplete blood count 
recovery (CRi) or MDS patients achieve a marrow complete response (Marrow CR) as 
defined in Section 9.0, decitabine administration may be decreased to 20 mg/m2/day for 
ten consecutive days every 6 weeks, to limit myelosuppressive toxicity and improve count 
recovery. After 2 cycles at 6 week intervals, the dose may be reduce to 20 mg/m2/day for 
five consecutive days in 6 week cycles, at the discretion of the treating physician.  After 
two cycles of five consecutive days of treatment, if the patient meets the definition of CR 
with complete cytogenetic response as defined in Section 9.0, administration may further 
be decreased to three consecutive days per cycle at the discretion of the treating physician. 
 
It will be a logistical goal of this trial consistently to return preliminary exome results to 
the treating physician by the end of Cycle 1 and validated results by the end of Cycle 2. 
However, this logistical goal is not a primary or secondary aim of this study. Given the 
limited data regarding mutation presence and drug response, we do not anticipate that the 
results will directly influence therapeutic decisions made by the treating physician. 
However, the development of comprehensive genomic pipelines and integration of this 
data into a clinical trial setting will be required before personalized medicine can be 
realized. 
 
 
 


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5.2 
Study Procedures 
 
5.2.1 Pre-study Procedures 
 
Pre-study procedures must take place no more than 14 days prior to the first dose 
of decitabine except where noted below. 
 
1. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments performed 
on bone marrow will include correlative studies (collected under HRPO# 
201011766), morphologic assessment, cytogenetics, and FISH.  
2. Bone marrow or peripheral blood for flow cytometry 
3. Skin biopsy sample (collected under HRPO# 201011766). There is no required 
time frame for this sample; it may have been collected months or even years 
prior to the first dose of decitabine.  
 
5.2.2 Baseline Evaluation 
 
Baseline evaluations must take place no more than 7 days prior to the first dose of 
decitabine.  
 
1. Medical history 
2. Physical exam including height, weight, and ECOG performance score. 
3. Record transfusion requirements for 8 weeks before first dose of study drug 
4. CBC with differential and platelets 
5. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
6. Urine βHCG pregnancy test if appropriate 
 
5.2.3 Day 1 of Each Cycle 
 
1. Physical exam, including weight  
2. CBC with differential and platelets 
3. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
 
5.2.4 Cycle 1 Days 1-10 
 
Decitabine administration, 20 mg/m2 per day given as a 1-hour continuous 
intravenous infusion.  The dose will be calculated based on actual patient weight at 
the beginning of each cycle.  Patients may be premedicated with lorazepam, 
prochlorperazine, palonosetron, or ondansetron at the treating physician’s 
discretion. 
 


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5.2.5 Cycle 1 Day 4 ± 1 
 
Obtain 3 mL peripheral blood for correlative studies. Sample must be obtained 45 
minutes ± 15 minutes after beginning the infusion, before the decitabine infusion is 
complete, and should be obtained from the opposite arm as the infusion.  The 
sample should be collected in a 3 mL K3EDTA tube pre-loaded with 8 mcL of 
tetrahydrouridine (THU 500 mcg/mL), an inhibitor of cytidine deaminase.  The 
sample should be processed by the Tissue Procurement Core Facility at the Siteman 
Cancer Center.  Plasma should be separated and stored per protocols. 
  
5.2.6 Cycle 1 Day 10 ± 1 
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH. 
 
5.2.7 Weekly 
 
CBC with differential and platelets. After the first cycle, this may be changed to 
every other week at the discretion of the treating physician. 
 
5.2.8 Every Other Week 
 
Serum chemistries, including bicarbonate, calcium, glucose, total protein, albumin, 
BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, ALT, alkaline 
phosphatase, LDH. After the first cycle, this may be changed to every month at the 
discretion of the treating physician. 
 
5.2.9 Cycles 1, 2, 4 and 6 Day 28 ± 4 days  
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include: correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH.  
 
Patients who achieve with clinical response (CRi or better) and are receiving cycles 
every 6 weeks, will undergo bone marrow biopsies on day 42 ± 4.  
 
5.2.10 End-of-Study Procedures  
 
1. Physical exam 
2. CBC with differential and platelets.  
3. Serum chemistries including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, and LDH.  


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4. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments performed 
on bone marrow will include morphologic assessment, cytogenetics, and FISH.  
 
5.2.11 Post-study Follow-up 
 
Patients are to be followed according to standard patient care procedures for 30 
days following the date of the last decitabine dose to monitor for toxicity, survival, 
and response. Thereafter, the investigator or designee will record information on 
subsequent therapy, disease progression, and/or death every 6 months for 2 years 
after last dose of study drug or until the patient is lost to follow-up or dies. 
 
5.3 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to the 
patient’s health and/or the patient no longer wishes to continue protocol therapy, the 
protocol therapy should be discontinued and the reason(s) for discontinuation documented 
in the case report forms. 
 
In the absence of treatment delays due to adverse events, treatment may continue 
indefinitely or until one of the following criteria applies: 
 
 Documented and confirmed disease progression 
 Death 
 Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
 General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
 Suspected pregnancy 
 Serious non-compliance with the study protocol 
 Lost to follow-up 
 Patient withdraws consent 
 Investigator removes the patient from study 
 The Siteman Cancer Center decides to close the study 
 
Patients who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar. 
 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
 
5.4.1 Chemotherapy 
 
Patients cannot receive any chemotherapy within 21 days of study enrollment and 
must have recovered from any treatment-related toxicities. However, patients may 
be receiving hydrea at enrollment and may continue on hydrea through Cycle 2 of 
decitabine. An indication for hydrea following 2 cycles of decitabine is evidence 


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that the patient is not responding and decitabine should be discontinued. Other than 
decitabine, patients cannot receive chemotherapy during this trial.  
 
5.4.2 Growth Factors 
 
Erythroid growth factors should not be routinely administered to patients during the 
study. 
 
Thrombopoietin receptor agonists should not be routinely administered to patients 
during the study. 
 
Filgrastim (G-CSF) or sargramostim (GM-CSF) may be administered to patients 
with septic shock or febrile neutropenia at the discretion of the treating physician. 
 
5.4.3 Transfusions 
 
 Packed red blood cells (RBCs) transfusion:   
RBC transfusions will be administered at the discretion of the treating 
physician.  
 
 Platelet transfusion:   
Platelet transfusions will be administered at the discretion of the treating 
physician  
5.4.4 Prophylactic Antimicrobial Agents  
 
Antimicrobial agents will be administered at the discretion of the treating physician. 
Common antimicrobial prophylaxis during cycles 1 and 2 include acyclovir, 
ciprofloxacin, and fluconazole. 
 
5.4.5 Radiotherapy 
 
Patients cannot receive radiotherapy within 14 days prior to study enrollment or 
while on study. 
 


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6.0 
DOSE MODIFICATIONS 
 
A criterion for discontinuation of the study is a delay of > 8 weeks between any two cycles.  
 
6.1 
Delay for Cytopenia or Infection 
 
As cytopenias are a defining feature of AML and MDS, their presence alone does not 
generally warrant dose modifications or delays. Patients will receive four cycles of therapy 
without adjustment for any cytopenia. Following Cycle 4, dose modifications or delays for 
cytopenias will be made at the discretion of the investigator.  
 
Patients with febrile neutropenia (temperature ≥ 38.5° with ANC < 1,000/l) or systemic 
infections should have treatment held until clinical resolution of the infection.  
 
6.2 
Delay for Organ Dysfunction 
 
If renal dysfunction (creatinine ≥ 3 X institutional upper limit of normal) or hepatic 
dysfunction (bilirubin ≥ 2.5 mg/dl or ALT/AST ≥ 3 X institutional upper limit of normal) 
occurs, therapy may not resume until the abnormalities have resolved to below the limits 
stated above. If renal or hepatic dysfunction occurs during a treatment course, the next dose 
of decitabine should be held, and serum chemistries should be checked twice per week.  
Decitabine can be resumed at full dose when creatinine, bilirubin, ALT and AST are below 
the limits listed above.  
 
For grade 3-4 non-hematologic toxicities related to decitabine, decitabine treatment should 
be delayed until recovery to baseline or to grade ≤ 1. Decitabine may be reintroduced with 
a 50% dose reduction, or at full dose at the discretion of the investigator. 
 
6.3 
Modification in Dose or Schedule for Treatment Response 
 
Once AML patients achieve morphologic complete remission with incomplete blood count 
recovery (CRi) or MDS patients achieve a marrow complete response (Marrow CR) as 
defined in Section 9.0, decitabine administration may be decreased to 20 mg/m2/day for 
ten consecutive days, and the cycles may be administered every 6 weeks to limit 
myelosuppressive toxicity, at the discretion of the treating physician. After two cycles at 
six week intervals, the dose may be reduced to 20 mg/m2/day for five consecutive days of 
treatment in six week intervals. If the patient meets the definition of CR with complete 
cytogenetic response as defined in Section 9.0, administration may further be decreased to 
three  consecutive days per cycle at the discretion of the treating physician. 
 


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7.0 
PHARMACEUTICAL INFORMATION 
 
7.1 
Decitabine 
 
Decitabine is an antimetabolite that can replace cytosine in DNA but, unlike cytosine, it 
cannot be methylated. The exact mechanism of decitabine activity has not been determined 
and may involve multiple pathways. Decitabine is thought primarily to act by correcting 
DNA methylation, a major mechanism for gene expression. In some cancer cells, 
hypermethylation blocks the activity of tumor suppressor genes, which regulate cell 
division and differentiation to prevent malignant transformation. When suppressor gene 
activity is blocked, cell division becomes unregulated, leading to the formation of 
neoplastic cells. Decitabine regulates DNA methylation (specifically targets the 
methyltransferase, DNMT1), effectively demethylating and reactivating different tumor 
suppressor genes. Because hypermethylation occurs early in the malignant transformation 
of cells, decitabine may have activity in chemoprevention. Other possible mechanisms for 
decitabine activity include cytotoxicity due to incorporation into DNA similar to other 
antimetabolites, methylation-independent induction of gene expression, and degradation of 
DNA methyltransferase 1. 
 
7.1.1 Study Drug Preparation  
 
OSHA Guidelines for handling cytotoxic drugs outlined in the American Journal 
of Hospital Pharmacy must be followed. 
 
Decitabine (5-aza-2’-deoxycytidine) is supplied as a lyophilized powder for 
injection, 50 mg in 20-ml vials. When reconstituted with 10 ml of sterile water for 
injection, each ml will contain 5 mg of decitabine and 6.8 mg of KH2PO4. The 
reconstituted solution should be diluted to 0.1-1.0 mg/mL with pre-chilled 0.9% 
sodium chloride intravenous infusion, which has been cooled to 2-8oC. 
Reconstitution of the powder results in a rapidly decomposing solution. Unless used 
within 15 minutes of reconstitution, the diluted solution must be prepared using 
cold (2—8 degrees C) infusion fluids and stored at 2—8 degrees C (36—46 degrees 
F) for a maximum of 7 hours. 
Decitabine will be administered per dosing schedule every 28 ± 4 days.  
 
Decitabine may be administered either as an inpatient or an outpatient.  
 
7.1.2 Supplier 
 
Decitabine is available commercially. 
 
 
  
 
  
 


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8.0 
CORRELATIVE STUDIES SAMPLE COLLECTIONS 
 
Bone marrow samples will be collected pre-study, on Cycle 1 Day 10 ± 1, and on Day 28 ± 4 of 
Cycle 1, 2, 4, and 6 for correlative studies. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. The bone 
marrow samples will be collected under HRPO# 201011766 (“Tissue Acquisition for Analysis of 
Genetic Progression Factors in Hematologic Diseases”) that all patients will be concurrently 
enrolled in.  
 
Skin biopsy sample will be collected as part of the patient’s participation in HRPO# 201011766 
(“Tissue Acquisition for Analysis of Genetic Progression Factors in Hematologic Malignancies”). 
There is no required time frame for this sample, and it may have been collected months or even 
years prior to the first dose of decitabine.  
 
Peripheral blood sample (3 mL) will be collected on Cycle 1 Day 4 ± 1.  Sample must be obtained 
45 minutes ± 15 minutes after beginning the infusion, before the decitabine infusion is complete, 
and should be obtained from the opposite arm as the infusion.  The sample should be collected in 
a 3 mL K3EDTA tube pre-loaded with 8 mcL of tetrahydrouridine (THU 500 mcg/mL), an 
inhibitor of cytidine deaminase.  The sample should be processed by the Tissue Procurement Core 
Facility at the Siteman Cancer Center.  Plasma should be separated and stored per protocols. 
 
 


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9.0 
EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
 
A secondary endpoint of this study is to estimate morphologic complete remission rate. 
  
9.1 
Response Criteria - AML 
 
AML Patients will be assessed for response according to the IWG criteria:1  
 
Morphologic complete remission (CR) – Defined as morphologic leukemia-free state, 
defined as less than 5% blasts in aspirate with marrow spicules and a count of ≥200 
nucleated cells and no blasts with Auer rods or any persistence of extramedullary disease. 
When erythroid precursors constitute less than 50% of bone marrow nucleated cells, the 
percentage of blasts is based on all nucleated cells; when there are 50% or more erythroid 
cells, the percentage blasts should be based on the nonerythroid cells. The presence of mild 
megaloblastoid changes may be permitted if they are thought to be consistent with 
treatment effect. However, persistence of pretreatment abnormalities (eg, pseudo-Pelger-
Hüet cells, ringed sideroblasts, dysplastic megakaryocytes) are not consistent with CR.  In 
addition, ANC >1000/μl and platelet count of ≥100,000/μl. Patient must be independent of 
transfusions. No duration requirement for this designation. 
 
Cytogenetic complete remission (CCR) – Defined as morphologic complete remission plus 
a reversion to a normal karyotype. For this study, reversion of a normal karyotype is 
defined as no clonal abnormalities detected in a minimum of 20 mitotic cells. 
 
Morphologic complete remission with incomplete blood count recovery (CRi) – Defined as 
morphologic complete recovery with the exception of neutropenia <1000/μl or 
thrombocytopenia <100,000/μl.  
 
Partial remission (PR) – Defined as meeting the above criteria for complete remission, 
with the following exceptions: decrease of ≥50% in the percentage of blast to 5-25% in the 
bone marrow aspirate. A value of 5% blasts in bone marrow with Auer rods present may 
also be considered a partial remission.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, CRi, PR, PD, or 
recurrence/morphologic relapse.   
 
Progressive disease (PD) – Defined as a clear increase in bone marrow blast count, 
typically a greater than 50% increase compared with baseline, or new extramedullary 
disease. Patients with a > 50% increase in peripheral blast count with an increase of the 
total peripheral white blood cell count to > 10,000/l should undergo evaluation by bone 
marrow biopsy to access for progressive disease.  
 
Recurrence/morphologic relapse - Defined as relapse following complete remission is 
defined as reappearance of blasts in the blood or the finding of ≥5% blasts in the bone 
marrow, not attributable to any other cause. New dysplastic changes is considered relapse. 


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If there are no blasts in the peripheral blood and 5-20% blasts in the bone marrow, bone 
marrow biopsy should be repeated in > 1 week to confirm relapse. 
 
9.2 
Response Criteria - MDS 
 
MDS Patients will be assessed for response according to the IWG criteria:69 
 
Complete remission (CR) – Defined as < 5% myeloblasts with normal maturation of all 
cell lines in the bone marrow and peripheral blood values of Hgb > 11g/dL, Platelets > 100 
x 109/L, Neutrophils > 1.0 x 109/L, and 0% blasts. Persistent dysplasia does not exclude 
CR but will be noted.  
 
Marrow Complete Response (Marrow CR) – Defined as < 5% myeloblasts in the bone 
marrow and a decrease by > 50% from pretreatment values, but not meeting the definition 
of CR above.  
 
Partial Remission (PR) – Defined as meeting the definition of CR above with a decrease 
of myeloeblasts in the bone marrow by > 50% from pretreatment values, but absolute 
myeloblasts still >5%.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, Marrow CR, PR, SD, 
PD, or recurrence/morphologic relapse.   
 
Progressive Disease//Relapse (PD) – Defined as ≥ 50% increase in blasts to > 5% blasts 
(for patients with less than 5% blasts at baseline only), ≥ 50% increase to > 10% blasts (for 
patients with 5-10% blasts at baseline only), ≥ 50% increase to > 20% blasts (for patients 
with l0-20% blasts at baseline only), ≥ 50% increase to > 30% blasts (for patients with 20-
30% blasts at baseline only) or any of the following: At least 50% decrement from 
maximum remission/response in granulocytes or platelets, reduction in Hgb by ≥ 2 g/dL, 
or New or worsened transfusion dependence not related to study drug toxicity. Or for 
patients with a CR, Marrow CR, or PR as defined above and subsequently development of 
one of the following: Return to pretreatment bone marrow blast percentage, decrement of 
≥ 50% from maximum remission/response levels in granulocytes or platelets, or reduction 
in Hgb concentration by ≥ 1.5 g/dL or transfusion dependence. 
 
9.2.1 Hematologic Improvement (HI) 
 
 
 
Progressive disease as defined above nullifies hematologic improvement.  
 
Erythroid response requires all of the following (only required if pretreatment HgB 
<11 g/dL): 
 Hgb increase by ≥ 1.5 g/dL 
 Relevant reduction of units of RBC transfusions by an absolute number of at 
least 4 RBC transfusions/8 wk compared with the pretreatment transfusion 
number in the previous 8 wk. Only RBC transfusions given for a Hgb of ≤ 9.0 
g/dL pretreatment will count in the RBC transfusion response evaluation 


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Platelet response requires one of the following (only required if pretreatment 
platelets < 100 x 109/L):  
 Absolute increase of ≥ 30 x 109/L (for patients starting with > 20 x 109/L 
platelets) 
 Increase from < 20 x 109/L to > 20 x 109/L and absolute increase > 100% (for 
patients starting with < 20 x 109/L) 
 
Neutrophil response requires the following (only required if pretreatment ANC 
<1.0 x 109/L):  
 At least 100% increase and an absolute increase > 0.5 x 109/L 
 
9.3.1 Cytogenetic Response  
 
Complete Cytogenetic Response – Defined as reversion to a normal karyotype. For 
this study, reversion of a normal karyotype is defined as no clonal abnormalities 
detected in a minimum of 20 mitotic cells. Progressive disease as defined above 
nullifies cytogenetic response.  
 
9.3 
Guidelines for Evaluation of Disease 
 
Bone marrow biopsy should be performed on Day 28 ± 4 of cycles 1, 2, 4 and 6 for clinical 
disease assessment. Following Cycle 6, bone marrow biopsies should be performed at the 
discretion of the treating physician to assess for relapse or progression. 
 
9.4 
Other Secondary Efficacy Measures 
 
9.4.1 Treatment Failure, Overall Survival and Event-free Survival 
 
Treatment failure is defined as failure to achieve a complete or partial remission 
following 4 cycles of therapy.  
 
Overall survival is defined as the date of first dose of study drug to the date of death 
from any cause.  Patients still alive at the time of the final analysis will be right-
censored. 
 
Event-free survival is defined as the interval from the date of first dose of study 
drug to date of treatment failure, recurrence, death due to any cause, or loss to 
follow up. 
 
9.4.2 Duration of Remission and Relapse-free Survival for Patients Having 
Complete Remission 
 
Duration of complete remission is defined as the interval from the date that 
complete remission is documented to the date of recurrence, death due to any cause, 


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or loss to follow-up.  
 
Relapse-free survival is defined only for those patients achieving a complete 
remission. It is defined as the interval from the date of first documentation of a 
leukemia-free state to the date of recurrence, death due to any cause, or loss to 
follow up.   
  
9.4.3 Safety 
 
Safety evaluation will include assessments of adverse experiences, medical history, 
physical examinations, vital signs, and laboratory assessments at baseline and 
throughout the study period. Local laboratories may be used for this study.  
 


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10.0 
ADVERSE EVENT REPORTING  
 
10.1 
Adverse Events (AEs) 
 
Definition:  any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading:  the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for all 
toxicity reporting.  A copy of the CTCAE version 4.0 can be downloaded from the CTEP 
web site:  http://ctep.cancer.gov/reporting/ctc.html.  Grade 1-2 events will be recorded for 
the first fifty enrolled patients. Subsequently, only events graded as 3 or greater will be 
recorded. Fifty patients should be sufficient to estimate the true incidence of any adverse 
event to within ± 14% with 95% confidence intervals. 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the terms 
listed that should be used are those provided by the Department of Health and Human 
Services’ Office for Human Research Protections (OHRP).  A copy of this guidance can 
be found on OHRP’s website: (http://www.hhs.gov/ohrp/policy/AdvEvntGuid.htm). 
 
10.2 
Unanticipated Problems 
 
Definition: 
 
 unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied; 
 related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the incident, 
experience, or outcome may have been caused by the procedures involved in the 
research); and 
 suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
 
10.3 
Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that govern 
human 
subjects 
research 
or 
failure 
to 
follow 
the 
determinations 
of 
the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate choice to 
ignore regulations, institutional policies, or determinations of the IRB. 
 
10.4 
Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial harm 
to subjects or others, or that materially compromises the rights or welfare of participants. 


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10.5 
Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under the research 
team’s control. Exceptions apply only to a single participant or a singular situation. 
 
Local IRB Pre-approval of all protocol exceptions must be obtained prior to the event.  For 
secondary sites, the Washington University PI will issue approval of the exception, but it 
must also be submitted to the local IRB with documentation of approval forwarded to 
Washington University.  HRPO approval is not required for protocol exceptions occurring 
at secondary sites. 
 
10.6 
Reporting to the Human Research Protection Office (HRPO) and the Quality 
Assurance and Safety Monitoring Committee (QASMC) at Washington 
University: 
 
The PI is required to promptly notify the IRB of the following events: 
 
 Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct of 
the study. 
 Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 
 Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence of the 
event or notification to the PI of the event.  The death of a research participant that qualifies 
as a reportable event should be reported within 1 working day of the occurrence of the 
event or notification to the PI of the event. 
 
10.7 
Reporting Requirements for Secondary Sites 
 
The research team at each secondary site is required to promptly notify the Washington 
University PI and research coordinator of all reportable events (as described in Section 7.6) 
within 1 working day of the occurrence of the event or notification of the secondary site’s 
PI of the event.  This notification may take place via email if there is not yet enough 
information for a formal written report (using either an FDA MedWatch form if required 
or an institutional SAE reporting form if not).  A formal written report must be sent to the 
Washington University PI and research coordinator within 10 working days of the 
occurrence of the event or notification of the secondary site’s PI of the event.  The death 
of a research participant that qualifies as a reportable event should be reported within 1 
working day of the occurrence of the event or notification of the secondary site’s PI of the 
event. 
 


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The research team at a secondary site is responsible for following its site’s guidelines for 
reporting applicable events to its site’s IRB according to its own institutional guidelines. 
 
10.8 
Reporting to Secondary Sites 
 
The Washington University PI (or designee) will notify the research team at each secondary 
site of all reportable events that have occurred at other sites (as described in Section 7.6) 
within 10 working days of the occurrence of the event or notification of the PI of the event.  
This includes events that take place both at Washington University and at other secondary 
sites, if applicable. 
 
10.9 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER 
FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the investigator to report any unanticipated problem to the FDA as 
follows: 
 
 Report any unexpected fatal or life-threatening adverse experiences associated with 
use of the drug (i.e., there is a reasonable possibility that the experience may have 
been caused by the drug) by telephone or fax no later than 7 calendar days after 
initial receipt of the information.  A life-threatening adverse experience is defined 
as any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that, had it occurred in a more severe form, might have caused 
death. 
 Report any serious, unexpected adverse experiences, as well as results from animal 
studies that suggest significant clinical risk within 15 calendar days after initial 
receipt of this information.  A serious adverse drug experience is defined as any 
adverse drug experience occurring at any dose that results in any of the following 
outcomes: 
o Death 
o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial disruption 
of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 
o Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed above 
An unexpected adverse drug experience is defined as any adverse drug experience, 
the specificity or severity of which is not consistent with the current investigator 
brochure (or risk information, if an IB is not required or available). 


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All MedWatch forms will be sent by the investigator or investigator’s team to the FDA at 
the following address or by fax: 
 
Food and Drug Administration 
Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
Secondary sites must submit a completed MedWatch form to the Washington University 
PI and research coordinator within 4 calendar days (for fatal or life-threatening adverse 
experiences) or 11 calendar days (for serious, unexpected adverse experiences).  The 
Washington University PI will be responsible for submitting all MedWatch forms from 
secondary sites to the FDA within the timeframes specified above. 
 
10.10 Timeframe for Reporting Required Events:  
 
Reportable adverse events will be tracked for 30 days following the last day of study 
treatment, or until the start of a new chemotherapy/treatment, whichever comes first. 
 
Deaths 
Any reportable death while on study or within 
30 days of study 
Immediately, within 24 hours, to PI 
and the IRB 
Any reportable death while off study  
Immediately, within 24 hours, to PI 
and the IRB  
Adverse Events/Unanticipated Problems 
Any reportable adverse events as described in 
Sections 10.1 and 10.2 (other than death) and 
10.7 
Immediately, within 24 hours to PI 
and within 10 working days to the 
IRB, and within 7 or 15 days to the 
FDA  
All adverse events are to be reported per 
protocol regardless of attribution should be 
submitted cumulatively  
Include in DSM report 
Noncompliance and Serious Noncompliance 
All noncompliance and serious noncompliance 
as described in Sections 10.3 and 10.4 
Immediately, within 24 hours, to PI 
and within 10 working days to the IRB 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 3 Version date: 11/13/14  
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11.0 
DATA SAFETY MONITORING PLAN 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, the 
Principal Investigator will provide a Data and Safety Monitoring (DSM) report to the Washington 
University Quality Assurance and Safety Monitoring Committee (QASMC) semi-annually 
beginning six months following activation of the first secondary site.  
 
The Principal Investigator will review all patient data at least every six months, and provide a 
semi-annual report to the QASMC. This report will include: 
 HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
 Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of study 
 History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol exceptions, 
error, or breach of confidentiality including start/stop dates and reason 
 Study-wide target accrual and study-wide actual accrual including numbers from 
participating sites 
 Protocol activation date at each participating site 
 Average rate of accrual observed in year 1, year 2, and subsequent years at each 
participating site 
 Expected accrual end date and accrual by site 
 Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
 Measures of efficacy 
 Early stopping rules with supporting data and list the number of participants who have 
met the early stopping rules 
 Summary of toxicities at all participating sites 
 Abstract submissions/publications 
 Summary of any recent literature that may affect the safety or ethics of the study  
 
Until such a time as the first secondary site activates this protocol, a semi-annual DSM report to 
be prepared by the study team will be submitted to the QASM Committee beginning 6 months 
after study activation at Washington University. 
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC according 
to institutional guidelines. 
 


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12.0 
AUDITING 
 
Since Washington University is the coordinating center, each site will be audited annually by 
Siteman Cancer Center personnel (QASMC) unless the outside institution has an auditing 
mechanism in place and can provide a report. The outside sites will be asked to send copies of all 
audit materials, including source documentation.  The audit notification will be sent to the 
Washington University Research Patient Coordinator, who will obtain the audit materials from the 
participating institution. 
 
Notification of an upcoming audit will be sent to the research team one month ahead of the audit. 
Once accrual numbers are confirmed, and approximately 30 days prior to the audit, a list of the 
cases selected for review (up to 10 for each site) will be sent to the research team. However, if 
during the audit the need arises to review cases not initially selected, the research team will be 
asked to provide the additional charts within two working days. 
 
Additional details regarding the Auditing Policies and procedures can be found at 
http://www.siteman.wustl.edu/uploadedFiles/Research_Programs/Clinical_Research_Resources/
Protocol_Review_and_Monitoring_Committee/QASMCQualityAssurcance.pdf  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 3 Version date: 11/13/14  
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13.0 
MULTICENTER REGULATORY REQUIREMENTS 
 
Washington University requires that each participating site sends its informed consent document 
to be reviewed and approved by the Washington University Regulatory Coordinator (or designee) 
prior to IRB/IEC submission.    
 
Each participating institution must have the following documents on file at Washington University 
prior to first subject enrollment: 
 Documentation of IRB approval of the study in the form of a letter or other official 
document from the participating institution’s IRB.  This documentation must show 
which version of the protocol was approved by the IRB. 
 Documentation of IRB approval of an informed consent form. The consent must 
include a statement that data will be shared with Washington University, including the 
Quality Assurance and Safety Monitoring Committee (QASMC), the DSMC (if 
applicable), and the Washington University study team. 
 Documentation of FWA, signed FDA Form 1572, and signed and dated CVs of all 
participating investigators. 
 Documentation of training in protection of human subjects by all participating 
investigators. 
 Protocol signature page signed and dated by the investigator at each participating site. 
 
The Principal Investigator is responsible for disseminating to the participating sites all study 
updates, amendments, reportable adverse events, etc.  There will be one current version of the 
protocol document at any given time and each participating institution will utilize that document.  
Protocol/consent modifications and IB updates will be forwarded electronically to the secondary 
sites within 2 weeks of obtaining Washington University IRB approval with acknowledgement of 
receipt requested.  Secondary sites are to submit protocol/consent/IB modifications to their local 
IRBs within 4 weeks of receipt, and confirmation of submission must be forwarded to the 
appropriate contact person on the Washington University study team at the time of submission.  
Upon the secondary sites obtaining local IRB approval, documentation of such shall be sent to the 
Washington University study team within 2 weeks of receipt of approval. 
 
Documentation of participating sites’ IRB approval of annual continuing reviews, protocol 
amendments or revisions, all SAE reports, and all protocol violations/deviations/exceptions must 
be kept on file at Washington University. 
 
The investigator or a designee from each institution must participate in a regular conference call 
to update and inform regarding the progress of the trial. 
 
 


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14.0 
STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
 
14.1 
Analysis Populations 
 
Intent-to-Treat - The intent-to-treat (ITT) population will be defined as all patients who 
receive at least one dose of decitabine.  
 
Efficacy-Evaluable - The efficacy-evaluable (EE) population will be defined as all patients 
who receive at least one cycle of treatment.   
 
Safety - The safety population will be defined as all patients with at least one dose of 
decitabine. 
 
14.2 
Statistical Analysis 
 
14.2.1 Descriptive Analyses 
 
Patient Disposition: The number of patients discontinued, the reasons for 
discontinuation, and the number of cycles administered will be summarized. 
 
Demographics and Baseline Characteristics: Descriptive summary statistics will 
be provided for demographic and important baseline characteristics.  For 
continuous variables, the number of patients, mean, standard deviation, median, 
minimum and maximum will be provided.  For categorical variables, the number 
and percent of patients in each demographic/characteristic category will be 
summarized. 
 
14.2.2 Primary Endpoint Analysis 
 
The primary end-point of this phase II study is the correlation biomarkers with 
overall response in AML/MDS patients to decitabine. 
 
We will correlate the presence of patient specific mutations (especially mutations 
in DNMT3A, IDH1, IDH2, and TET2) with overall response rates. Mutation rates 
in these genes and in other patient-specific genes will be correlated with overall 
response and their respective 95% confidence intervals will be provided. 
 
Statistical considerations. The relationship between AML/MDS-associated 
mutations and the responsiveness to decitabine will be described by contingency 
tables and assessed using Chi-square test or Fisher’s exact test as appropriate. The 
differentially expressed genes between responders and non-responders will be 
determined using the significance analysis of microarray (SAM). Multivariate 
logistic regression will also be fit to adjust the potential confounding effects of other 
demographic and clinical characteristics. The resultant critical P values will be 
corrected for multiple testing.  Since the Bonferroni correction is known to be too 
conservative, a permutation analysis (e.g., 10,000 random assignments for 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 3 Version date: 11/13/14  
Page 40 of 50 
responsive status) will be used to derive an empirical critical P value.  The new 
critical p-value is the number of permutation tests which yield a lower p-value than 
the original test divided by the total number of permutation tests.[61]   
 
A power analysis was performed using Chi-square test for the association between 
mutation and response to decitabine. The frequency of DNMT3A, IDH1/2, and 
TET2 mutations in AML and MDS patients are each between 15% and 30% in 
multiple cohorts.[29-37] Table 3 shows the minimal detectable differences of 
response rates between mutant and wild-type cases, assuming an expected overall 
45% CR/CRi rate and 80% power. For an average of 20% frequency of a mutated 
gene, for example, 100 patients will provide 80% power to detect 30% difference 
at a 2-sided 0.1 alpha error. The purpose of this study is to determine whether any 
of these mutations result in a 
clinically 
significant 
association 
that 
would 
warrant pre-treatment testing. 
We believe that a reasonable 
cut off would be if at least 
30% of clinical response 
could be attributed to the 
presence 
of 
a 
specific 
mutation. 
However, 
we 
anticipate that more power 
could be achieved as we 
assess patient groups with 
combined characteristics (e.g. 
DNMT3A mutation or loss of 
DNMT3A 
expression). 
In 
addition, we expect that the 
majority of the enrolled 125 
patients can be used in the analysis (since all of our molecular information will be 
acquired within 30 days of enrollment, we anticipate a drop-out rate of 20% or less). 
 
14.2.3 Secondary Endpoint Analyses 
 
The overall response rate (CR/CRi/PR) and complete response rate (CR/CRi) will 
be compared with historical controls[2], and the 95% confidence intervals will be 
provided. 
 
The rate of patient specific mutation clearance after 10, 28, and 56 days of therapy 
will be determined using an Illumina based platform. We will compare the rate of 
mutation clearance between the patients who achieve a CR/CRi after 4 cycles and 
those who do not. We anticipate that we will identify between 6 and 25 exonic 
SNVs per case (the range obtained with >200 AML samples sequenced to date with 
whole genome or exome sequencing), and that the expected 95% confidence 
interval of the mean variant allele frequency will be ±2.56% and ±1.25%, 
Table 3. Detectable mutation associated response rates (80% power, α = 0.05) 
 
Total 
N 
% of patients 
with a given 
mutation  
Detectable 
response 
difference 
Sensitivity of mutation to 
predict response at minimum 
detectable response difference* 
100 
15%  
20%  
30% 
40% 
50% 
0.40 
0.35 
0.30 
0.27 
0.26 
80% 
75% 
67% 
61% 
58% 
* Assuming the overall response  rate (CR/Cri) of 45%   


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 3 Version date: 11/13/14  
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respectively, if we assume an anticipated standard deviation of 3.2% within the 
same clone. The change of tumor burden over time and its association with clinical 
response will  be assessed using 2-way ANOVA for repeated measurement data, 
followed by the ad-hoc multiple comparisons for between-group differences (e.g., 
responders vs. non-responders) at given time points as well as the between-time 
differences within each group. The resultant critical P values will also be corrected 
using permutation analysis for multiple testing. 
 
The steady-state plasma decitabine concentration on day 4 ± 1 will be measured 
and correlated with clinical overall response. 
 
The percent decrease in bone marrow methylcytosine will be calculated on day 
10±1. We will compare this reduction with previously published data and between 
the patients who achieve CR/CRi and those who do not. The correlation of peak 
plasma levels of decitabine with response will be assessed using 2-sample t-test or 
Mann-Whitney non-parametric rank-sum test as appropriate. The change of total 
bone marrow DNA methylcytosine from baseline and its association with both 
steady-state plasma drug levels and response will be assessed using 2-way ANOVA 
for repeated measurement data, followed by the ad-hoc multiple comparisons for 
between-group differences (e.g., responders vs. non-responders) and between-time 
differences. Similar analyses will be performed for the correlation of expression 
profiles and SNP analysis with pharmacokinetic data.   
 
Statistical power justification.  A power analysis was performed using two-sample 
t-test for between-group difference and paired t-test for the change over time. For 
tumor clearance, this will provide > 99% power to detect a 10% difference in the 
means at two separate time points for clones with 6 or more variants, and > 98% 
power to detect a 15% difference if there are 3 or more variants per clone with α = 
0.05 (power will decrease to 91% and 82%, respectively, in cases where the 
standard deviation is 4.7%, the maximum we have observed thus far). For 
pharmacologic correlations, we anticipate 85% power to determine a 25 ng/ml 
difference between the average Cmax of 45 evaluable responders and 55 evaluable 
non-responders with an anticipated average Cmax of 70 ng/ml and a standard 
deviation of 40 ng/ml within the entire cohort, and with α = 0.05.[22] We believe a 
less significant difference would not be sufficient to warrant application in clinical 
decision making (i.e. whether to continue therapy for more than 1 cycle). We 
anticipate 99% power to determine a difference of 20% in the reduction of bone 
marrow methylcytosine between 45 evaluable responders and 55 evaluable non-
responders with an expected standard deviation of 15% and α = 0.05.[28, 66-68] 
The difference in methylcytosine reduction previously observed was 30% between 
patients achieving CR/PR and those with only hematologic improvement.[28]  
 
Time to relapse and time to death will also be listed for each patient. 
 
14.2.4 Safety Analysis 
 


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Extent of Exposure – Duration (number of cycles/infusions) of decitabine doses 
(cumulative and intensity) will be summarized. 
 
Adverse Events - All adverse events will be coded using NCI CTCAE v. 4.0.  The 
incidence of treatment-emergent adverse events (number and percent of patients 
reporting the adverse event at least once during the study) will be summarized.  In 
addition, adverse events will be summarized by investigator attribution of 
relationship to study medication and by grade.  Similar summaries will be provided 
by treatment cycle. 
 
Clinical Laboratory Evaluation – Applicable laboratory parameters will be graded 
according to NCI CTCAE version 4.0. The incidence of maximum grade (number 
and percent of patients experiencing the maximum grade during the study) will be 
summarized for each laboratory parameter.  Similar summaries will be provided by 
treatment cycle.  Graphical displays of select hematological parameters will also be 
provided. 
 
Vital Signs, Physical Findings, and Other Safety Observations – Vital signs, 
findings from physical examinations, ECOG scores and weights will be presented 
in listing format. 
 
14.2.5 Description of Planned Subgroup Analyses 
 
The following subgroups (if sufficient numbers of patients) will be explored with 
respect to the primary and overall survival endpoints. 
 Age (60-69 years old vs ≥ 70 years old) 
 Cytogenetics 
 Patients with AML-associated mutations in DNMT3A, TET2, IDH1, or 
IDH2 vs those who do not have somatic variants in these genes. 
 
14.3 
Sample Size 
 
We anticipate initially enrolling 25 patients at Washington University. Once we establish 
a routine exome analysis with validation and calculation of the rate of patient-specific 
mutation clearance, we will enroll an additional 100 patients from at least 2 additional 
centers.  
 


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15.0 
DATA SUBMISSION SCHEDULE 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to registration 
Patient Registration Form 
Disease Diagnosis Form 
Inclusion Criteria Worksheet 
Exclusion Criteria Worksheet 
 
Prior to starting treatment 
Treatment Record Form 
To be maintained throughout a patient’s treatment 
cycle.  The complete record is due at completion of 
treatment. 
Bone Marrow Sample Collection 
Form 
To be maintained throughout the course of the 
study at designated bone marrow sample 
collections time points (See Appendix 2). 
Pathology: Bone Marrow Biopsy 
To be completed upon the return of pathology 
report for each bone marrow biopsy. 
Study Calendar Form 
To be completed with the date of each time point 
upon occurrence. 
Labs Form 
To be completed upon the return of laboratory 
reports for each study time point (See Appendix 2) 
Dose Modifications 
To be maintained throughout the course of the 
study if dose modifications are performed. 
Therapy Response Form 
To be filled out upon remission (prior to, and/or on 
therapy) 
End of Study Form 
 
Due at the completion of the EOS visit, at the time 
of death, and/or at the time that the patient is lost to 
follow-up 
Follow Up Form 
 30 days post date of last decitabine dose. 
 Every 6 months for 2 years after the last dose of 
study drug. 
Record of Adverse Events 
At the time of any AE 
 
Any queries generated by Washington University must be responded to within 28 days of receipt 
by the participating site.  The Washington University research team will conduct a regular review 
of data status at all secondary sites, with appropriate corrective action to be requested as needed. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 3 Version date: 11/13/14  
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Appendix 1 
ECOG Performance Scale 
Activity Status 
Description 
0 
Asymptomatic, fully active, and able to carry on all predisease 
performance without restrictions. 
 
1 
Symptomatic, fully ambulatory but restricted in physically strenuous 
activity and able to carry out performance of a light or sedentary 
nature, eg, light housework, office work. 
 
2 
Symptomatic, ambulatory and capable of all self-care but unable to 
carry out any work activities.  Up and about more than 50% of 
waking hours: in bed less than 50% of day. 
 
3 
Symptomatic, capable of only limited self-care, confined to bed or 
chair more than 50% of waking hours, but not bedridden. 
 
4 
Completely disabled.  Cannot carry on any self-care.   Totally 
bedridden. 
 
5 
Dead 
 
 


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Appendix 2 
Study Calendar 
Study Calendar 
Test 
Pre-
study1 
Base-
line2 
All 
Cycles 
Day 1 
All Cycles 
Days 1-103 
Cycle 1 Day 
4 ± 1 
 
Cycle 1 
Day 10 ± 1 
and 28 ± 4 
Every 
week 
Every 
2 
weeks 
Even 
cycles 
Day 28±4 
End of 
Study 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Medical History 
 
X 
 
 
 
 
 
 
 
 
Physical Exam 
 
Xa 
Xb 
 
 
 
 
 
 
X 
Transfusion 
Requirementsc 
 
Xc 
Ongoing 
Bone Marrow 
Aspirate and Biopsy 
Xd 
 
 
 
 
Xd 
 
 
Xd 
Xd 
Flow Cytometrye 
X 
 
 
 
 
 
 
 
 
 
Peripheral blood for 
correlative studies 
 
 
 
 
X 
 
 
 
 
 
Pregnancy test 
 
X 
 
 
 
 
 
 
 
 
CBC with differential 
and platelets 
 
X 
X 
 
 
 
Xf 
 
 
X 
Serum Chemistriesg 
 
X 
X 
 
 
 
 
Xf 
 
X 
Decitabine 
administrationh 
 
 
 
X 
 
 
 
 
 
 
Adverse Events  
 
 
Ongoing 
Post-study follow-up 
 
 
 
 
 
 
 
 
 
Xi 
1 To be performed within 14 days of the first dose of decitabine.  
2 To be performed within 7 days of the first dose of decitabine.  
3 Decitabine may be reduced to Days 1-5 or Days 1-3. See Section 6.3 for details.  
a To include height, weight, and ECOG. 
b To include weight. 
c Care should be given to record the frequency of transfusions from 8 weeks prior to first dose of study drug through 
the duration of the study. 
d Bone marrow biopsy and aspirate should be performed at baseline, at Cycle 1 Day 10 ± 1, and at  Day 28 ± 4 of Cycles 
1, 2, 4 and 6 for clinical disease assessment. Patients who achieve with clinical response (CRi or better) and are receiving 
cycles every 6 weeks, will undergo bone marrow biopsies on day 42 ± 4. Following Cycle 6, bone marrow biopsies 
should be performed at the discretion of the treating physician to assess for relapse or progression. Standard bone marrow 
procurement procedures will be followed for collection of the tissue. To include: correlative studies (collected under 
HRPO# 201011766), morphologic assessment, cytogenetics and FISH at all time points. A skin biopsy sample (collected 
under HRPO# 201011766) is required at pre-study, but may have been collected months or even years prior to first dose 
of decitabine.  
e On bone marrow or peripheral blood.  
f After Cycle 1, these labs may be obtained every other week (CBC) or once a month (Chemistries) at the discretion of 
the treating physician. 
g Serum chemistries include bicarbonate, calcium, glucose, total protein, albumin, BUN, potassium, chloride, sodium, 
creatinine, total bilirubin, AST, ALT, alkaline phosphatase, and LDH. 
h Decitabine treatment will be 20 mg/m2 over a 1-hour IV infusion on consecutive Days 1-10 every 28-day cycle.   The 
dose will be calculated based on actual patient weight at the beginning of each cycle. Patients may be premedicated 
with lorazepam, prochlorperazine, palonosetron, or ondansetron at the treating physician’s discretion. 
i Patients will be followed for  30 days following the date of the last decitabine dose to monitor for toxicity, survival 
and response. Thereafter, the investigator or designee will record information on subsequent therapy, disease 
progression, and death, every 6 months for 2 years after first dose of study drug or until the patient is lost to follow-up.


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myelofibrosis. Leukemia, 2010. 24(7): p. 1302-9. 
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Ley, T.J., et al., DNMT3A mutations in acute myeloid leukemia. N Engl J Med, 
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31. 
Mardis, E.R., et al., Recurring mutations found by sequencing an acute myeloid 
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Genomic Predictors of Decitabine Response in AML/MDS 
 
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32. 
Abdel-Wahab, O., J. Patel, and R.L. Levine, Clinical Implications of Novel 
Mutations in Epigenetic Modifiers in AML. Hematol Oncol Clin North Am, 2011. 
25(6): p. 1119-33. 
33. 
Reuther, G.W., Recurring mutations in myeloproliferative neoplasms alter 
epigenetic regulation of gene expression. Am J Cancer Res, 2011. 1(6): p. 752-62. 
34. 
Paschka, P., et al., IDH1 and IDH2 mutations are frequent genetic alterations in 
acute myeloid leukemia and confer adverse prognosis in cytogenetically normal 
acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem 
duplication. J Clin Oncol, 2010. 28(22): p. 3636-43. 
35. 
Shen, Y., et al., Gene mutation patterns and their prognostic impact in a cohort of 
1185 patients with acute myeloid leukemia. Blood, 2011. 118(20): p. 5593-603. 
36. 
Tefferi, A., et al., Detection of mutant TET2 in myeloid malignancies other than 
myeloproliferative neoplasms: CMML, MDS, MDS/MPN and AML. Leukemia, 
2009. 23(7): p. 1343-5. 
37. 
Walter, M.J., et al., Recurrent DNMT3A mutations in patients with 
myelodysplastic syndromes. Leukemia, 2011. 25(7): p. 1153-8. 
38. 
Lin, J., et al., Recurrent DNMT3A R882 Mutations in Chinese Patients with Acute 
Myeloid Leukemia and Myelodysplastic Syndrome. PLoS One, 2011. 6(10): p. 
e26906. 
39. 
Bejar, R., et al., Clinical effect of point mutations in myelodysplastic syndromes. 
N Engl J Med, 2011. 364(26): p. 2496-506. 
40. 
Walter, M.J., et al., Clonal architecture of secondary acute myeloid leukemia. N 
Engl J Med, 2012. 366(12): p. 1090-8. 
41. 
Kantarjian, H.M., et al., Survival advantage with decitabine versus intensive 
chemotherapy in patients with higher risk myelodysplastic syndrome: comparison 
with historical experience. Cancer, 2007. 109(6): p. 1133-7. 
42. 
Lubbert, M., et al., Continued Low-Dose Decitabine (DAC) Is an Active First-
Line Treatment in All Cytogenetic Subgroups of Older AML Patients: Results of 
the FR00331 Multicenter Phase II Study. ASH Annual Meeting Abstracts, 2007. 
110(11): p. 300-. 
43. 
Blum, W., et al., Phase I Study of Decitabine Alone or in Combination With 
Valproic Acid in Acute Myeloid Leukemia. J Clin Oncol, 2007. 25(25): p. 3884-
3891. 
44. 
Issa, J.-P.J., et al., Phase 1 study of low-dose prolonged exposure schedules of the 
hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in hematopoietic 
malignancies. Blood, 2004. 103(5): p. 1635-1640. 
45. 
Garcia-Manero, G., et al., Phase 1/2 study of the combination of 5-aza-2'-
deoxycytidine with valproic acid in patients with leukemia. Blood, 2006. 108(10): 
p. 3271-3279. 
46. 
Issa, J.P., S.B. Baylin, and J.G. Herman, DNA methylation changes in 
hematologic malignancies: biologic and clinical implications. Leukemia, 1997. 
11 Suppl 1: p. S7-11. 
47. 
Ganetzky, R.D., et al., Differences Between Normal and Leukemic Stem Cell-
Specific Methylome Indicates Aberrantly Silenced Genes Involved in the 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 3 Version date: 11/13/14  
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Pathogenesis of Malignant Evolution. ASH Annual Meeting Abstracts, 2008. 
112(11): p. 599-. 
48. 
Christman, J.K., 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA 
methylation: mechanistic studies and their implications for cancer therapy. 
Oncogene, 2002. 21(35): p. 5483-95. 
49. 
Lubbert, M., DNA methylation inhibitors in the treatment of leukemias, 
myelodysplastic syndromes and hemoglobinopathies: clinical results and possible 
mechanisms of action. Curr Top Microbiol Immunol, 2000. 249: p. 135-64. 
50. 
Kantarjian, H., et al., Results of a randomized study of 3 schedules of low-dose 
decitabine in higher-risk myelodysplastic syndrome and chronic myelomonocytic 
leukemia. Blood, 2007. 109(1): p. 52-57. 
51. 
Steensma, D.P., et al., Multicenter Study of Decitabine Administered Daily for 5 
Days Every 4 Weeks to Adults With Myelodysplastic Syndromes: The Alternative 
Dosing for Outpatient Treatment (ADOPT) Trial. J Clin Oncol, 2009: p. 
JCO.2008.19.6550. 
52. 
Cashen, A.F., et al., Preliminary Results of a Multicenter Phase II Trial of 5-Day 
Decitabine as Front-Line Therapy for Elderly Patients with Acute Myeloid 
Leukemia (AML). ASH Annual Meeting Abstracts, 2008. 112(11): p. 560-. 
53. 
Jabbour, E., et al., Evolution of decitabine development: accomplishments, 
ongoing investigations, and future strategies. Cancer, 2008. 112(11): p. 2341-51. 
54. 
Kantarjian, H., et al., Results of a randomized study of 3 schedules of low-dose 
decitabine in higher-risk myelodysplastic syndrome and chronic myelomonocytic 
leukemia. Blood, 2007. 109(1): p. 52-7. 
55. 
Blum, W.G., Klisovic, R., Liu, S., Kefauver, C., Grever, M. R., Schaaf, L., Chan, 
K., Byrd, J. C., Villalona-Calero, M., Marcucci,  G. Efficacy of a novel schedule 
of decitabine in previously untreated AML, age 60 or older. in ASCO Annual 
Meeting. 2009. 
56. 
van Groeningen, C.J., et al., Phase I and pharmacokinetic study of 5-aza-2'-
deoxycytidine (NSC 127716) in cancer patients. Cancer Res, 1986. 46(9): p. 
4831-6. 
57. 
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leukemia. Leuk Res, 1981. 5(6): p. 453-62. 
58. 
Lin, K.T., R.L. Momparler, and G.E. Rivard, Sample preparation for the 
determination of 5-aza-2'-deoxycytidine in plasma by high-performance liquid 
chromatography. J Chromatogr, 1985. 345(1): p. 162-7. 
59. 
Momparler, R.L., G.E. Rivard, and M. Gyger, Clinical trial on 5-aza-2'-
deoxycytidine in patients with acute leukemia. Pharmacol Ther, 1985. 30(3): p. 
277-86. 
60. 
Raska, K., Jurovcik, M., Sormova., Z., On the metabolism of 5-azacytidine and 5-
aza-2'-deoxycytidine in mice. Coll Czech Chem Commun, 1965. 30: p. 3001-
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61. 
Momparler, R.L., et al., Pilot phase I-II study on 5-aza-2'-deoxycytidine 
(Decitabine) in patients with metastatic lung cancer. Anticancer Drugs, 1997. 
8(4): p. 358-68. 


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62. 
Aparicio, A., et al., Phase I trial of continuous infusion 5-aza-2'-deoxycytidine. 
Cancer Chemother Pharmacol, 2003. 51(3): p. 231-9. 
63. 
Bryan, J., et al., Pharmacokinetic evaluation of decitabine for the treatment of 
leukemia. Expert Opin Drug Metab Toxicol, 2011. 7(5): p. 661-72. 
64. 
Scandura, J.M., et al., Phase 1 study of epigenetic priming with decitabine prior 
to standard induction chemotherapy for patients with AML. Blood, 2011. 118(6): 
p. 1472-80. 
65. 
Westfall, P.H. and S.A. Young, Resampling-based multiple testing: examples and 
methods for p-value adjustment. 1993, New York: John Wiley & Sons, Inc. 
66. 
Samlowski, W.E., et al., Evaluation of a 7-day continuous intravenous infusion of 
decitabine: inhibition of promoter-specific and global genomic DNA methylation. 
J Clin Oncol, 2005. 23(17): p. 3897-905. 
67. 
Blum, W., et al., Phase I study of decitabine alone or in combination with 
valproic acid in acute myeloid leukemia. J Clin Oncol, 2007. 25(25): p. 3884-91. 
68. 
Yang, A.S., et al., DNA methylation changes after 5-aza-2'-deoxycytidine therapy 
in patients with leukemia. Cancer Res, 2006. 66(10): p. 5495-503. 
69.       Cheson, B.D., et al., Clinical application and proposal for modification of the 
International Working Group (IWG) response criteria in myelodysplasia. Blood, 
2006. 108(2): p. 419-25. 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
 
NCT# NCT01687400 
 
 
Document Version Date:  09/11/2014 
 
IRB Approval Date:  10/08/2014 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
Page 1 of 51 
 
 
Genomic Predictors of Decitabine Response in AML/MDS 
 
Washington University School of Medicine 
Division of Oncology 
660 South Euclid Avenue, Box 8056 
St. Louis, MO 63110 
 
Protocol #: 201210102 
Protocol Version Date: 09/11/14 
 
Coordinating Center: Washington University School of Medicine 
 
Principal Investigator 
John Welch, M.D./Ph.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-2626, Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Sub-Investigator 
Timothy Ley, M.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-8832, Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Pathology 
Jeffery M. Klco, M.D., Ph.D. 
Department of Anatomic and Molecular Pathology 
Telephone: (314) 362-8832 
Email: klcoj@wustl.edu 
 
Statistician 
Feng Gao, Ph.D. 
Division of Biostatistics 
Telephone: (314) 362-3682 
E-mail: feng@wubios.wustl.edu 
 
IND# 116548 
Clinical Trials.gov # NCT01687400 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to obtain 
informed consent, may not be disclosed to another party unless law or regulations require such disclosure.  Persons 
to whom the information is disclosed must be informed that the information is confidential and may not be further 
disclosed by them. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
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Genomic Predictors of Decitabine Response in AML/MDS  
Principal Investigator Signature Page 
 
 
Principal Investigator: 
 
John Welch, M.D., Ph.D. 
 
 
 
 
 
Signature of Investigator 
 
Date 
 
 
 
Printed Name of Investigator 
 
 
 
By my signature, I agree to personally supervise the 
conduct of this study and to ensure its conduct in 
compliance with the protocol, informed consent, 
IRB/HRPO procedures, the Declaration of Helsinki, ICH 
Good Clinical Practices guidelines, and the applicable 
parts of the United States Code of Federal Regulations or 
local regulations governing the conduct of clinical 
studies. 
 
 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
Page 3 of 51 
 
Study Schema: 
 
 
Patients with progressive disease after two cycles (defined as a clear increase in bone marrow blast count, typically a 
> 50% increase in bone marrow blasts over baseline) should be removed from study. 
Patients with stable disease may continue on study and should undergo a diagnostic bone marrow biopsy at the end 
of even numbered cycles. 
Once AML patients achieve morphologic complete remission with incomplete blood count recovery (CRi) or MDS 
patients achieve a marrow complete response (Marrow CR) as defined in Section 9.0, decitabine 
administration may be decreased to 20 mg/m2/day for ten consecutive days every 6 weeks, to limit 
myelosuppressive toxicity and improve count recovery. After 2 cycles at 6 week intervals, the dose may be 
reduce to 20 mg/m2/day for five consecutive days in 6 week cycles, at the discretion of the treating 
physcian. After two cycles of five consecutive days of treatment, if the patient meets the definition of CR 
with complete cytogenetic response as defined in Section 9.0, administration may further be decreased to 
three consecutive days per cycle at the discretion of the treating physician. 
Sample collection for correlative studies: 
Bone marrow biopsies should be performed at baseline, at Cycle 1 Day 10 ± 1, and at Day 28 ± 4 of Cycles 
1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be performed at the discretion of the 
treating physician to assess for relapse or progression.  
Peripheral blood samples should be collected on Day 4± 1 of Cycle 1. Collection should be obtained 45 ± 
15 minutes after decitabine infusion is initiated.
20 
mg/m
2/day  
Cycle 1 
20 
mg/m
2/day 
Cycle 2+ 
Day 1 
Day 28 
BM Bx 
PB 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
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Genomic Predictors of Decitabine Response in AML/MDS 
Table of Contents 
Section 
Page 
Section 4.21.0 ........................................................................... INTRODUCTION 
7 
1.1 
Rationale ............................................................................................ 7 
1.2 
Decitabine and Hypomethylating agents in AML/MDS ................... 9 
1.2.1 
Pharmacokinetics and Metabolism of Decitabine ................... 11 
1.2.2 
Experience with extended dosing schedules ........................... 12 
1.3 
Correlative Studies .......................................................................... 13 
2.0 OBJECTIVES ....................................................................................... 15 
2.1 
Primary Objective ............................................................................ 15 
2.2 
Secondary Objectives ...................................................................... 15 
3.0 PATIENT SELECTION ....................................................................... 16 
3.1 
Inclusion Criteria ............................................................................. 16 
3.2 
Exclusion Criteria ............................................................................ 16 
3.3 
Inclusion of Women and Minorities ................................................ 17 
4.0 REGISTRATION PROCEDURES ...................................................... 18 
4.1 
Confirmation of Patient Eligibility .................................................. 18 
4.2 
Patient Registration in the Siteman Cancer Center Database .......... 18 
4.3 
Assignment of UPN ......................................................................... 18 
5.0 INVESTIGATIONAL PLAN .............................................................. 20 
5.1 
Summary of Study Design............................................................... 20 
5.2 
Study Procedures ............................................................................. 20 
5.2.1 
Pre-study Procedures ............................................................... 20 
5.2.2 
Baseline Evaluation ................................................................. 21 
5.2.3 
Day 1 of Each Cycle ............................................................... 21 
5.2.4 
Cycle 1 Days 1-10 ................................................................... 21 
5.2.5 
Cycle 1 Day 4 ± 1 ................................................................... 21 
5.2.6 
Cycle 1 Day 10 ± 1 ................................................................. 22 
5.2.7 
Weekly .................................................................................... 22 
5.2.8 
Every Other Week ................................................................... 22 
5.2.9 
Cycles 1, 2, 4 and 6Day 28 ± 4 days ....................................... 22 
5.2.10 
End-of-Study Procedures ........................................................ 22 
5.2.11 
Post-study Follow-up .............................................................. 23 
5.3 
Duration of Therapy ........................................................................ 23 


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5.4 
Concomitant Therapy and Supportive Care Guidelines .................. 23 
5.4.1 
Chemotherapy ......................................................................... 23 
5.4.2 
Growth Factors ........................................................................ 24 
5.4.3 
Transfusions ............................................................................ 24 
5.4.4 
Prophylactic Antimicrobial Agents ......................................... 24 
5.4.5 
Radiotherapy ........................................................................... 24 
6.0 DOSE MODIFICATIONS ................................................................... 25 
6.1 
Delay for Cytopenia or Infection ..................................................... 25 
6.2 
Delay for Organ Dysfunction .......................................................... 25 
6.3 
Modification in Dose or Schedule for Treatment Response ........... 25 
7.0 PHARMACEUTICAL INFORMATION ............................................ 26 
7.1 
Decitabine ........................................................................................ 26 
7.1.1 
Study Drug Preparation ........................................................... 26 
7.1.2 
Supplier ................................................................................... 26 
7.1.3 
Toxicities ................................................................................. 26 
8.0 CORRELATIVE STUDIES SAMPLE COLLECTIONS .................... 28 
9.0 EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES29 
9.1 
Response Criteria - AML ................................................................ 29 
9.2 
Response Criteria - MDS................................................................. 30 
9.2.1 
Hematologic Improvement (HI) .............................................. 30 
9.3.1 Cytogenetic Response ....................................................................... 31 
9.3 
Guidelines for Evaluation of Disease .............................................. 31 
9.4 
Other Secondary Efficacy Measures ............................................... 31 
9.4.1 
Treatment Failure, Overall Survival and Event-free Survival 31 
9.4.2 
Duration of Remission and Relapse-free Survival for 
Patients Having Complete Remission ..................................... 31 
9.4.3 
Safety ...................................................................................... 32 
10.0 ADVERSE EVENT REPORTING ...................................................... 33 
10.1 Adverse Events (AEs) ..................................................................... 33 
10.2 Unanticipated Problems................................................................... 33 
10.3 Noncompliance ................................................................................ 33 
10.4 Serious Noncompliance ................................................................... 33 
10.5 Protocol Exceptions ......................................................................... 34 
10.6 Reporting to the Human Research Protection Office (HRPO) and 
the Quality Assurance and Safety Monitoring Committee 
(QASMC) at Washington University: ............................................. 34 
10.7 Reporting Requirements for Secondary Sites .................................. 34 
10.8 Reporting to Secondary Sites .......................................................... 35 


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10.9 Reporting to the FDA ...................................................................... 35 
10.10 Timeframe for Reporting Required Events: .................................... 36 
11.0 DATA SAFETY MONITORING PLAN ............................................. 37 
12.0 AUDITING ........................................................................................... 38 
13.0 MULTICENTER REGULATORY REQUIREMENTS ...................... 39 
14.0 STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE ........ 40 
14.1 Analysis Populations ....................................................................... 40 
14.2 Statistical Analysis .......................................................................... 40 
14.2.1 
Descriptive Analyses ............................................................... 40 
14.2.2 
Primary Endpoint Analysis ..................................................... 40 
14.2.3 
Secondary Endpoint Analyses ................................................ 41 
14.2.4 
Safety Analysis ....................................................................... 43 
14.2.5 
Description of Planned Subgroup Analyses ............................ 43 
14.3 Sample Size ..................................................................................... 43 
15.0 DATA SUBMISSION SCHEDULE .................................................... 44 
16.0 REFERENCES ..................................................................................... 47 
  


Genomic Predictors of Decitabine Response in AML/MDS 
 
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1.0 
INTRODUCTION 
 
1.1 
Rationale   
 
Personalized therapy for cancer requires that 1) patient-specific factors (e.g. patient-
specific mutations, expression profiles, or pharmacogenomics) influence treatment 
algorithms, and 2) once therapy is initiated, in vivo responsiveness is used to guide 
further therapy. The modern era of genomics has provided significant insights into cancer 
pathogenesis by describing patterns of germline and somatic variants, as well as defining 
disease-specific patterns of gene expression. However, the application and integration of 
this data with therapeutics remains a difficult task. 
 
Decitabine is a commonly used single-agent therapy for patients with acute myeloid 
leukemia (AML) and/or myelodysplastic syndrome (MDS). Historically, it has yielded 
clinical complete responses in approximately 25% of patients across diverse clinical 
trials, it is well tolerated, and it can be given as in the outpatient setting. However, the 
majority of patients do not respond to decitabine, the molecular etiology of decitabine 
sensitivity is unknown, and there are no algorithms to predict patient responsiveness a 
priori or even within one month of starting therapy.  
 
AML and MDS are two closely related hematological disorders characterized by 
ineffective hematopoiesis and malignant expansion of clonal myeloid cells.  Both AML 
and MDS frequently occur in elderly patients and some cases are secondary and/or 
related to long-term bone marrow damage after radiation or chemotherapy. Current 
therapy for AML consists of cytotoxic chemotherapy followed by consolidation 
chemotherapy or stem cell transplantation. Using these approaches, current cure rates 
may be as high as 60% for patients younger than 60 with good risk cytogenetics (e.g. 
t(15;17), t(8;21), inv(16)).[4]  Outcomes in patients ≥ 60 years old and with relapsed 
disease remain poor with 2 year survival of only ~25%.[5] Historically, the available 
treatment for MDS has been symptomatic and has not been shown to be effective in 
producing sustained improvement in hematopoiesis or in delaying leukemic evolution[6, 
7].  
 
Current AML therapy is poorly tolerated in elderly and pre-treated patients due to 
hematologic and non-hematologic toxicities; following such therapy, patients typically 
require 3-4 weeks of hospitalization for treatment of neutropenic fevers and for regular 
transfusions. Even among the relatively healthy patients enrolled in clinical trials, 
induction mortality in the elderly can be as high as 20-40% following standard induction 
therapy,[8-11] and moderate to severe non-hematologic toxicity occurs in at least an 
additional 30% of patients.[9, 12-14] Consequently, many older AML patients are not 
offered traditional intensive chemotherapy, but receive supportive care alone.[15]  More 
effective and less toxic therapies are needed to provide durable remissions in a significant 
proportion of elderly AML in populations. 
 
Decitabine has been studied in MDS patients and in elderly and/or relapsed AML 
patients. It has been found to be reasonably well tolerated, can be given in the outpatient 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
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setting, and has overall response rates of 20-45%.[2, 3, 16-20] Our own experience has 
been of CR/PR rates of 25%, [2] and this is typical of many other groups for both MDS 
and AML patients (reviewed in [21]  Decitabine is an S-phase depended drug.  [22-24] 
However, decitabine is usually administered in one hour daily infusions during 3 to 5 
consecutive days (too short to expose decitabine to most AML blasts during at least 2 
cycles of replication, which divide every 2 to 7 days).[25-27]  Recent experience with 
increasing the number of infusions per cycle from 5 consecutive days to 10 consecutive 
days has improved overall response rates to 67%.[3] Response to decitabine can be slow, 
sometimes requiring 3 to 5 cycles before maximum response is observed.[2] As a 
consequence, many patients are exposed to prolonged therapy, gaining little benefit, 
before determining resistance. There are currently no biomarkers that predict response to 
decitabine. 
 
Decitabine is a cytosine analogue that cannot be methylated. Incorporation of decitabine 
into DNA prevents DNA methylation at cytosine residues and derepresses epigenetically 
silenced gene expression. Therefore, the reduction in total genomic DNA methylcytosine 
content acts as a marker of effective decitabine dosing. Indeed, two groups have recently 
correlated effective methylcytosine reduction and gene expression derepression to 
response (Figure 1.1).[3, 28] 
 
 
 
   
Figure 1.1. From Shen et al JCO (2010) 28:611 and Blum et al JCO (2007) 25: 3884. 
 
We and others have recently used whole genome sequencing to identify recurrent, 
somatic mutations in AML and MDS patients in four genes that are involved in 
methylcytosine regulation: DNMT3A, IDH1, IDH2, and TET2.[29-37] Collectively, 
nearly half of all AML/MDS patients have a mutation in one of these four genes.  
DNMT3A mutations are associated with an adverse outcome in AML patients treated with 
cytotoxic chemotherapy.[35, 38] However, it is not yet clear whether the presence of 
mutations in one or more of these genes is associated with response or resistance to 
decitabine. 
 
 


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Amendment 2 Version date: 09/11/14  
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In this study, we will treat patients with the current state-of-the-art decitabine 
schedule.[3] We will perform exome sequencing on all patients and correlate response 
with mutation status, rate of mutation clearance, expression profile, plasma decitabine 
levels, and DNA methylcytosine content reduction. This approach will comprehensively 
assess genomic and pharmacologic parameters that could clinically predict patient 
response to decitabine.  
 
In this study, we will combine analysis of patients with AML and MDS. Recent genomic 
analysis has shown that AML and MDS have shared mutations in many of the same 
genes, shared patterns of mutation acquisition, and shared patterns of mutation 
evolution.[39, 40] Furthermore, MDS, like AML, presents with clonal hematopoiesis, 
even when the morphologic blast count is zero.[36]Because this study will identify and 
track the rate of clearance of patient specific mutations, and because decitabine has been 
shown to have efficacy in both AML and MDS patients, [2, 3, 16-19, 41-45] combining 
analysis of AML and MDS patients is reasonable.  Asymptomatic MDS patients are 
excluded because these patients are typically followed with watchful-waiting rather than 
with chemotherapy. 
  
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
 
Methylation of cytosine in the CpG dinucleotide by DNA methyltransferase leads to 
transcriptional silencing of genes during normal development and has emerged as a 
significant mechanism for the loss of tumor suppressor gene expression in human cancer, 
including AML and MDS.[46, 47] Like azacitidine, decitabine (5-aza-2’-deoxycitidine) is 
incorporated into DNA during S phase and forms covalent adducts with DNA 
methyltransferase. The irreversible inhibition of this enzyme results in loss of 
methylation and is associated with differentiation.[48]  Decitabine is an approximately 5-
fold more potent inhibitor of DNA methyltransferase than azacitidine, which might 
explain why decitabine can induce differentiation of myeloid progenitors in vitro at lower 
doses than azacitidine. In addition to their differentiation inducing activity at low doses, 
both agents have a direct cytotoxic effect at higher doses (1-2 µM).[49]  
 
Decitabine has been investigated in hematologic malignancies using a range of doses and 
schedules.  It is currently FDA approved for the treatment of adults with MDS at a dose 
of 15 mg/m2 every 8 hours for 3 days, based on a Phase III study that demonstrated 
improved outcomes in the treatment arm compared to patients treated with supportive 
care alone.[15]  However, the overall response rate in this trial was modest (17%). Of 
note, 18% of the patients in this trial had 20-30% bone marrow blasts, meeting the 
current WHO criteria for AML.  Subsequent phase I and II studies in AML, using a 
variety of low-dose decitabine regimens, have reported overall response rates of 22-
49%.[2, 3, 16-19, 41-45] Comparisons of multiple low dose schedules have noted 
improved efficacy in the MDS population using a dose of 20 mg/m2 on Days 1-5 of a 28-
day cycle.[50, 51] Because of these results, we have recently explored this dosing 
schedule in the treatment of elderly patients with AML.[52] In this study, we observed an 
overall response rate of 24%, and an additional 44% of patients achieved stable disease 
during a median of 5 monthly cycles.[48] In this study, we noted that decitabine does not 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
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act quickly and the median time to response was 3 months. These results are similar to 
the recently published experience in Germany treating 227 patients with 15 mg/m2 over 3 
hours TID for 3 days every 6 weeks, which resulted in a CR/PR rate of 26%.[16]  
 
A recent study using a prolonged administration schedule (20 mg/m2 IV over 1 hour on 
consecutive days 1-10) showed improved response (64% CR/PR) and improved time to 
respond (50% of patients had bone marrow blasts < 5% after 1 cycle of therapy).[3]  
These results are an improvement over previous experience by other groups using 
decitabine to treat elderly AML patients or patients with relapsed AML, and this 
experience is summarized below: 
Trials of decitabine in AML 
Status 
Other Therapy 
DAC Dose 
(mg x days) 
Evaluable 
CR 
de novo 
daunomycin1 
90 mg/m2 x 5 
6 
100% 
Salvage 
None4 
0.75-80 mg/kg 
16 
  
None5 
37-81 mg/kg 
6 
33% 
None7.  Ara-C 
Refractory 
Various 
4 
0 
AMSA7 
250-500 mg/m2 x 6 
10 
80% 
idarubicin or 
AMSA3 
250 mg/m2 x 6 
56 
39% 
Elderly 
None6 
270-360 mg/m2 x 3 
10 
30% 
None2 
45 mg/m2 x 3 
4 
50% 
None8 
20 mg/m2day 1-5,8-12 
50 
23% 
None9 
20 mg/m2 x 5 
55 
25% 
 
None10 
50 mg/m2 CIVI x 3 
29 
48% 
 
None11 
20 mg/m2 x 10 
53 
64% 
 
ATRA cycle 212 
15 mg/m2 TID x 3 
227 
26% 
1 Schwartsmann G.  Leukemia 1997; 11(Suppl 1):S28-31. 
2 Lubbert M. ASH 2002 Abstract #4601. 
3 Willemze R.  Leukemia.  1997; 11(Suppl 1):S24-27 
4 Rivard GE.  Leukemia Research 1981; 5:453-462. 
5 Momparler RL.  Pharmac Therapy 1985; 30:277-286.  
6 Petti MC.  Leukemia.  1993; 7(Suppl 1):36-41.  
7 Richel DJ.  Br. J. Cancer 1991; 64:144-148 
8 Issa, JP. Blood 2004. 103: 1635. 
9 Cashen AF. ASH Annual Meeting. 2008 abstract 560. 
10 Wijermans PW. Leukemia 1997. 11: Suppl 1, 19-23. 
11 Blum, W. PNAS 2010; 107:7473. 
12 Lubbert. Haematologica 2011. ePub 11/04/2011. 
 
In general, decitabine is well tolerated. Unlike conventional cytotoxic chemotherapy, 
decitabine can be administered in the outpatient setting. However, AML and MDS are 
aggressive hematologic malignancies. Cytopenias commonly result from AML/MDS 
inhibition of normal hematopoiesis, and often do not resolve until a complete response is 
achieved. Thus, neutropenic sepsis and thrombocytopenic bleeding remain significant 
problems regardless of therapy, especially during the first 4-8 weeks. In our experience 
treating elderly AML patients, neutropenia occurred in 24%, sepsis in 9%, and 
bacteremia in 7%.  Similar results have been observed in MDS patients.[20, 53, 54] As a 


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result, death is not uncommon in the first 8 weeks of therapy, occurring in 15-17% of 
patients, and most commonly resulting from infection and cytopenias related to disease 
and therapy.[16, 52, 55] While unsettling, these numbers are better than many historical 
patients treated with cytotoxic chemotherapy, and suggest a need for continued 
improvements in therapy.[9, 12]  
 
1.2.1 Pharmacokinetics and Metabolism of Decitabine 
 
In human subjects, after IV administration, decitabine displayed a distribution 
phase with a half-life of 7 minutes and a terminal phase elimination half-life of 10-
35 minutes as measured by bioassay.[56, 57]   The short plasma half-life is due to 
rapid inactivation of decitabine by deamination by liver cytidine deaminase.[23] 
Total body clearance (Clp) in humans was high with a mean value of 126 
mL/min/kg.[58]  
 
Among five adult patients with advanced solid tumors administered seven courses 
of 100 mg/m2 of decitabine by 1-hour infusions,[56] maximum plasma 
concentration ranged from 1 to 4 µM. Disappearance from plasma was biphasic 
with a rapid initial decline to less than 0.1 µM during the first hour post infusion 
and a slower decline over the next two hours to 6 nM, the level of detectability. 
Plasma half-life (meanSE) was estimated to be approximately 71 minutes in 
the initial phase (t½α) and 355 minutes in the second phase (t½β). The area under 
the plasma concentration-time curve was 40888 ng.hour/mL for these patients, 
with a peak plasma concentration of 2.010.44 µM (0.440.1 µg/mL), while mean 
volume of distribution (Vdss) and CLp was 4.591.42 L/kg and 12621 
mL/min/kg, respectively. The time course of DAC concentrations was similar in 1 
of 2 patients administered 75 mg/m2, while DAC was usually undetectable in the 
plasma of patients administered 25 to 60 mg/m2. 
 
Using a longer infusion time (up to 40 hours), Rivard et al.[57] reported a plasma 
concentration of 0.1 to 0.4 g/mL. With infusion times of 40-60 hours, at an 
infusion rate of 1 mg/kg/h, plasma concentrations of 0.43-0.76 µg/mL were 
achieved.[59]  The steady-state plasma concentration at an infusion rate of 
1 mg/kg/h was estimated to be 0.2-0.5 µg/mL. The half-life after discontinuing the 
infusion was 12-20 min.[57]  
 
Urinary excretion of unchanged decitabine was low, ranging from less than 0.01% 
to 0.9% of the total dose, and there was no relationship between excretion and dose 
or plasma drug levels. High clearance values and a total urinary excretion of <1% 
of the administered dose suggest that decitabine is eliminated rapidly and largely by 
metabolic processes, including extrahepatic metabolism.[60] 
 
The steady state plasma concentrations (Css) ranged from 0.31 to 0.39 µg/mL 
(1.27 to 1.60 µM) during the infusion of 100 mg/m² decitabine over a 6-hour 
period.[61] The range of concentrations during a 600 mg/m2 infusion over an 8-
hour period was 0.41-16 g/mL(1.68 to 4.76 µM).[60] These plasma 


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concentrations are in the same range as the in vitro concentrations that activated 
the expression of tumor suppressor gene p16 in three human non-small-cell lung 
cancer (NSCLC) cell lines.[61]  
 
Steady state decitabine levels of 0.1 to 0.2 µM were achieved in solid tumor 
patients dosed with 30 mg/m²/day and 40 mg/m²/day by continuous intravenous 
infusion over 72 hours.[62]  
 
Summary of published steady state serum levels are below and are in proportion to 
infusion rate (R2 = 0.98, p < 0.0001):[63] 
 
Rate 
Serum 
Concentration 
mg/m2/hr 
ng/ml 
nM 
5 
55 
241 
7.5 
80 
351 
15 
180 
789 
21 
182.4 
800 
22.5 
350 
1535 
42 
456 
2000 
84 
1140 
5000 
 
In our experience treating primary patient blasts in vitro, concentrations of 100 – 
500 nM are required maximize methylcytosine reduction without causing cellular 
toxicity. Because decitabine incorporation into DNA is S-phase dependent, 
exposure of cells during cell division is necessary for efficacy. 
  
 
 
 
1.2.2 Experience with extended dosing schedules 
 
Blum et al recently published their experience treating elderly AML patients with 
a 10-day course of decitabine 20 mg/m2/day as a 1-hour infusion.[3] This 
 


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prolonged exposure course was designed to increase the number of AML blast 
divisions exposed to decitabine per cycle. Toxicities were very similar to a similar 
regiment given as a 3 and 5 day course,[2, 16] but the overall response (CR/PR) 
was much improved (64% vs 25%). In all three studies, the toxicities were 
primarily myeloid suppression and the resultant infectious and bleeding 
complications associated with leukemic and pharmacologic hematopoietic 
suppression. Furthermore, Blum et al noted that the percent methylcytosine 
reduction was improved (22% vs 11%) as was the time to blast clearance (50% of 
patients who ultimately achieved a CR had ≤ 5% bone marrow blasts after 1 cycle 
vs 0% of patients after 2 cycles). Finally, like Shen et al, they observed a 
correlation between the extent of methylcytosine reduction and response.[28]  
This study suggests that improvement in response rates and time to response can 
be achieved by prolonged administration of decitabine. Further, that DNA 
methylcytosine reduction can be achieved with prolonged exposure to decitabine 
and that this might be a patient-specific biological marker of adequate dosing. 
  
Scandura et al. recently published a study of infusional decitabine prior to 
cytarabine + daunorubicin induction therapy for AML.[64] They escalated the 
duration of infusional therapy and reached their planned goal of 20 mg/m2/day x 7 
days of continuous therapy given prior to cytarabine and daunorubicin. They 
noted similar toxicities (primarily hematopoietic and the associated neutropenic 
infections), but that these were not different from their experience giving 
cytarabine + daunorubicin alone. 
 
1.3 
Correlative Studies 
 
With exception of the peripheral blood sample collected on Cycle 1 Day 4, all correlative 
studies will be collected under HRPO# 201011766 (“Tissue Acquisition for Analysis of 
Genetic Progression Factors in Hematologic Diseases”) that all patients will be 
concurrently enrolled in. The goals of the correlative studies include: 
 
1. To correlate patient-specific mutations with response to decitabine. 
We will perform exome sequencing of each patient’s pre-study bone marrow sample 
and skin sample. We will determine AML/MDS-associated variants found in the bone 
marrow, but not skin sample. We will correlate the presence of mutations with 
response (especially mutations in DNMT3A, IDH1, IDH2, and TET2). This study is 
powered to identify mutations with >80% sensitivity to predict decitabine response if 
used as a clinical assay. 
 
2. To correlate the velocity and depth of response with response to decitabine. 
We will assess tumor burden by allele frequency of patient-specific mutations in bone 
marrow samples collected pre study, Cycle 1 Day 10±1, Cycle 1 Day 28±4, and 
Cycle 2 Day 28±4. We will assess mutations in at least 24 recurrently mutated genes 
(including, but not limited to: FLT3, DNMT3A, NPM1, TET2, WT1, IDH1, IDH2, 
RUNX1, NRAS, TP53, U2AF1, KRAS, PHF6, MSTP9, PTPN11, KIT, SMC3, 
PRUNE2, ETV6, CEBPA, SMC1A, ASXL1, STAG2, RAD21) as well as all patient-


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specific mutations discovered during exome sequencing. The leukemic allele 
frequency will be measured using the Illumnia MySeq or HiSeq platform using DNA 
capture by gene specific biotinylated-oligonucleotides. Patient bone marrow samples 
will be assessed pre-study, Cycle 1 Day 10 ± 1, Cycle 1 Day 28 ± 4, and Cycle 2 Day 
28 ± 4 and compared with patient specific skin samples (normal control). The 
velocity of response will be calculated as the natural log rate of patient-specific 
mutation allele frequency clearance. Depth of response will be defined as the lowest 
patient-specific mutation allele frequency in a bone marrow sample. The velocity and 
depth of tumor response will be correlated to: 
 Achievement of clinical CR, 
 Time to best response, 
 Mutational status, 
 Time to disease progression. 
Because all patient-specific exome-discovered mutations will be assessed a second 
time in the primary bone marrow sample as well as the  Cycle 1 Day 10 ± 1, Cycle 1 
Day 28 ± 4, and Cycle 2 Day 28 ± 4 bone marrow samples, this study will also serve 
to validate all exome-discovered mutations. This is important, because exome 
sequencing is associated with a 20-50% false-discovery rate depending on type I error 
tolerated (mutation-calling algorithms are typically set for greater sensitivity than 
specificity).  Thus, many mutations discovered during exome sequencing are 
incorrect.  This second analysis of these mutations will server to exclude these false 
positive calls. 
 
3. To correlate the bone marrow expression profile from pre-study and Cycle 1 Day 10 
± 1 with response to decitabine. 
We will correlate expression signatures, and changes in expression signatures, with: 
 Achievement of clinical CR, 
 Mutation status. 
 
4. To correlate steady-state plasma decitabine concentration on Cycle 1 Day 4 ± 1 with: 
 
Achievement of CR. 
 
5. To correlate the extent of bone marrow DNA hypomethylation on Cycle 1 Day 10 ± 1 
with response to decitabine. 
We will assess the percent reduction in bone marrow methylcytosine content using 
LC-MS. Patients will be evaluated pre-study and Cycle 1 Day 10 ± 1. The change in 
DNA methylcytosine content will be correlated to: 
 Achievement of clinical CR, 
 Change in expression signature. 
This will determine whether inadequate blast exposure to decitabine correlates with 
drug resistance. 
 


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2.0 
OBJECTIVES 
 
2.1 
Primary Objective 
 
To determine whether patient-specific mutations correlate with clinical overall response 
to decitabine in patients with AML or MDS. 
 
2.2 
Secondary Objectives 
 
- 
To determine whether 10 consecutive days per cycle of decitabine improves 
outcomes compared with historical controls treated with 5 consecutive days per cycle 
of decitabine. 
 
- 
To determine whether other biomarkers correlate with clinical overall response to 
decitabine in patients with AML or MDS. These will include:  
 
1. The velocity and depth of patient-specific mutation clearance; 
2. The bone marrow expression profile and change in expression profile during 
decitabine treatment; 
3. The steady-state plasma decitabine concentrations; 
4. The decrease in bone marrow methylcytosine content in response to decitabine.  
 
 


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3.0 
PATIENT SELECTION 
 
3.1 
Inclusion Criteria 
 
One of the following: 
 Patient must have non-M3 AML and be > 60 years of age OR 
 non-M3 AML with relapsed disease OR 
 Symptomatic MDS with one of the following: 
□ Symptomatic anemia with either hemoglobin < 10.0 g/dL or requiring RBC 
transfusion 
□ Thrombocytopenia with a history of two or more platelet counts < 
50,000/mcL or a significant hemorrhage requiring platelet transfusions 
□ Neutropenia with two or more ANC < 1,000/mcL 
 
All of the following: 
1. Patient must have an ECOG performance status ≤ 2. 
2. Patient must have >10% disease burden measured by cytomorphology, flow 
cytometry, or cytogenetics. 
3. Patient must have peripheral white blood cell count < 50,000/mcl. 
4. Patient must have adequate organ function, defined as: 
a. 
Total bilirubin < 1.5 x ULN 
b. 
AST/ALT < 2.5 x ULN 
c. 
Serum creatinine < 2.0 x ULN  
5. Patient must have undergone ≤ 2 cycles of prior hypomethylating agent 
(decitabine or azacitidine). 
6. Patient must be enrolled in HRPO# 201011766 (“Tissue Acquisition for Analysis 
of Genetic Progression Factors in Hematologic Diseases”). 
7. Patient must be > 18 years of age. 
8. Patient must be able to understand and willing to sign an IRB-approved written 
informed consent document. 
 
3.2 
Exclusion Criteria 
 
1. Patient must not be pregnant or nursing. 
2. Patient must not have known CNS leukemia.  
3. Patient must not have a history of positive HIV serology.  
4. Patient must not have a history of positive Hepatitis C serology. 
5. Patient must not have undergone a prior allogeneic stem cell transplant. 
6. Patient must not have any uncontrolled intercurrent illness including, but not 
limited to, ongoing or active infection, ongoing or active GVHD, congestive heart 
failure of NYHA class 3 or 4, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situation that would limit compliance with study 
requirements. 
7. Patient must not have had radiation therapy within 14 days of enrollment. 
8. Patient must not have received any chemotherapy within 21 days of enrollment, 
and any acute treatment-related toxicities must have returned to baseline. Patients 


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may be receiving hydrea at time of enrollment.    
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible for this 
trial. 


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4.0 
REGISTRATION PROCEDURES 
 
Patients must not start any protocol intervention prior to registration through the Siteman 
Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility by Washington University 
2. Registration of patient in the Siteman Cancer Center database 
3. Assignment of unique patient number (UPN) 
 
Once the patient has been entered in the Siteman Cancer Center database, the WUSM 
coordinator will forward verification of enrollment and the UPN via email. 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility collecting the information listed below and scanning and 
emailing it to the research coordinator listed in the Siteman Cancer Center Clinical Trials 
Core Protocol Procedures for Secondary Sites packet at least one business day prior to 
registering patient: 
1. Your name and contact information (telephone number, fax number, and email 
address) 
2. Your site PI’s name, the registering MD’s name, and your institution name 
3. Patient’s race, sex, and DOB 
4. Three letters (or two letters and a dash) for the patient’s initials 
5. Currently approved protocol version date 
6. Copy of signed consent form (patient name may be blacked out) 
7. Planned date of enrollment 
8. Completed eligibility checklist, signed and dated by a member of the study team 
9. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center Database 
 
Registrations may be submitted Monday through Friday between 8am and 5pm CT. 
Urgent late afternoon or early morning enrollments should be planned in advance and 
coordinated with the Washington University research coordinator.  Registration will be 
confirmed by the research coordinator or his/her delegate by email within one business 
day. Verification of eligibility and registration should be kept in the patient chart. 
 
All patients at all sites must be registered through the Siteman Cancer Center database at 
Washington University. 
 
4.3 
Assignment of UPN 
 


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Each patient will be identified with a unique patient number (UPN) for this study.  
Patients will also be identified by first, middle, and last initials.  If the patient has no 
middle initial, a dash will be used on the case report forms (CRFs).  All data will be 
recorded with this identification number on the appropriate CRFs. 
 


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5.0 
INVESTIGATIONAL PLAN 
 
5.1 
Summary of Study Design 
 
This is an open-label, single-arm, Phase II, biomarker study of patients with AML or 
MDS who are treated with 10 consecutive days of decitabine. FAB-M3 AML is excluded 
from this trial, because other well-tolerated options exist for the treatment of FAB-M3 
acute promyelocytic leukemia. AML patients who are < 60 years old and have not 
received cytotoxic induction chemotherapy are also excluded, because cytotoxic therapy 
offers a curative possibility for these patients.  MDS patients without cytopenias are also 
excluded because these patients can be followed with watchful-waiting. 
 
All patients will undergo bone marrow biopsies pre-study, on Cycle 1 Day 10 ± 1, and on 
Day 28 ± 4 of Cycles 1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. 
Patients will provide a skin biopsy sample pre-study. Patients will also provide peripheral 
blood samples on Cycle 1 Day 4 ± 1 for pharmacokinetic studies. See Section 8.0 for 
details on correlative studies.  
 
Patients will receive decitabine 20 mg/m2/day as a 1-hour infusion on consecutive Days 
1-10 in 28-day cycles.  The dose will be calculated based on actual patient weight at the 
beginning of each cycle. 
 
Once AML patients achieve morphologic complete remission with incomplete blood 
count recovery (CRi) or MDS patients achieve a marrow complete response (Marrow 
CR) as defined in Section 9.0, decitabine administration may be decreased to five 
consecutive days to limit myelosuppressive toxicity at the discretion of the treating 
physician. After two cycles of five consecutive days of treatment, if the patient meets the 
definition of CR with complete cytogenetic response as defined in Section 9.0, 
administration may further be decreased to three consecutive days per cycle at the 
discretion of the treating physician. 
 
It will be a logistical goal of this trial consistently to return preliminary exome results to 
the treating physician by the end of Cycle 1 and validated results by the end of Cycle 2. 
However, this logistical goal is not a primary or secondary aim of this study. Given the 
limited data regarding mutation presence and drug response, we do not anticipate that the 
results will directly influence therapeutic decisions made by the treating physician. 
However, the development of comprehensive genomic pipelines and integration of this 
data into a clinical trial setting will be required before personalized medicine can be 
realized. 
 
5.2 
Study Procedures 
 
5.2.1 Pre-study Procedures 
 


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Pre-study procedures must take place no more than 14 days prior to the first dose 
of decitabine except where noted below. 
 
1. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments 
performed on bone marrow will include correlative studies (collected under 
HRPO# 201011766), morphologic assessment, cytogenetics, and FISH.  
2. Bone marrow or peripheral blood for flow cytometry 
3. Skin biopsy sample (collected under HRPO# 201011766). There is no 
required time frame for this sample; it may have been collected months or 
even years prior to the first dose of decitabine.  
 
5.2.2 Baseline Evaluation 
 
Baseline evaluations must take place no more than 7 days prior to the first dose of 
decitabine.  
 
1. Medical history 
2. Physical exam including height, weight, and ECOG performance score. 
3. Record transfusion requirements for 8 weeks before first dose of study drug 
4. CBC with differential and platelets 
5. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
6. Urine βHCG pregnancy test if appropriate 
 
5.2.3 Day 1 of Each Cycle 
 
1. Physical exam, including weight  
2. CBC with differential and platelets 
3. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
 
5.2.4 Cycle 1 Days 1-10 
 
Decitabine administration, 20 mg/m2 per day given as a 1-hour continuous 
intravenous infusion.  The dose will be calculated based on actual patient weight 
at the beginning of each cycle.  Patients may be premedicated with lorazepam, 
prochlorperazine, palonosetron, or ondansetron at the treating physician’s 
discretion. 
 
5.2.5 Cycle 1 Day 4 ± 1 
 
Obtain 3 mL peripheral blood for correlative studies. Sample must be obtained 45 
minutes ± 15 minutes after beginning the infusion, before the decitabine infusion 


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is complete, and should be obtained from the opposite arm as the infusion.  The 
sample should be collected in a 3 mL K3EDTA tube pre-loaded with 8 mcL of 
tetrahydrouridine (THU 500 mcg/mL), an inhibitor of cytidine deaminase.  The 
sample should be processed by the Tissue Procurement Core Facility at the 
Siteman Cancer Center.  Plasma should be separated and stored per protocols. 
  
5.2.6 Cycle 1 Day 10 ± 1 
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH. 
 
5.2.7 Weekly 
 
CBC with differential and platelets. After the first cycle, this may be changed to 
every other week at the discretion of the treating physician. 
 
5.2.8 Every Other Week 
 
Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH. After the first cycle, this may be changed to 
every month at the discretion of the treating physician. 
 
5.2.9 Cycles 1, 2, 4 and 6 Day 28 ± 4 days  
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include: correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH.  
 
Patients who achieve with clinical response (CRi or better) and are receiving 
cycles every 6 weeks, will undergo bone marrow biopsies on day 42 ± 4.  
 
5.2.10 End-of-Study Procedures  
 
1. Physical exam 
2. CBC with differential and platelets.  
3. Serum chemistries including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, and LDH.  
4. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments 
performed on bone marrow will include morphologic assessment, 
cytogenetics, and FISH.  


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5.2.11 Post-study Follow-up 
 
Patients are to be followed according to standard patient care procedures for 30 
days following the date of the last decitabine dose to monitor for toxicity, 
survival, and response. Thereafter, the investigator or designee will record 
information on subsequent therapy, disease progression, and/or death every 6 
months for 2 years after last dose of study drug or until the patient is lost to 
follow-up or dies. 
 
5.3 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to the 
patient’s health and/or the patient no longer wishes to continue protocol therapy, the 
protocol therapy should be discontinued and the reason(s) for discontinuation 
documented in the case report forms. 
 
In the absence of treatment delays due to adverse events, treatment may continue 
indefinitely or until one of the following criteria applies: 
 
 Documented and confirmed disease progression 
 Death 
 Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
 General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
 Suspected pregnancy 
 Serious non-compliance with the study protocol 
 Lost to follow-up 
 Patient withdraws consent 
 Investigator removes the patient from study 
 The Siteman Cancer Center decides to close the study 
 
Patients who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar. 
 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
 
5.4.1 Chemotherapy 
 
Patients cannot receive any chemotherapy within 21 days of study enrollment and 
must have recovered from any treatment-related toxicities. However, patients may 
be receiving hydrea at enrollment and may continue on hydrea through Cycle 2 of 
decitabine. An indication for hydrea following 2 cycles of decitabine is evidence 
that the patient is not responding and decitabine should be discontinued. Other 
than decitabine, patients cannot receive chemotherapy during this trial.  


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5.4.2 Growth Factors 
 
Erythroid growth factors should not be routinely administered to patients during 
the study. 
 
Thrombopoietin receptor agonists should not be routinely administered to patients 
during the study. 
 
Filgrastim (G-CSF) or sargramostim (GM-CSF) may be administered to patients 
with septic shock or febrile neutropenia at the discretion of the treating physician. 
 
5.4.3 Transfusions 
 
 Packed red blood cells (RBCs) transfusion:   
RBC transfusions will be administered at the discretion of the treating 
physician.  
 
 Platelet transfusion:   
Platelet transfusions will be administered at the discretion of the treating 
physician  
5.4.4 Prophylactic Antimicrobial Agents  
 
Antimicrobial agents will be administered at the discretion of the treating 
physician. Common antimicrobial prophylaxis during cycles 1 and 2 include 
acyclovir, ciprofloxacin, and fluconazole. 
 
5.4.5 Radiotherapy 
 
Patients cannot receive radiotherapy within 14 days prior to study enrollment or 
while on study. 
 


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6.0 
DOSE MODIFICATIONS 
 
A criterion for discontinuation of the study is a delay of > 8 weeks between any two 
cycles.  
 
6.1 
Delay for Cytopenia or Infection 
 
As cytopenias are a defining feature of AML and MDS, their presence alone does not 
generally warrant dose modifications or delays. Patients will receive four cycles of 
therapy without adjustment for any cytopenia. Following Cycle 4, dose modifications or 
delays for cytopenias will be made at the discretion of the investigator.  
 
Patients with febrile neutropenia (temperature ≥ 38.5° with ANC < 1,000/l) or systemic 
infections should have treatment held until clinical resolution of the infection.  
 
6.2 
Delay for Organ Dysfunction 
 
If renal dysfunction (creatinine ≥ 3 X institutional upper limit of normal) or hepatic 
dysfunction (bilirubin ≥ 2.5 mg/dl or ALT/AST ≥ 3 X institutional upper limit of normal) 
occurs, therapy may not resume until the abnormalities have resolved to below the limits 
stated above. If renal or hepatic dysfunction occurs during a treatment course, the next 
dose of decitabine should be held, and serum chemistries should be checked twice per 
week.  Decitabine can be resumed at full dose when creatinine, bilirubin, ALT and AST 
are below the limits listed above.  
 
For grade 3-4 non-hematologic toxicities related to decitabine, decitabine treatment 
should be delayed until recovery to baseline or to grade ≤ 1. Decitabine may be 
reintroduced with a 50% dose reduction, or at full dose at the discretion of the 
investigator. 
 
6.3 
Modification in Dose or Schedule for Treatment Response 
 
Once AML patients achieve morphologic complete remission with incomplete blood 
count recovery (CRi) or MDS patients achieve a marrow complete response (Marrow 
CR) as defined in Section 9.0, decitabine administration may be decreased to 20 
mg/m2/day for ten consecutive days, and the cycles may be administered every 6 weeks 
to limit myelosuppressive toxicity, at the discretion of the treating physician. After two 
cycles at six week intervals, the dose may be reduced to 20 mg/m2/day for five 
consecutive days of treatment in six week intervals. If the patient meets the definition of 
CR with complete cytogenetic response as defined in Section 9.0, administration may 
further be decreased to three  consecutive days per cycle at the discretion of the treating 
physician. 
 


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7.0 
PHARMACEUTICAL INFORMATION 
 
7.1 
Decitabine 
 
Decitabine is an antimetabolite that can replace cytosine in DNA but, unlike cytosine, it 
cannot be methylated. The exact mechanism of decitabine activity has not been 
determined and may involve multiple pathways. Decitabine is thought primarily to act by 
correcting DNA methylation, a major mechanism for gene expression. In some cancer 
cells, hypermethylation blocks the activity of tumor suppressor genes, which regulate cell 
division and differentiation to prevent malignant transformation. When suppressor gene 
activity is blocked, cell division becomes unregulated, leading to the formation of 
neoplastic cells. Decitabine regulates DNA methylation (specifically targets the 
methyltransferase, DNMT1), effectively demethylating and reactivating different tumor 
suppressor genes. Because hypermethylation occurs early in the malignant transformation 
of cells, decitabine may have activity in chemoprevention. Other possible mechanisms 
for decitabine activity include cytotoxicity due to incorporation into DNA similar to other 
antimetabolites, methylation-independent induction of gene expression, and degradation 
of DNA methyltransferase 1. 
 
7.1.1 Study Drug Preparation  
 
OSHA Guidelines for handling cytotoxic drugs outlined in the American Journal 
of Hospital Pharmacy must be followed. 
 
Decitabine (5-aza-2’-deoxycytidine) is supplied as a lyophilized powder for 
injection, 50 mg in 20-ml vials. When reconstituted with 10 ml of sterile water for 
injection, each ml will contain 5 mg of decitabine and 6.8 mg of KH2PO4. The 
reconstituted solution should be diluted to 0.1-1.0 mg/mL with pre-chilled 0.9% 
sodium chloride intravenous infusion, which has been cooled to 2-8oC. 
Reconstitution of the powder results in a rapidly decomposing solution. Unless 
used within 15 minutes of reconstitution, the diluted solution must be prepared 
using cold (2—8 degrees C) infusion fluids and stored at 2—8 degrees C (36—46 
degrees F) for a maximum of 7 hours. 
Decitabine will be administered per dosing schedule every 28 ± 4 days.  
 
Decitabine may be administered either as an inpatient or an outpatient.  
 
7.1.2 Supplier 
 
Decitabine is available commercially. 
 
7.1.3 Toxicities 
 
Likely (>15%) 
Cytopenias: 
neutropenia, 
thrombocytopenia, 
anemia, 
leukopenia, 
febrile 
neutropenia.  


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Bleeding: petechiae, ecchymosis. 
Infections: pneumonia. 
Constitutional: fatigue, pallor, fever, rigors, arthralgia, cough. 
GI: nausea/vomiting, constipation, diarrhea, decreased appetite. 
Electrolyte abnormalities: hyperglycemia, hypoalbuminemia, hypomagnesemia, 
hypokalemia, hyponatremia.  
Cardiac: cardiac murmur. 
Neurologic: headache, insomnia, dizziness. 
Dermatologic: rash. 
 
Less likely (1-15%)  
Hematologic: thrombocythemia, hematoma. 
Other infections: cellulitis, candidal infection, urinary tract infection, sinusitis. 
GI: abdominal 
pain, hyperbilirubinemia, 
stomatitis, 
dyspepsia, ascites, 
hemorrhoids, oral ulceration, dysphagia, abdominal distention, gastro-esophageal 
reflux disease/pyrosis (heartburn), and glossodynia. 
Electrolyte abnormalities: hyperkalemia, and dehydration. 
Urologic: dysuria, increased urinary frequency.  
Dermatologic: pruritus, alopecia, urticaria, edema. 
 
Rare (<1%) 
Cardiac: arrest/respiratory arrest, atrial fibrillation, and tachycardia. 
 
  
 
  
 


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8.0 
CORRELATIVE STUDIES SAMPLE COLLECTIONS 
 
Bone marrow samples will be collected pre-study, on Cycle 1 Day 10 ± 1, and on Day 28 ± 4 of 
Cycle 1, 2, 4, and 6 for correlative studies. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. The 
bone marrow samples will be collected under HRPO# 201011766 (“Tissue Acquisition for 
Analysis of Genetic Progression Factors in Hematologic Diseases”) that all patients will be 
concurrently enrolled in.  
 
Skin biopsy sample will be collected as part of the patient’s participation in HRPO# 201011766 
(“Tissue Acquisition for Analysis of Genetic Progression Factors in Hematologic 
Malignancies”). There is no required time frame for this sample, and it may have been collected 
months or even years prior to the first dose of decitabine.  
 
Peripheral blood sample (3 mL) will be collected on Cycle 1 Day 4 ± 1.  Sample must be 
obtained 45 minutes ± 15 minutes after beginning the infusion, before the decitabine infusion is 
complete, and should be obtained from the opposite arm as the infusion.  The sample should be 
collected in a 3 mL K3EDTA tube pre-loaded with 8 mcL of tetrahydrouridine (THU 500 
mcg/mL), an inhibitor of cytidine deaminase.  The sample should be processed by the Tissue 
Procurement Core Facility at the Siteman Cancer Center.  Plasma should be separated and stored 
per protocols. 
 
 


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9.0 
EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
 
A secondary endpoint of this study is to estimate morphologic complete remission rate. 
  
9.1 
Response Criteria - AML 
 
AML Patients will be assessed for response according to the IWG criteria:1  
 
Morphologic complete remission (CR) – Defined as morphologic leukemia-free state, 
defined as less than 5% blasts in aspirate with marrow spicules and a count of ≥200 
nucleated cells and no blasts with Auer rods or any persistence of extramedullary disease. 
When erythroid precursors constitute less than 50% of bone marrow nucleated cells, the 
percentage of blasts is based on all nucleated cells; when there are 50% or more erythroid 
cells, the percentage blasts should be based on the nonerythroid cells. The presence of 
mild megaloblastoid changes may be permitted if they are thought to be consistent with 
treatment effect. However, persistence of pretreatment abnormalities (eg, pseudo-Pelger-
Hüet cells, ringed sideroblasts, dysplastic megakaryocytes) are not consistent with CR.  
In addition, ANC >1000/μl and platelet count of ≥100,000/μl. Patient must be 
independent of transfusions. No duration requirement for this designation. 
 
Cytogenetic complete remission (CCR) – Defined as morphologic complete remission 
plus a reversion to a normal karyotype. For this study, reversion of a normal karyotype is 
defined as no clonal abnormalities detected in a minimum of 20 mitotic cells. 
 
Morphologic complete remission with incomplete blood count recovery (CRi) – Defined 
as morphologic complete recovery with the exception of neutropenia <1000/μl or 
thrombocytopenia <100,000/μl.  
 
Partial remission (PR) – Defined as meeting the above criteria for complete remission, 
with the following exceptions: decrease of ≥50% in the percentage of blast to 5-25% in 
the bone marrow aspirate. A value of 5% blasts in bone marrow with Auer rods present 
may also be considered a partial remission.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, CRi, PR, PD, or 
recurrence/morphologic relapse.   
 
Progressive disease (PD) – Defined as a clear increase in bone marrow blast count, 
typically a greater than 50% increase compared with baseline, or new extramedullary 
disease. Patients with a > 50% increase in peripheral blast count with an increase of the 
total peripheral white blood cell count to > 10,000/l should undergo evaluation by bone 
marrow biopsy to access for progressive disease.  
 
Recurrence/morphologic relapse - Defined as relapse following complete remission is 
defined as reappearance of blasts in the blood or the finding of ≥5% blasts in the bone 
marrow, not attributable to any other cause. New dysplastic changes is considered 


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relapse. If there are no blasts in the peripheral blood and 5-20% blasts in the bone 
marrow, bone marrow biopsy should be repeated in > 1 week to confirm relapse. 
 
9.2 
Response Criteria - MDS 
 
MDS Patients will be assessed for response according to the IWG criteria:69 
 
Complete remission (CR) – Defined as < 5% myeloblasts with normal maturation of all 
cell lines in the bone marrow and peripheral blood values of Hgb > 11g/dL, Platelets > 
100 x 109/L, Neutrophils > 1.0 x 109/L, and 0% blasts. Persistent dysplasia does not 
exclude CR but will be noted.  
 
Marrow Complete Response (Marrow CR) – Defined as < 5% myeloblasts in the bone 
marrow and a decrease by > 50% from pretreatment values, but not meeting the 
definition of CR above.  
 
Partial Remission (PR) – Defined as meeting the definition of CR above with a decrease 
of myeloeblasts in the bone marrow by > 50% from pretreatment values, but absolute 
myeloblasts still >5%.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, Marrow CR, PR, 
SD, PD, or recurrence/morphologic relapse.   
 
Progressive Disease//Relapse (PD) – Defined as ≥ 50% increase in blasts to > 5% blasts 
(for patients with less than 5% blasts at baseline only), ≥ 50% increase to > 10% blasts 
(for patients with 5-10% blasts at baseline only), ≥ 50% increase to > 20% blasts (for 
patients with l0-20% blasts at baseline only), ≥ 50% increase to > 30% blasts (for patients 
with 20-30% blasts at baseline only) or any of the following: At least 50% decrement 
from maximum remission/response in granulocytes or platelets, reduction in Hgb by ≥ 2 
g/dL, or New or worsened transfusion dependence not related to study drug toxicity. Or 
for patients with a CR, Marrow CR, or PR as defined above and subsequently 
development of one of the following: Return to pretreatment bone marrow blast 
percentage, decrement of ≥ 50% from maximum remission/response levels in 
granulocytes or platelets, or reduction in Hgb concentration by ≥ 1.5 g/dL or transfusion 
dependence. 
 
9.2.1 Hematologic Improvement (HI) 
 
 
 
Progressive disease as defined above nullifies hematologic improvement.  
 
Erythroid response requires all of the following (only required if pretreatment 
HgB <11 g/dL): 
 Hgb increase by ≥ 1.5 g/dL 
 Relevant reduction of units of RBC transfusions by an absolute number of at 
least 4 RBC transfusions/8 wk compared with the pretreatment transfusion 


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number in the previous 8 wk. Only RBC transfusions given for a Hgb of ≤ 9.0 
g/dL pretreatment will count in the RBC transfusion response evaluation 
 
Platelet response requires one of the following (only required if pretreatment 
platelets < 100 x 109/L):  
 Absolute increase of ≥ 30 x 109/L (for patients starting with > 20 x 109/L 
platelets) 
 Increase from < 20 x 109/L to > 20 x 109/L and absolute increase > 100% (for 
patients starting with < 20 x 109/L) 
 
Neutrophil response requires the following (only required if pretreatment ANC 
<1.0 x 109/L):  
 At least 100% increase and an absolute increase > 0.5 x 109/L 
 
9.3.1 Cytogenetic Response  
 
Complete Cytogenetic Response – Defined as reversion to a normal karyotype. 
For this study, reversion of a normal karyotype is defined as no clonal 
abnormalities detected in a minimum of 20 mitotic cells. Progressive disease as 
defined above nullifies cytogenetic response.  
 
9.3 
Guidelines for Evaluation of Disease 
 
Bone marrow biopsy should be performed on Day 28 ± 4 of cycles 1, 2, 4 and 6 for 
clinical disease assessment. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. 
 
9.4 
Other Secondary Efficacy Measures 
 
9.4.1 Treatment Failure, Overall Survival and Event-free Survival 
 
Treatment failure is defined as failure to achieve a complete or partial remission 
following 4 cycles of therapy.  
 
Overall survival is defined as the date of first dose of study drug to the date of 
death from any cause.  Patients still alive at the time of the final analysis will be 
right-censored. 
 
Event-free survival is defined as the interval from the date of first dose of study 
drug to date of treatment failure, recurrence, death due to any cause, or loss to 
follow up. 
 
9.4.2 Duration of Remission and Relapse-free Survival for Patients Having 
Complete Remission 
 


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Duration of complete remission is defined as the interval from the date that 
complete remission is documented to the date of recurrence, death due to any 
cause, or loss to follow-up.  
 
Relapse-free survival is defined only for those patients achieving a complete 
remission. It is defined as the interval from the date of first documentation of a 
leukemia-free state to the date of recurrence, death due to any cause, or loss to 
follow up.   
  
9.4.3 Safety 
 
Safety evaluation will include assessments of adverse experiences, medical 
history, physical examinations, vital signs, and laboratory assessments at baseline 
and throughout the study period. Local laboratories may be used for this study.  
 


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10.0 
ADVERSE EVENT REPORTING  
 
10.1 
Adverse Events (AEs) 
 
Definition:  any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading:  the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for all 
toxicity reporting.  A copy of the CTCAE version 4.0 can be downloaded from the CTEP 
web site:  http://ctep.cancer.gov/reporting/ctc.html.  Grade 1-2 events will be recorded for 
the first fifty enrolled patients. Subsequently, only events graded as 3 or greater will be 
recorded. Fifty patients should be sufficient to estimate the true incidence of any adverse 
event to within ± 14% with 95% confidence intervals. 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the terms 
listed that should be used are those provided by the Department of Health and Human 
Services’ Office for Human Research Protections (OHRP).  A copy of this guidance can 
be found on OHRP’s website: (http://www.hhs.gov/ohrp/policy/AdvEvntGuid.htm). 
 
10.2 
Unanticipated Problems 
 
Definition: 
 
 unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied; 
 related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the 
incident, experience, or outcome may have been caused by the procedures 
involved in the research); and 
 suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
 
10.3 
Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that govern 
human 
subjects 
research 
or 
failure 
to 
follow 
the 
determinations 
of 
the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate choice to 
ignore regulations, institutional policies, or determinations of the IRB. 
 
10.4 
Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial harm 
to subjects or others, or that materially compromises the rights or welfare of participants. 


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10.5 
Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under the research 
team’s control. Exceptions apply only to a single participant or a singular situation. 
 
Local IRB Pre-approval of all protocol exceptions must be obtained prior to the event.  
For secondary sites, the Washington University PI will issue approval of the exception, 
but it must also be submitted to the local IRB with documentation of approval forwarded 
to Washington University.  HRPO approval is not required for protocol exceptions 
occurring at secondary sites. 
 
10.6 
Reporting to the Human Research Protection Office (HRPO) and the Quality 
Assurance and Safety Monitoring Committee (QASMC) at Washington 
University: 
 
The PI is required to promptly notify the IRB of the following events: 
 
 Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct 
of the study. 
 Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 
 Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence of 
the event or notification to the PI of the event.  The death of a research participant that 
qualifies as a reportable event should be reported within 1 working day of the occurrence 
of the event or notification to the PI of the event. 
 
10.7 
Reporting Requirements for Secondary Sites 
 
The research team at each secondary site is required to promptly notify the Washington 
University PI and research coordinator of all reportable events (as described in Section 
7.6) within 1 working day of the occurrence of the event or notification of the secondary 
site’s PI of the event.  This notification may take place via email if there is not yet 
enough information for a formal written report (using either an FDA MedWatch form if 
required or an institutional SAE reporting form if not).  A formal written report must be 
sent to the Washington University PI and research coordinator within 10 working days 
of the occurrence of the event or notification of the secondary site’s PI of the event.  The 
death of a research participant that qualifies as a reportable event should be reported 
within 1 working day of the occurrence of the event or notification of the secondary 
site’s PI of the event. 
 


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The research team at a secondary site is responsible for following its site’s guidelines for 
reporting applicable events to its site’s IRB according to its own institutional guidelines. 
 
10.8 
Reporting to Secondary Sites 
 
The Washington University PI (or designee) will notify the research team at each 
secondary site of all reportable events that have occurred at other sites (as described in 
Section 7.6) within 10 working days of the occurrence of the event or notification of the 
PI of the event.  This includes events that take place both at Washington University and 
at other secondary sites, if applicable. 
 
10.9 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER 
FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the investigator to report any unanticipated problem to the FDA as 
follows: 
 
 Report any unexpected fatal or life-threatening adverse experiences associated 
with use of the drug (i.e., there is a reasonable possibility that the experience may 
have been caused by the drug) by telephone or fax no later than 7 calendar days 
after initial receipt of the information.  A life-threatening adverse experience is 
defined as any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it 
does not include a reaction that, had it occurred in a more severe form, might have 
caused death. 
 Report any serious, unexpected adverse experiences, as well as results from 
animal studies that suggest significant clinical risk within 15 calendar days after 
initial receipt of this information.  A serious adverse drug experience is defined as 
any adverse drug experience occurring at any dose that results in any of the 
following outcomes: 
o Death 
o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial 
disruption of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 
o Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical 
intervention to prevent one of the outcomes listed above 
An unexpected adverse drug experience is defined as any adverse drug 
experience, the specificity or severity of which is not consistent with the current 
investigator brochure (or risk information, if an IB is not required or available). 


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All MedWatch forms will be sent by the investigator or investigator’s team to the FDA at 
the following address or by fax: 
 
Food and Drug Administration 
Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
Secondary sites must submit a completed MedWatch form to the Washington University 
PI and research coordinator within 4 calendar days (for fatal or life-threatening adverse 
experiences) or 11 calendar days (for serious, unexpected adverse experiences).  The 
Washington University PI will be responsible for submitting all MedWatch forms from 
secondary sites to the FDA within the timeframes specified above. 
 
10.10 Timeframe for Reporting Required Events:  
 
Reportable adverse events will be tracked for 30 days following the last day of study 
treatment, or until the start of a new chemotherapy/treatment, whichever comes first. 
 
Deaths 
Any reportable death while on study or within 
30 days of study 
Immediately, within 24 hours, to PI 
and the IRB 
Any reportable death while off study  
Immediately, within 24 hours, to PI 
and the IRB  
Adverse Events/Unanticipated Problems 
Any reportable adverse events as described in 
Sections 10.1 and 10.2 (other than death) and 
10.7 
Immediately, within 24 hours to PI 
and within 10 working days to the 
IRB, and within 7 or 15 days to the 
FDA  
All adverse events are to be reported per 
protocol regardless of attribution should be 
submitted cumulatively  
Include in DSM report 
Noncompliance and Serious Noncompliance 
All noncompliance and serious noncompliance 
as described in Sections 10.3 and 10.4 
Immediately, within 24 hours, to PI 
and within 10 working days to the IRB 


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11.0 
DATA SAFETY MONITORING PLAN 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, 
the Principal Investigator will provide a Data and Safety Monitoring (DSM) report to the 
Washington University Quality Assurance and Safety Monitoring Committee (QASMC) semi-
annually beginning six months following activation of the first secondary site.  
 
The Principal Investigator will review all patient data at least every six months, and provide a 
semi-annual report to the QASMC. This report will include: 
 HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
 Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of 
study 
 History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol 
exceptions, error, or breach of confidentiality including start/stop dates and reason 
 Study-wide target accrual and study-wide actual accrual including numbers from 
participating sites 
 Protocol activation date at each participating site 
 Average rate of accrual observed in year 1, year 2, and subsequent years at each 
participating site 
 Expected accrual end date and accrual by site 
 Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
 Measures of efficacy 
 Early stopping rules with supporting data and list the number of participants who 
have met the early stopping rules 
 Summary of toxicities at all participating sites 
 Abstract submissions/publications 
 Summary of any recent literature that may affect the safety or ethics of the study  
 
Until such a time as the first secondary site activates this protocol, a semi-annual DSM report to 
be prepared by the study team will be submitted to the QASM Committee beginning 6 months 
after study activation at Washington University. 
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC 
according to institutional guidelines. 
 


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12.0 
AUDITING 
 
Since Washington University is the coordinating center, each site will be audited annually by 
Siteman Cancer Center personnel (QASMC) unless the outside institution has an auditing 
mechanism in place and can provide a report. The outside sites will be asked to send copies of all 
audit materials, including source documentation.  The audit notification will be sent to the 
Washington University Research Patient Coordinator, who will obtain the audit materials from 
the participating institution. 
 
Notification of an upcoming audit will be sent to the research team one month ahead of the audit. 
Once accrual numbers are confirmed, and approximately 30 days prior to the audit, a list of the 
cases selected for review (up to 10 for each site) will be sent to the research team. However, if 
during the audit the need arises to review cases not initially selected, the research team will be 
asked to provide the additional charts within two working days. 
 
Additional details regarding the Auditing Policies and procedures can be found at 
http://www.siteman.wustl.edu/uploadedFiles/Research_Programs/Clinical_Research_Resources/
Protocol_Review_and_Monitoring_Committee/QASMCQualityAssurcance.pdf  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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13.0 
MULTICENTER REGULATORY REQUIREMENTS 
 
Washington University requires that each participating site sends its informed consent document 
to be reviewed and approved by the Washington University Regulatory Coordinator (or 
designee) prior to IRB/IEC submission.    
 
Each participating institution must have the following documents on file at Washington 
University prior to first subject enrollment: 
 Documentation of IRB approval of the study in the form of a letter or other official 
document from the participating institution’s IRB.  This documentation must show 
which version of the protocol was approved by the IRB. 
 Documentation of IRB approval of an informed consent form. The consent must 
include a statement that data will be shared with Washington University, including 
the Quality Assurance and Safety Monitoring Committee (QASMC), the DSMC (if 
applicable), and the Washington University study team. 
 Documentation of FWA, signed FDA Form 1572, and signed and dated CVs of all 
participating investigators. 
 Documentation of training in protection of human subjects by all participating 
investigators. 
 Protocol signature page signed and dated by the investigator at each participating site. 
 
The Principal Investigator is responsible for disseminating to the participating sites all study 
updates, amendments, reportable adverse events, etc.  There will be one current version of the 
protocol document at any given time and each participating institution will utilize that document.  
Protocol/consent modifications and IB updates will be forwarded electronically to the secondary 
sites within 2 weeks of obtaining Washington University IRB approval with acknowledgement 
of receipt requested.  Secondary sites are to submit protocol/consent/IB modifications to their 
local IRBs within 4 weeks of receipt, and confirmation of submission must be forwarded to the 
appropriate contact person on the Washington University study team at the time of submission.  
Upon the secondary sites obtaining local IRB approval, documentation of such shall be sent to 
the Washington University study team within 2 weeks of receipt of approval. 
 
Documentation of participating sites’ IRB approval of annual continuing reviews, protocol 
amendments or revisions, all SAE reports, and all protocol violations/deviations/exceptions must 
be kept on file at Washington University. 
 
The investigator or a designee from each institution must participate in a regular conference call 
to update and inform regarding the progress of the trial. 
 
 


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14.0 
STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
 
14.1 
Analysis Populations 
 
Intent-to-Treat - The intent-to-treat (ITT) population will be defined as all patients who 
receive at least one dose of decitabine.  
 
Efficacy-Evaluable - The efficacy-evaluable (EE) population will be defined as all 
patients who receive at least one cycle of treatment.   
 
Safety - The safety population will be defined as all patients with at least one dose of 
decitabine. 
 
14.2 
Statistical Analysis 
 
14.2.1 Descriptive Analyses 
 
Patient Disposition: The number of patients discontinued, the reasons for 
discontinuation, and the number of cycles administered will be summarized. 
 
Demographics and Baseline Characteristics: Descriptive summary statistics will 
be provided for demographic and important baseline characteristics.  For 
continuous variables, the number of patients, mean, standard deviation, median, 
minimum and maximum will be provided.  For categorical variables, the number 
and percent of patients in each demographic/characteristic category will be 
summarized. 
 
14.2.2 Primary Endpoint Analysis 
 
The primary end-point of this phase II study is the correlation biomarkers with 
overall response in AML/MDS patients to decitabine. 
 
We will correlate the presence of patient specific mutations (especially mutations 
in DNMT3A, IDH1, IDH2, and TET2) with overall response rates. Mutation rates 
in these genes and in other patient-specific genes will be correlated with overall 
response and their respective 95% confidence intervals will be provided. 
 
Statistical considerations. The relationship between AML/MDS-associated 
mutations and the responsiveness to decitabine will be described by contingency 
tables and assessed using Chi-square test or Fisher’s exact test as appropriate. The 
differentially expressed genes between responders and non-responders will be 
determined using the significance analysis of microarray (SAM). Multivariate 
logistic regression will also be fit to adjust the potential confounding effects of 
other demographic and clinical characteristics. The resultant critical P values will 
be corrected for multiple testing.  Since the Bonferroni correction is known to be 
too conservative, a permutation analysis (e.g., 10,000 random assignments for 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
Page 41 of 51 
responsive status) will be used to derive an empirical critical P value.  The new 
critical p-value is the number of permutation tests which yield a lower p-value 
than the original test divided by the total number of permutation tests.[61]   
 
A power analysis was performed using Chi-square test for the association 
between mutation and response to decitabine. The frequency of DNMT3A, 
IDH1/2, and TET2 mutations in AML and MDS patients are each between 15% 
and 30% in multiple cohorts.[29-37] Table 3 shows the minimal detectable 
differences of response rates between mutant and wild-type cases, assuming an 
expected overall 45% CR/CRi rate and 80% power. For an average of 20% 
frequency of a mutated gene, for example, 100 patients will provide 80% power 
to detect 30% difference at a 2-sided 0.1 alpha error. The purpose of this study is 
to determine whether any of 
these mutations result in a 
clinically 
significant 
association 
that 
would 
warrant 
pre-treatment 
testing. We believe that a 
reasonable cut off would be 
if at least 30% of clinical 
response could be attributed 
to the presence of a specific 
mutation. 
However, 
we 
anticipate that more power 
could be achieved as we 
assess patient groups with 
combined 
characteristics 
(e.g. DNMT3A mutation or 
loss 
of 
DNMT3A 
expression). In addition, we 
expect that the majority of the enrolled 125 patients can be used in the analysis 
(since all of our molecular information will be acquired within 30 days of 
enrollment, we anticipate a drop-out rate of 20% or less). 
 
14.2.3 Secondary Endpoint Analyses 
 
The overall response rate (CR/CRi/PR) and complete response rate (CR/CRi) will 
be compared with historical controls[2], and the 95% confidence intervals will be 
provided. 
 
The rate of patient specific mutation clearance after 10, 28, and 56 days of therapy 
will be determined using an Illumina based platform. We will compare the rate of 
mutation clearance between the patients who achieve a CR/CRi after 4 cycles and 
those who do not. We anticipate that we will identify between 6 and 25 exonic 
SNVs per case (the range obtained with >200 AML samples sequenced to date 
with whole genome or exome sequencing), and that the expected 95% confidence 
Table 3. Detectable mutation associated response rates (80% power, α = 0.05) 
 
Total 
N 
% of patients 
with a given 
mutation  
Detectable 
response 
difference 
Sensitivity of mutation to 
predict response at minimum 
detectable response difference* 
100 
15%  
20%  
30% 
40% 
50% 
0.40 
0.35 
0.30 
0.27 
0.26 
80% 
75% 
67% 
61% 
58% 
* Assuming the overall response  rate (CR/Cri) of 45%   


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
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interval of the mean variant allele frequency will be ±2.56% and ±1.25%, 
respectively, if we assume an anticipated standard deviation of 3.2% within the 
same clone. The change of tumor burden over time and its association with 
clinical response will  be assessed using 2-way ANOVA for repeated 
measurement data, followed by the ad-hoc multiple comparisons for between-
group differences (e.g., responders vs. non-responders) at given time points as 
well as the between-time differences within each group. The resultant critical P 
values will also be corrected using permutation analysis for multiple testing. 
 
The steady-state plasma decitabine concentration on day 4 ± 1 will be measured 
and correlated with clinical overall response. 
 
The percent decrease in bone marrow methylcytosine will be calculated on day 
10±1. We will compare this reduction with previously published data and between 
the patients who achieve CR/CRi and those who do not. The correlation of peak 
plasma levels of decitabine with response will be assessed using 2-sample t-test or 
Mann-Whitney non-parametric rank-sum test as appropriate. The change of total 
bone marrow DNA methylcytosine from baseline and its association with both 
steady-state plasma drug levels and response will be assessed using 2-way 
ANOVA for repeated measurement data, followed by the ad-hoc multiple 
comparisons for between-group differences (e.g., responders vs. non-responders) 
and between-time differences. Similar analyses will be performed for the 
correlation of expression profiles and SNP analysis with pharmacokinetic data.   
 
Statistical power justification.  A power analysis was performed using two-
sample t-test for between-group difference and paired t-test for the change over 
time. For tumor clearance, this will provide > 99% power to detect a 10% 
difference in the means at two separate time points for clones with 6 or more 
variants, and > 98% power to detect a 15% difference if there are 3 or more 
variants per clone with α = 0.05 (power will decrease to 91% and 82%, 
respectively, in cases where the standard deviation is 4.7%, the maximum we 
have observed thus far). For pharmacologic correlations, we anticipate 85% 
power to determine a 25 ng/ml difference between the average Cmax of 45 
evaluable responders and 55 evaluable non-responders with an anticipated 
average Cmax of 70 ng/ml and a standard deviation of 40 ng/ml within the entire 
cohort, and with α = 0.05.[22] We believe a less significant difference would not 
be sufficient to warrant application in clinical decision making (i.e. whether to 
continue therapy for more than 1 cycle). We anticipate 99% power to determine a 
difference of 20% in the reduction of bone marrow methylcytosine between 45 
evaluable responders and 55 evaluable non-responders with an expected standard 
deviation of 15% and α = 0.05.[28, 66-68] The difference in methylcytosine 
reduction previously observed was 30% between patients achieving CR/PR and 
those with only hematologic improvement.[28]  
 
Time to relapse and time to death will also be listed for each patient. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
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14.2.4 Safety Analysis 
 
Extent of Exposure – Duration (number of cycles/infusions) of decitabine doses 
(cumulative and intensity) will be summarized. 
 
Adverse Events - All adverse events will be coded using NCI CTCAE v. 4.0.  The 
incidence of treatment-emergent adverse events (number and percent of patients 
reporting the adverse event at least once during the study) will be summarized.  In 
addition, adverse events will be summarized by investigator attribution of 
relationship to study medication and by grade.  Similar summaries will be 
provided by treatment cycle. 
 
Clinical Laboratory Evaluation – Applicable laboratory parameters will be 
graded according to NCI CTCAE version 4.0. The incidence of maximum grade 
(number and percent of patients experiencing the maximum grade during the 
study) will be summarized for each laboratory parameter.  Similar summaries will 
be provided by treatment cycle.  Graphical displays of select hematological 
parameters will also be provided. 
 
Vital Signs, Physical Findings, and Other Safety Observations – Vital signs, 
findings from physical examinations, ECOG scores and weights will be presented 
in listing format. 
 
14.2.5 Description of Planned Subgroup Analyses 
 
The following subgroups (if sufficient numbers of patients) will be explored with 
respect to the primary and overall survival endpoints. 
 Age (60-69 years old vs ≥ 70 years old) 
 Cytogenetics 
 Patients with AML-associated mutations in DNMT3A, TET2, IDH1, or 
IDH2 vs those who do not have somatic variants in these genes. 
 
14.3 
Sample Size 
 
We anticipate initially enrolling 25 patients at Washington University. Once we establish 
a routine exome analysis with validation and calculation of the rate of patient-specific 
mutation clearance, we will enroll an additional 100 patients from at least 2 additional 
centers.  
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
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15.0 
DATA SUBMISSION SCHEDULE 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to registration 
Patient Registration Form 
Disease Diagnosis Form 
Inclusion Criteria Worksheet 
Exclusion Criteria Worksheet 
 
Prior to starting treatment 
Treatment Record Form 
To be maintained throughout a patient’s treatment 
cycle.  The complete record is due at completion of 
treatment. 
Bone Marrow Sample Collection 
Form 
To be maintained throughout the course of the 
study at designated bone marrow sample 
collections time points (See Appendix 2). 
Pathology: Bone Marrow Biopsy 
To be completed upon the return of pathology 
report for each bone marrow biopsy. 
Study Calendar Form 
To be completed with the date of each time point 
upon occurrence. 
Labs Form 
To be completed upon the return of laboratory 
reports for each study time point (See Appendix 2) 
Dose Modifications 
To be maintained throughout the course of the 
study if dose modifications are performed. 
Therapy Response Form 
To be filled out upon remission (prior to, and/or on 
therapy) 
End of Study Form 
 
Due at the completion of the EOS visit, at the time 
of death, and/or at the time that the patient is lost to 
follow-up 
Follow Up Form 
 30 days post date of last decitabine dose. 
 Every 6 months for 2 years after the last dose of 
study drug. 
Record of Adverse Events 
At the time of any AE 
 
Any queries generated by Washington University must be responded to within 28 days of receipt 
by the participating site.  The Washington University research team will conduct a regular 
review of data status at all secondary sites, with appropriate corrective action to be requested as 
needed. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
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Appendix 1 
ECOG Performance Scale 
Activity Status 
Description 
0 
Asymptomatic, fully active, and able to carry on all predisease 
performance without restrictions. 
 
1 
Symptomatic, fully ambulatory but restricted in physically strenuous 
activity and able to carry out performance of a light or sedentary 
nature, eg, light housework, office work. 
 
2 
Symptomatic, ambulatory and capable of all self-care but unable to 
carry out any work activities.  Up and about more than 50% of 
waking hours: in bed less than 50% of day. 
 
3 
Symptomatic, capable of only limited self-care, confined to bed or 
chair more than 50% of waking hours, but not bedridden. 
 
4 
Completely disabled.  Cannot carry on any self-care.   Totally 
bedridden. 
 
5 
Dead 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
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Appendix 2 
Study Flow Chart 
Study Flow Chart 
Test 
Pre-
study1 
Base-
line2 
All 
Cycles 
Day 1 
All Cycles 
Days 1-103 
Cycle 1 Day 
4 ± 1 
 
Cycle 1 
Day 10 ± 1 
and 28 ± 4 
Every 
week 
Every 
2 
weeks 
Even 
cycles 
Day 28±4 
End of 
Study 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Medical History 
 
X 
 
 
 
 
 
 
 
 
Physical Exam 
 
Xa 
Xb 
 
 
 
 
 
 
X 
Transfusion 
Requirementsc 
 
Xc 
Ongoing 
Bone Marrow 
Aspirate and Biopsy 
Xd 
 
 
 
 
Xd 
 
 
Xd 
Xd 
Flow Cytometrye 
X 
 
 
 
 
 
 
 
 
 
Peripheral blood for 
correlative studies 
 
 
 
 
X 
 
 
 
 
 
Pregnancy test 
 
X 
 
 
 
 
 
 
 
 
CBC with differential 
and platelets 
 
X 
X 
 
 
 
Xf 
 
 
X 
Serum Chemistriesg 
 
X 
X 
 
 
 
 
Xf 
 
X 
Decitabine 
administrationh 
 
 
 
X 
 
 
 
 
 
 
Adverse Events  
 
 
Ongoing 
Post-study follow-up 
 
 
 
 
 
 
 
 
 
Xi 
1 To be performed within 14 days of the first dose of decitabine.  
2 To be performed within 7 days of the first dose of decitabine.  
3 Decitabine may be reduced to Days 1-5 or Days 1-3. See Section 6.3 for details.  
a To include height, weight, and ECOG. 
b To include weight. 
c Care should be given to record the frequency of transfusions from 8 weeks prior to first dose of study drug through 
the duration of the study. 
d Bone marrow biopsy and aspirate should be performed at baseline, at Cycle 1 Day 10 ± 1, and at  Day 28 ± 4 of 
Cycles 1, 2, 4 and 6 for clinical disease assessment. Patients who achieve with clinical response (CRi or better) and are 
receiving cycles every 6 weeks, will undergo bone marrow biopsies on day 42 ± 4. Following Cycle 6, bone marrow 
biopsies should be performed at the discretion of the treating physician to assess for relapse or progression. Standard 
bone marrow procurement procedures will be followed for collection of the tissue. To include: correlative studies 
(collected under HRPO# 201011766), morphologic assessment, cytogenetics and FISH at all time points. A skin 
biopsy sample (collected under HRPO# 201011766) is required at pre-study, but may have been collected months or 
even years prior to first dose of decitabine.  
e On bone marrow or peripheral blood.  
f After Cycle 1, these labs may be obtained every other week (CBC) or once a month (Chemistries) at the discretion of 
the treating physician. 
g Serum chemistries include bicarbonate, calcium, glucose, total protein, albumin, BUN, potassium, chloride, sodium, 
creatinine, total bilirubin, AST, ALT, alkaline phosphatase, and LDH. 
h Decitabine treatment will be 20 mg/m2 over a 1-hour IV infusion on consecutive Days 1-10 every 28-day cycle.   The 
dose will be calculated based on actual patient weight at the beginning of each cycle. Patients may be premedicated 
with lorazepam, prochlorperazine, palonosetron, or ondansetron at the treating physician’s discretion. 
i Patients will be followed for  30 days following the date of the last decitabine dose to monitor for toxicity, survival 
and response. Thereafter, the investigator or designee will record information on subsequent therapy, disease 
progression, and death, every 6 months for 2 years after first dose of study drug or until the patient is lost to follow-up.


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
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(Decitabine) in patients with metastatic lung cancer. Anticancer Drugs, 1997. 
8(4): p. 358-68. 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 09/11/14  
Page 51 of 51 
62. 
Aparicio, A., et al., Phase I trial of continuous infusion 5-aza-2'-deoxycytidine. 
Cancer Chemother Pharmacol, 2003. 51(3): p. 231-9. 
63. 
Bryan, J., et al., Pharmacokinetic evaluation of decitabine for the treatment of 
leukemia. Expert Opin Drug Metab Toxicol, 2011. 7(5): p. 661-72. 
64. 
Scandura, J.M., et al., Phase 1 study of epigenetic priming with decitabine prior 
to standard induction chemotherapy for patients with AML. Blood, 2011. 118(6): 
p. 1472-80. 
65. 
Westfall, P.H. and S.A. Young, Resampling-based multiple testing: examples and 
methods for p-value adjustment. 1993, New York: John Wiley & Sons, Inc. 
66. 
Samlowski, W.E., et al., Evaluation of a 7-day continuous intravenous infusion of 
decitabine: inhibition of promoter-specific and global genomic DNA methylation. 
J Clin Oncol, 2005. 23(17): p. 3897-905. 
67. 
Blum, W., et al., Phase I study of decitabine alone or in combination with 
valproic acid in acute myeloid leukemia. J Clin Oncol, 2007. 25(25): p. 3884-91. 
68. 
Yang, A.S., et al., DNA methylation changes after 5-aza-2'-deoxycytidine therapy 
in patients with leukemia. Cancer Res, 2006. 66(10): p. 5495-503. 
69.       Cheson, B.D., et al., Clinical application and proposal for modification of the 
International Working Group (IWG) response criteria in myelodysplasia. Blood, 
2006. 108(2): p. 419-25. 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
 
NCT# NCT01687400 
 
 
Document Version Date:  03/10/2014 
 
IRB Approval Date:  03/14/2014 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
 
 
Genomic Predictors of Decitabine Response in AML/MDS 
 
Washington University School of Medicine 
Division of Oncology 
660 South Euclid Avenue, Box 8056 
St. Louis, MO 63110 
 
Protocol #: 201210102 
Protocol Version Date: 03/10/14 
 
Principal Investigator 
John Welch, M.D./Ph.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-2626 
Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Sub-Investigator 
Timothy Ley, M.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-8832 
 
Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Pathology 
Jeffery M. Klco, M.D., Ph.D. 
Department of Anatomic and Molecular Pathology 
Telephone: (314) 362-8832 
Email: klcoj@wustl.edu 
 
Statistician 
Feng Gao, Ph.D. 
Division of Biostatistics 
Telephone: (314) 362-3682 
E-mail: feng@wubios.wustl.edu 
 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to obtain 
informed consent, may not be disclosed to another party unless law or regulations require such disclosure.  Persons 
to whom the information is disclosed must be informed that the information is confidential and may not be further 
disclosed by them. 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
Study Schema: 
 
 
Patients with progressive disease after two cycles (defined as a clear increase in bone marrow blast count, typically a 
> 50% increase in bone marrow blasts over baseline) should be removed from study. 
Patients with stable disease may continue on study and should undergo a diagnostic bone marrow biopsy at the end 
of even numbered cycles. 
Once AML patients achieve morphologic complete remission with incomplete blood count recovery (CRi) or MDS 
patients achieve a marrow complete response (Marrow CR) as defined in Section 9.0, decitabine 
administration may be decreased to five consecutive days to limit myelosuppressive toxicityat the 
discretion of the treating physician. After two cycles of five consecutive days of treatment, if the patient 
meets the definition of CR with complete cytogenetic response as defined in Section 9.0, administration 
may further be decreased to three consecutive days per cycle at the discretion of the treating physician. 
Sample collection for correlative studies: 
Bone marrow biopsies should be performed at baseline, at Cycle 1 Day 10 ± 1, and at Day 28 ± 4 of Cycles 
1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be performed at the discretion of the 
treating physician to assess for relapse or progression.  
Peripheral blood samples should be collected on Day 4± 1 of Cycle 1. Collection should be obtained 45 ± 
15 minutes after decitabine infusion is initiated.
20 
mg/m
2/day
Cycle 1 
20 
mg/m
2/day
Cycle 2+ 
Day 1 
Day 28 
BM Bx 
PB 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
Genomic Predictors of Decitabine Response in AML/MDS 
Table of Contents 
Section 
Page 
1.0 INTRODUCTION ...................................................................................................................6 
1.1 
Rationale .............................................................................................................................6 
1.2 
Decitabine and Hypomethylating agents in AML/MDS ....................................................8 
1.2.1 
Pharmacokinetics and Metabolism of Decitabine ....................................................10 
1.2.2 
Experience with extended dosing schedules ............................................................11 
1.3 
Correlative Studies ...........................................................................................................12 
2.0 OBJECTIVES ........................................................................................................................14 
2.1 
Primary Objective .............................................................................................................14 
2.2 
Secondary Objectives .......................................................................................................14 
3.0 PATIENT SELECTION ........................................................................................................15 
3.1 
Inclusion Criteria ..............................................................................................................15 
3.2 
Exclusion Criteria .............................................................................................................15 
3.3 
Inclusion of Women and Minorities .................................................................................16 
4.0 REGISTRATION PROCEDURES .......................................................................................17 
4.1 
Confirmation of Patient Eligibility ...................................................................................17 
4.2 
Patient Registration in the Siteman Cancer Center Database ...........................................17 
4.3 
Assignment of UPN ..........................................................................................................17 
5.0 INVESTIGATIONAL PLAN ...............................................................................................18 
5.1 
Summary of Study Design................................................................................................18 
5.2 
Study Procedures ..............................................................................................................18 
5.2.1 
Pre-study Procedures ................................................................................................18 
5.2.2 
Baseline Evaluation ..................................................................................................19 
5.2.3 
Day 1 of Each Cycle ................................................................................................19 
5.2.4 
Cycle 1 Days 1-10 ....................................................................................................19 
5.2.5 
Cycle 1 Day 4 ± 1 ....................................................................................................19 
5.2.6 
Cycle 1 Day 10 ± 1 ..................................................................................................20 
5.2.7 
Weekly .....................................................................................................................20 
5.2.8 
Every Other Week ....................................................................................................20 
5.2.9 
Cycles 1, 2, 4 and 6Day 28 ± 4 days ........................................................................20 
5.2.10 
End-of-Study Procedures .........................................................................................20 
5.2.11 
Post-study Follow-up ...............................................................................................20 
5.3 
Duration of Therapy .........................................................................................................21 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
5.4 
Concomitant Therapy and Supportive Care Guidelines ...................................................21 
5.4.1 
Chemotherapy ..........................................................................................................21 
5.4.2 
Growth Factors .........................................................................................................21 
5.4.3 
Transfusions .............................................................................................................22 
5.4.4 
Prophylactic Antimicrobial Agents ..........................................................................22 
5.4.5 
Radiotherapy ............................................................................................................22 
6.0 DOSE MODIFICATIONS ....................................................................................................23 
6.1 
Delay for Cytopenia or Infection ......................................................................................23 
6.2 
Delay for Organ Dysfunction ...........................................................................................23 
6.3 
Modification in Dose or Schedule for Treatment Response ............................................23 
7.0 PHARMACEUTICAL INFORMATION .............................................................................24 
7.1 
Decitabine .........................................................................................................................24 
7.1.1 
Study Drug Preparation ............................................................................................24 
7.1.2 
Supplier ....................................................................................................................24 
7.1.3 
Toxicities ..................................................................................................................24 
8.0 CORRELATIVE STUDIES SAMPLE COLLECTIONS .....................................................26 
9.0 EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES................................27 
9.1 
Response Criteria - AML .................................................................................................27 
9.2 
Response Criteria - MDS..................................................................................................28 
9.2.1 
Hematologic Improvement (HI) ...............................................................................28 
9.3.1 Cytogenetic Response ........................................................................................................29 
9.3 
Guidelines for Evaluation of Disease ...............................................................................29 
9.4 
Other Secondary Efficacy Measures ................................................................................29 
9.4.1 
Treatment Failure, Overall Survival and Event-free Survival .................................29 
9.4.2 
Duration of Remission and Relapse-free Survival for 
Patients Having Complete Remission ......................................................................29 
9.4.3 
Safety .......................................................................................................................30 
10.0 ADVERSE EVENT REPORTING .......................................................................................31 
10.1 Adverse Events (AEs) ......................................................................................................31 
10.2 Unanticipated Problems....................................................................................................31 
10.3 Noncompliance .................................................................................................................31 
10.4 Serious Noncompliance ....................................................................................................31 
10.5 Protocol Exceptions ..........................................................................................................32 
10.6 Reporting to the Human Research Protection Office (HRPO) and 
the Quality Assurance and Safety Monitoring Committee 
(QASMC) at Washington University: ..............................................................................32 
10.7 Reporting to the FDA .......................................................................................................32 
10.8 Timeframe for Reporting Required Events: .....................................................................33 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
11.0 DATA SAFETY MONITORING PLAN ..............................................................................34 
12.0 STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE .........................................35 
12.1 Analysis Populations ........................................................................................................35 
12.2 Statistical Analysis ...........................................................................................................35 
12.2.1 
Descriptive Analyses ................................................................................................35 
12.2.2 
Primary Endpoint Analysis ......................................................................................35 
12.2.3 
Secondary Endpoint Analyses .................................................................................36 
12.2.4 
Safety Analysis ........................................................................................................38 
12.2.5 
Description of Planned Subgroup Analyses .............................................................38 
12.3 Sample Size ......................................................................................................................38 
13.0 DATA SUBMISSION SCHEDULE .....................................................................................39 
14.0 REFERENCES ......................................................................................................................42 
  


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
1.0 
INTRODUCTION 
 
1.1 
Rationale   
 
Personalized therapy for cancer requires that 1) patient-specific factors (e.g. patient-
specific mutations, expression profiles, or pharmacogenomics) influence treatment 
algorithms, and 2) once therapy is initiated, in vivo responsiveness is used to guide 
further therapy. The modern era of genomics has provided significant insights into cancer 
pathogenesis by describing patterns of germline and somatic variants, as well as defining 
disease-specific patterns of gene expression. However, the application and integration of 
this data with therapeutics remains a difficult task. 
 
Decitabine is a commonly used single-agent therapy for patients with acute myeloid 
leukemia (AML) and/or myelodysplastic syndrome (MDS). Historically, it has yielded 
clinical complete responses in approximately 25% of patients across diverse clinical 
trials, it is well tolerated, and it can be given as in the outpatient setting. However, the 
majority of patients do not respond to decitabine, the molecular etiology of decitabine 
sensitivity is unknown, and there are no algorithms to predict patient responsiveness a 
priori or even within one month of starting therapy.  
 
AML and MDS are two closely related hematological disorders characterized by 
ineffective hematopoiesis and malignant expansion of clonal myeloid cells.  Both AML 
and MDS frequently occur in elderly patients and some cases are secondary and/or 
related to long-term bone marrow damage after radiation or chemotherapy. Current 
therapy for AML consists of cytotoxic chemotherapy followed by consolidation 
chemotherapy or stem cell transplantation. Using these approaches, current cure rates 
may be as high as 60% for patients younger than 60 with good risk cytogenetics (e.g. 
t(15;17), t(8;21), inv(16)).[4]  Outcomes in patients ≥ 60 years old and with relapsed 
disease remain poor with 2 year survival of only ~25%.[5] Historically, the available 
treatment for MDS has been symptomatic and has not been shown to be effective in 
producing sustained improvement in hematopoiesis or in delaying leukemic evolution[6, 
7].  
 
Current AML therapy is poorly tolerated in elderly and pre-treated patients due to 
hematologic and non-hematologic toxicities; following such therapy, patients typically 
require 3-4 weeks of hospitalization for treatment of neutropenic fevers and for regular 
transfusions. Even among the relatively healthy patients enrolled in clinical trials, 
induction mortality in the elderly can be as high as 20-40% following standard induction 
therapy,[8-11] and moderate to severe non-hematologic toxicity occurs in at least an 
additional 30% of patients.[9, 12-14] Consequently, many older AML patients are not 
offered traditional intensive chemotherapy, but receive supportive care alone.[15]  More 
effective and less toxic therapies are needed to provide durable remissions in a significant 
proportion of elderly AML in populations. 
 
Decitabine has been studied in MDS patients and in elderly and/or relapsed AML 
patients. It has been found to be reasonably well tolerated, can be given in the outpatient 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
setting, and has overall response rates of 20-45%.[2, 3, 16-20] Our own experience has 
been of CR/PR rates of 25%, [2] and this is typical of many other groups for both MDS 
and AML patients (reviewed in [21]  Decitabine is an S-phase depended drug.  [22-24] 
However, decitabine is usually administered in one hour daily infusions during 3 to 5 
consecutive days (too short to expose decitabine to most AML blasts during at least 2 
cycles of replication, which divide every 2 to 7 days).[25-27]  Recent experience with 
increasing the number of infusions per cycle from 5 consecutive days to 10 consecutive 
days has improved overall response rates to 67%.[3] Response to decitabine can be slow, 
sometimes requiring 3 to 5 cycles before maximum response is observed.[2] As a 
consequence, many patients are exposed to prolonged therapy, gaining little benefit, 
before determining resistance. There are currently no biomarkers that predict response to 
decitabine. 
 
Decitabine is a cytosine analogue that cannot be methylated. Incorporation of decitabine 
into DNA prevents DNA methylation at cytosine residues and derepresses epigenetically 
silenced gene expression. Therefore, the reduction in total genomic DNA methylcytosine 
content acts as a marker of effective decitabine dosing. Indeed, two groups have recently 
correlated effective methylcytosine reduction and gene expression derepression to 
response (Figure 1.1).[3, 28] 
 
 
 
   
Figure 1.1. From Shen et al JCO (2010) 28:611 and Blum et al JCO (2007) 25: 3884. 
 
We and others have recently used whole genome sequencing to identify recurrent, 
somatic mutations in AML and MDS patients in four genes that are involved in 
methylcytosine regulation: DNMT3A, IDH1, IDH2, and TET2.[29-37] Collectively, 
nearly half of all AML/MDS patients have a mutation in one of these four genes.  
DNMT3A mutations are associated with an adverse outcome in AML patients treated with 
cytotoxic chemotherapy.[35, 38] However, it is not yet clear whether the presence of 
mutations in one or more of these genes is associated with response or resistance to 
decitabine. 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
In this study, we will treat patients with the current state-of-the-art decitabine 
schedule.[3] We will perform exome sequencing on all patients and correlate response 
with mutation status, rate of mutation clearance, expression profile, plasma decitabine 
levels, and DNA methylcytosine content reduction. This approach will comprehensively 
assess genomic and pharmacologic parameters that could clinically predict patient 
response to decitabine.  
 
In this study, we will combine analysis of patients with AML and MDS. Recent genomic 
analysis has shown that AML and MDS have shared mutations in many of the same 
genes, shared patterns of mutation acquisition, and shared patterns of mutation 
evolution.[39, 40] Furthermore, MDS, like AML, presents with clonal hematopoiesis, 
even when the morphologic blast count is zero.[36]Because this study will identify and 
track the rate of clearance of patient specific mutations, and because decitabine has been 
shown to have efficacy in both AML and MDS patients, [2, 3, 16-19, 41-45] combining 
analysis of AML and MDS patients is reasonable.  Asymptomatic MDS patients are 
excluded because these patients are typically followed with watchful-waiting rather than 
with chemotherapy. 
  
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
 
Methylation of cytosine in the CpG dinucleotide by DNA methyltransferase leads to 
transcriptional silencing of genes during normal development and has emerged as a 
significant mechanism for the loss of tumor suppressor gene expression in human cancer, 
including AML and MDS.[46, 47] Like azacitidine, decitabine (5-aza-2’-deoxycitidine) is 
incorporated into DNA during S phase and forms covalent adducts with DNA 
methyltransferase. The irreversible inhibition of this enzyme results in loss of 
methylation and is associated with differentiation.[48]  Decitabine is an approximately 5-
fold more potent inhibitor of DNA methyltransferase than azacitidine, which might 
explain why decitabine can induce differentiation of myeloid progenitors in vitro at lower 
doses than azacitidine. In addition to their differentiation inducing activity at low doses, 
both agents have a direct cytotoxic effect at higher doses (1-2 µM).[49]  
 
Decitabine has been investigated in hematologic malignancies using a range of doses and 
schedules.  It is currently FDA approved for the treatment of adults with MDS at a dose 
of 15 mg/m2 every 8 hours for 3 days, based on a Phase III study that demonstrated 
improved outcomes in the treatment arm compared to patients treated with supportive 
care alone.[15]  However, the overall response rate in this trial was modest (17%). Of 
note, 18% of the patients in this trial had 20-30% bone marrow blasts, meeting the 
current WHO criteria for AML.  Subsequent phase I and II studies in AML, using a 
variety of low-dose decitabine regimens, have reported overall response rates of 22-
49%.[2, 3, 16-19, 41-45] Comparisons of multiple low dose schedules have noted 
improved efficacy in the MDS population using a dose of 20 mg/m2 on Days 1-5 of a 28-
day cycle.[50, 51] Because of these results, we have recently explored this dosing 
schedule in the treatment of elderly patients with AML.[52] In this study, we observed an 
overall response rate of 24%, and an additional 44% of patients achieved stable disease 
during a median of 5 monthly cycles.[48] In this study, we noted that decitabine does not 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
act quickly and the median time to response was 3 months. These results are similar to 
the recently published experience in Germany treating 227 patients with 15 mg/m2 over 3 
hours TID for 3 days every 6 weeks, which resulted in a CR/PR rate of 26%.[16]  
 
A recent study using a prolonged administration schedule (20 mg/m2 IV over 1 hour on 
consecutive days 1-10) showed improved response (64% CR/PR) and improved time to 
respond (50% of patients had bone marrow blasts < 5% after 1 cycle of therapy).[3]  
These results are an improvement over previous experience by other groups using 
decitabine to treat elderly AML patients or patients with relapsed AML, and this 
experience is summarized below: 
Trials of decitabine in AML 
Status 
Other Therapy 
DAC Dose 
(mg x days) 
Evaluable 
CR 
de novo 
daunomycin1 
90 mg/m2 x 5 
6 
100% 
Salvage 
None4 
0.75-80 mg/kg 
16 
  
None5 
37-81 mg/kg 
6 
33% 
None7.  Ara-C 
Refractory 
Various 
4 
0 
AMSA7 
250-500 mg/m2 x 6 
10 
80% 
idarubicin or 
AMSA3 
250 mg/m2 x 6 
56 
39% 
Elderly 
None6 
270-360 mg/m2 x 3 
10 
30% 
None2 
45 mg/m2 x 3 
4 
50% 
None8 
20 mg/m2day 1-5,8-12 
50 
23% 
None9 
20 mg/m2 x 5 
55 
25% 
 
None10 
50 mg/m2 CIVI x 3 
29 
48% 
 
None11 
20 mg/m2 x 10 
53 
64% 
 
ATRA cycle 212 
15 mg/m2 TID x 3 
227 
26% 
1 Schwartsmann G.  Leukemia 1997; 11(Suppl 1):S28-31. 
2 Lubbert M. ASH 2002 Abstract #4601. 
3 Willemze R.  Leukemia.  1997; 11(Suppl 1):S24-27 
4 Rivard GE.  Leukemia Research 1981; 5:453-462. 
5 Momparler RL.  Pharmac Therapy 1985; 30:277-286.  
6 Petti MC.  Leukemia.  1993; 7(Suppl 1):36-41.  
7 Richel DJ.  Br. J. Cancer 1991; 64:144-148 
8 Issa, JP. Blood 2004. 103: 1635. 
9 Cashen AF. ASH Annual Meeting. 2008 abstract 560. 
10 Wijermans PW. Leukemia 1997. 11: Suppl 1, 19-23. 
11 Blum, W. PNAS 2010; 107:7473. 
12 Lubbert. Haematologica 2011. ePub 11/04/2011. 
 
In general, decitabine is well tolerated. Unlike conventional cytotoxic chemotherapy, 
decitabine can be administered in the outpatient setting. However, AML and MDS are 
aggressive hematologic malignancies. Cytopenias commonly result from AML/MDS 
inhibition of normal hematopoiesis, and often do not resolve until a complete response is 
achieved. Thus, neutropenic sepsis and thrombocytopenic bleeding remain significant 
problems regardless of therapy, especially during the first 4-8 weeks. In our experience 
treating elderly AML patients, neutropenia occurred in 24%, sepsis in 9%, and 
bacteremia in 7%.  Similar results have been observed in MDS patients.[20, 53, 54] As a 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
result, death is not uncommon in the first 8 weeks of therapy, occurring in 15-17% of 
patients, and most commonly resulting from infection and cytopenias related to disease 
and therapy.[16, 52, 55] While unsettling, these numbers are better than many historical 
patients treated with cytotoxic chemotherapy, and suggest a need for continued 
improvements in therapy.[9, 12]  
 
1.2.1 Pharmacokinetics and Metabolism of Decitabine 
 
In human subjects, after IV administration, decitabine displayed a distribution 
phase with a half-life of 7 minutes and a terminal phase elimination half-life of 10-
35 minutes as measured by bioassay.[56, 57]   The short plasma half-life is due to 
rapid inactivation of decitabine by deamination by liver cytidine deaminase.[23] 
Total body clearance (Clp) in humans was high with a mean value of 126 
mL/min/kg.[58]  
 
Among five adult patients with advanced solid tumors administered seven courses 
of 100 mg/m2 of decitabine by 1-hour infusions,[56] maximum plasma 
concentration ranged from 1 to 4 µM. Disappearance from plasma was biphasic 
with a rapid initial decline to less than 0.1 µM during the first hour post infusion 
and a slower decline over the next two hours to 6 nM, the level of detectability. 
Plasma half-life (meanSE) was estimated to be approximately 71 minutes in 
the initial phase (t½α) and 355 minutes in the second phase (t½β). The area under 
the plasma concentration-time curve was 40888 ng.hour/mL for these patients, 
with a peak plasma concentration of 2.010.44 µM (0.440.1 µg/mL), while mean 
volume of distribution (Vdss) and CLp was 4.591.42 L/kg and 12621 
mL/min/kg, respectively. The time course of DAC concentrations was similar in 1 
of 2 patients administered 75 mg/m2, while DAC was usually undetectable in the 
plasma of patients administered 25 to 60 mg/m2. 
 
Using a longer infusion time (up to 40 hours), Rivard et al.[57] reported a plasma 
concentration of 0.1 to 0.4 g/mL. With infusion times of 40-60 hours, at an 
infusion rate of 1 mg/kg/h, plasma concentrations of 0.43-0.76 µg/mL were 
achieved.[59]  The steady-state plasma concentration at an infusion rate of 
1 mg/kg/h was estimated to be 0.2-0.5 µg/mL. The half-life after discontinuing the 
infusion was 12-20 min.[57]  
 
Urinary excretion of unchanged decitabine was low, ranging from less than 0.01% 
to 0.9% of the total dose, and there was no relationship between excretion and dose 
or plasma drug levels. High clearance values and a total urinary excretion of <1% 
of the administered dose suggest that decitabine is eliminated rapidly and largely by 
metabolic processes, including extrahepatic metabolism.[60] 
 
The steady state plasma concentrations (Css) ranged from 0.31 to 0.39 µg/mL 
(1.27 to 1.60 µM) during the infusion of 100 mg/m² decitabine over a 6-hour 
period.[61] The range of concentrations during a 600 mg/m2 infusion over an 8-
hour period was 0.41-16 g/mL(1.68 to 4.76 µM).[60] These plasma 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
concentrations are in the same range as the in vitro concentrations that activated 
the expression of tumor suppressor gene p16 in three human non-small-cell lung 
cancer (NSCLC) cell lines.[61]  
 
Steady state decitabine levels of 0.1 to 0.2 µM were achieved in solid tumor 
patients dosed with 30 mg/m²/day and 40 mg/m²/day by continuous intravenous 
infusion over 72 hours.[62]  
 
Summary of published steady state serum levels are below and are in proportion to 
infusion rate (R2 = 0.98, p < 0.0001):[63] 
 
Rate 
Serum 
Concentration 
mg/m2/hr 
ng/ml 
nM 
5 
55 
241 
7.5 
80 
351 
15 
180 
789 
21 
182.4 
800 
22.5 
350 
1535 
42 
456 
2000 
84 
1140 
5000 
 
In our experience treating primary patient blasts in vitro, concentrations of 100 – 
500 nM are required maximize methylcytosine reduction without causing cellular 
toxicity. Because decitabine incorporation into DNA is S-phase dependent, 
exposure of cells during cell division is necessary for efficacy. 
  
 
 
 
1.2.2 Experience with extended dosing schedules 
 
Blum et al recently published their experience treating elderly AML patients with 
a 10-day course of decitabine 20 mg/m2/day as a 1-hour infusion.[3] This 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
prolonged exposure course was designed to increase the number of AML blast 
divisions exposed to decitabine per cycle. Toxicities were very similar to a similar 
regiment given as a 3 and 5 day course,[2, 16] but the overall response (CR/PR) 
was much improved (64% vs 25%). In all three studies, the toxicities were 
primarily myeloid suppression and the resultant infectious and bleeding 
complications associated with leukemic and pharmacologic hematopoietic 
suppression. Furthermore, Blum et al noted that the percent methylcytosine 
reduction was improved (22% vs 11%) as was the time to blast clearance (50% of 
patients who ultimately achieved a CR had ≤ 5% bone marrow blasts after 1 cycle 
vs 0% of patients after 2 cycles). Finally, like Shen et al, they observed a 
correlation between the extent of methylcytosine reduction and response.[28]  
This study suggests that improvement in response rates and time to response can 
be achieved by prolonged administration of decitabine. Further, that DNA 
methylcytosine reduction can be achieved with prolonged exposure to decitabine 
and that this might be a patient-specific biological marker of adequate dosing. 
  
Scandura et al. recently published a study of infusional decitabine prior to 
cytarabine + daunorubicin induction therapy for AML.[64] They escalated the 
duration of infusional therapy and reached their planned goal of 20 mg/m2/day x 7 
days of continuous therapy given prior to cytarabine and daunorubicin. They 
noted similar toxicities (primarily hematopoietic and the associated neutropenic 
infections), but that these were not different from their experience giving 
cytarabine + daunorubicin alone. 
 
1.3 
Correlative Studies 
 
With exception of the peripheral blood sample collected on Cycle 1 Day 4, all correlative 
studies will be collected under HRPO# 201011766 (“Tissue Acquisition for Analysis of 
Genetic Progression Factors in Hematologic Diseases”) that all patients will be 
concurrently enrolled in. The goals of the correlative studies include: 
 
1. To correlate patient-specific mutations with response to decitabine. 
We will perform exome sequencing of each patient’s pre-study bone marrow sample 
and skin sample. We will determine AML/MDS-associated variants found in the bone 
marrow, but not skin sample. We will correlate the presence of mutations with 
response (especially mutations in DNMT3A, IDH1, IDH2, and TET2). This study is 
powered to identify mutations with >80% sensitivity to predict decitabine response if 
used as a clinical assay. 
 
2. To correlate the velocity and depth of response with response to decitabine. 
We will assess tumor burden by allele frequency of patient-specific mutations in bone 
marrow samples collected pre study, Cycle 1 Day 10±1, Cycle 1 Day 28±4, and 
Cycle 2 Day 28±4. We will assess mutations in at least 24 recurrently mutated genes 
(including, but not limited to: FLT3, DNMT3A, NPM1, TET2, WT1, IDH1, IDH2, 
RUNX1, NRAS, TP53, U2AF1, KRAS, PHF6, MSTP9, PTPN11, KIT, SMC3, 
PRUNE2, ETV6, CEBPA, SMC1A, ASXL1, STAG2, RAD21) as well as all patient-


Genomic Predictors of Decitabine Response in AML/MDS 
 
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specific mutations discovered during exome sequencing. The leukemic allele 
frequency will be measured using the Illumnia MySeq or HiSeq platform using DNA 
capture by gene specific biotinylated-oligonucleotides. Patient bone marrow samples 
will be assessed pre-study, Cycle 1 Day 10 ± 1, Cycle 1 Day 28 ± 4, and Cycle 2 Day 
28 ± 4 and compared with patient specific skin samples (normal control). The 
velocity of response will be calculated as the natural log rate of patient-specific 
mutation allele frequency clearance. Depth of response will be defined as the lowest 
patient-specific mutation allele frequency in a bone marrow sample. The velocity and 
depth of tumor response will be correlated to: 
 Achievement of clinical CR, 
 Time to best response, 
 Mutational status, 
 Time to disease progression. 
Because all patient-specific exome-discovered mutations will be assessed a second 
time in the primary bone marrow sample as well as the  Cycle 1 Day 10 ± 1, Cycle 1 
Day 28 ± 4, and Cycle 2 Day 28 ± 4 bone marrow samples, this study will also serve 
to validate all exome-discovered mutations. This is important, because exome 
sequencing is associated with a 20-50% false-discovery rate depending on type I error 
tolerated (mutation-calling algorithms are typically set for greater sensitivity than 
specificity).  Thus, many mutations discovered during exome sequencing are 
incorrect.  This second analysis of these mutations will server to exclude these false 
positive calls. 
 
3. To correlate the bone marrow expression profile from pre-study and Cycle 1 Day 10 
± 1 with response to decitabine. 
We will correlate expression signatures, and changes in expression signatures, with: 
 Achievement of clinical CR, 
 Mutation status. 
 
4. To correlate steady-state plasma decitabine concentration on Cycle 1 Day 4 ± 1 with: 
 
Achievement of CR. 
 
5. To correlate the extent of bone marrow DNA hypomethylation on Cycle 1 Day 10 ± 1 
with response to decitabine. 
We will assess the percent reduction in bone marrow methylcytosine content using 
LC-MS. Patients will be evaluated pre-study and Cycle 1 Day 10 ± 1. The change in 
DNA methylcytosine content will be correlated to: 
 Achievement of clinical CR, 
 Change in expression signature. 
This will determine whether inadequate blast exposure to decitabine correlates with 
drug resistance. 
 


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2.0 
OBJECTIVES 
 
2.1 
Primary Objective 
 
To determine whether patient-specific mutations correlate with clinical overall response 
to decitabine in patients with AML or MDS. 
 
2.2 
Secondary Objectives 
 
- 
To determine whether 10 consecutive days per cycle of decitabine improves 
outcomes compared with historical controls treated with 5 consecutive days per cycle 
of decitabine. 
 
- 
To determine whether other biomarkers correlate with clinical overall response to 
decitabine in patients with AML or MDS. These will include:  
 
1. The velocity and depth of patient-specific mutation clearance; 
2. The bone marrow expression profile and change in expression profile during 
decitabine treatment; 
3. The steady-state plasma decitabine concentrations; 
4. The decrease in bone marrow methylcytosine content in response to decitabine.  
 
 


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3.0 
PATIENT SELECTION 
 
3.1 
Inclusion Criteria 
 
One of the following: 
 Patient must have non-M3 AML and be > 60 years of age OR 
 non-M3 AML with relapsed disease OR 
 Symptomatic MDS with one of the following: 
□ Symptomatic anemia with either hemoglobin < 10.0 g/dL or requiring RBC 
transfusion 
□ Thrombocytopenia with a history of two or more platelet counts < 
50,000/mcL or a significant hemorrhage requiring platelet transfusions 
□ Neutropenia with two or more ANC < 1,000/mcL 
 
All of the following: 
1. Patient must have an ECOG performance status ≤ 2. 
2. Patient must have >10% disease burden measured by cytomorphology, flow 
cytometry, or cytogenetics. 
3. Patient must have peripheral white blood cell count < 50,000/mcl. 
4. Patient must have adequate organ function, defined as: 
a. 
Total bilirubin < 1.5 x ULN 
b. 
AST/ALT < 2.5 x ULN 
c. 
Serum creatinine < 2.0 x ULN  
5. Patient must have undergone ≤ 2 cycles of prior hypomethylating agent 
(decitabine or azacitidine). 
6. Patient must be enrolled in HRPO# 201011766 (“Tissue Acquisition for Analysis 
of Genetic Progression Factors in Hematologic Diseases”). 
7. Patient must be > 18 years of age. 
8. Patient must be able to understand and willing to sign an IRB-approved written 
informed consent document. 
 
3.2 
Exclusion Criteria 
 
1. Patient must not be pregnant or nursing. 
2. Patient must not have known CNS leukemia.  
3. Patient must not have a history of positive HIV serology.  
4. Patient must not have a history of positive Hepatitis C serology. 
5. Patient must not have undergone a prior allogeneic stem cell transplant. 
6. Patient must not have any uncontrolled intercurrent illness including, but not 
limited to, ongoing or active infection, ongoing or active GVHD, congestive heart 
failure of NYHA class 3 or 4, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situation that would limit compliance with study 
requirements. 
7. Patient must not have had radiation therapy within 14 days of enrollment. 
8. Patient must not have received any chemotherapy within 21 days of enrollment, 
and any acute treatment-related toxicities must have returned to baseline. Patients 


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may be receiving hydrea at time of enrollment.    
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible for this 
trial. 


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4.0 
REGISTRATION PROCEDURES 
 
Patients must not start any protocol intervention prior to registration through the Siteman 
Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility 
2. Registration of patient in the Siteman Cancer Center database 
3. Assignment of unique patient number (UPN) 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility collecting the information listed below  
1. The registering MD’s name 
2. Patient’s race, sex, and DOB 
3. Three letters (or two letters and a dash) for the patient’s initials 
4. Copy of signed consent form 
5. Completed eligibility checklist, signed and dated by a member of the study team 
6. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center Database 
 
All patients must be registered through the Siteman Cancer Center database. 
 
4.3 
Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  
Patients will also be identified by first, middle, and last initials.  If the patient has no 
middle initial, a dash will be used on the case report forms (CRFs).  All data will be 
recorded with this identification number on the appropriate CRFs. 
 


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5.0 
INVESTIGATIONAL PLAN 
 
5.1 
Summary of Study Design 
 
This is an open-label, single-arm, Phase II, biomarker study of patients with AML or 
MDS who are treated with 10 consecutive days of decitabine. FAB-M3 AML is excluded 
from this trial, because other well-tolerated options exist for the treatment of FAB-M3 
acute promyelocytic leukemia. AML patients who are < 60 years old and have not 
received cytotoxic induction chemotherapy are also excluded, because cytotoxic therapy 
offers a curative possibility for these patients.  MDS patients without cytopenias are also 
excluded because these patients can be followed with watchful-waiting. 
 
All patients will undergo bone marrow biopsies pre-study, on Cycle 1 Day 10 ± 1, and on 
Day 28 ± 4 of Cycles 1, 2, 4, and 6. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. 
Patients will provide a skin biopsy sample pre-study. Patients will also provide peripheral 
blood samples on Cycle 1 Day 4 ± 1 for pharmacokinetic studies. See Section 8.0 for 
details on correlative studies.  
 
Patients will receive decitabine 20 mg/m2/day as a 1-hour infusion on consecutive Days 
1-10 in 28-day cycles.  The dose will be calculated based on actual patient weight at the 
beginning of each cycle. 
 
Once AML patients achieve morphologic complete remission with incomplete blood 
count recovery (CRi) or MDS patients achieve a marrow complete response (Marrow 
CR) as defined in Section 9.0, decitabine administration may be decreased to five 
consecutive days to limit myelosuppressive toxicity at the discretion of the treating 
physician. After two cycles of five consecutive days of treatment, if the patient meets the 
definition of CR with complete cytogenetic response as defined in Section 9.0, 
administration may further be decreased to three consecutive days per cycle at the 
discretion of the treating physician. 
 
It will be a logistical goal of this trial consistently to return preliminary exome results to 
the treating physician by the end of Cycle 1 and validated results by the end of Cycle 2. 
However, this logistical goal is not a primary or secondary aim of this study. Given the 
limited data regarding mutation presence and drug response, we do not anticipate that the 
results will directly influence therapeutic decisions made by the treating physician. 
However, the development of comprehensive genomic pipelines and integration of this 
data into a clinical trial setting will be required before personalized medicine can be 
realized. 
 
5.2 
Study Procedures 
 
5.2.1 Pre-study Procedures 
 


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Pre-study procedures must take place no more than 14 days prior to the first dose 
of decitabine except where noted below. 
 
1. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments 
performed on bone marrow will include correlative studies (collected under 
HRPO# 201011766), morphologic assessment, cytogenetics, and FISH.  
2. Bone marrow or peripheral blood for flow cytometry 
3. Skin biopsy sample (collected under HRPO# 201011766). There is no 
required time frame for this sample; it may have been collected months or 
even years prior to the first dose of decitabine.  
 
5.2.2 Baseline Evaluation 
 
Baseline evaluations must take place no more than 7 days prior to the first dose of 
decitabine.  
 
1. Medical history 
2. Physical exam including height, weight, and ECOG performance score. 
3. Record transfusion requirements for 8 weeks before first dose of study drug 
4. CBC with differential and platelets 
5. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
6. Urine βHCG pregnancy test if appropriate 
 
5.2.3 Day 1 of Each Cycle 
 
1. Physical exam, including weight  
2. CBC with differential and platelets 
3. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
 
5.2.4 Cycle 1 Days 1-10 
 
Decitabine administration, 20 mg/m2 per day given as a 1-hour continuous 
intravenous infusion.  The dose will be calculated based on actual patient weight 
at the beginning of each cycle.  Patients may be premedicated with lorazepam, 
prochlorperazine, palonosetron, or ondansetron at the treating physician’s 
discretion. 
 
5.2.5 Cycle 1 Day 4 ± 1 
 
Obtain 3 mL peripheral blood for correlative studies. Sample must be obtained 45 
minutes ± 15 minutes after beginning the infusion, before the decitabine infusion 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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is complete, and should be obtained from the opposite arm as the infusion.  The 
sample should be collected in a 3 mL K3EDTA tube pre-loaded with 8 mcL of 
tetrahydrouridine (THU 500 mcg/mL), an inhibitor of cytidine deaminase.  The 
sample should be processed by the Tissue Procurement Core Facility at the 
Siteman Cancer Center.  Plasma should be separated and stored per protocols. 
  
5.2.6 Cycle 1 Day 10 ± 1 
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH. 
 
5.2.7 Weekly 
 
CBC with differential and platelets. After the first cycle, this may be changed to 
every other week at the discretion of the treating physician. 
 
5.2.8 Every Other Week 
 
Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH. After the first cycle, this may be changed to 
every month at the discretion of the treating physician. 
 
5.2.9 Cycles 1, 2, 4 and 6Day 28 ± 4 days  
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include: correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH. 
 
5.2.10 End-of-Study Procedures  
 
1. Physical exam 
2. CBC with differential and platelets.  
3. Serum chemistries including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, and LDH.  
4. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments 
performed on bone marrow will include morphologic assessment, 
cytogenetics, and FISH.  
 
5.2.11 Post-study Follow-up 
 


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Patients are to be followed according to standard patient care procedures for 30 
days following the date of the last decitabine dose to monitor for toxicity, 
survival, and response. Thereafter, the investigator or designee will record 
information on subsequent therapy, disease progression, and/or death every 6 
months for 2 years after last dose of study drug or until the patient is lost to 
follow-up or dies. 
 
5.3 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to the 
patient’s health and/or the patient no longer wishes to continue protocol therapy, the 
protocol therapy should be discontinued and the reason(s) for discontinuation 
documented in the case report forms. 
 
In the absence of treatment delays due to adverse events, treatment may continue 
indefinitely or until one of the following criteria applies: 
 
 Documented and confirmed disease progression 
 Death 
 Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
 General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
 Suspected pregnancy 
 Serious non-compliance with the study protocol 
 Lost to follow-up 
 Patient withdraws consent 
 Investigator removes the patient from study 
 The Siteman Cancer Center decides to close the study 
 
Patients who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar. 
 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
 
5.4.1 Chemotherapy 
 
Patients cannot receive any chemotherapy within 21 days of study enrollment and 
must have recovered from any treatment-related toxicities. However, patients may 
be receiving hydrea at enrollment and may continue on hydrea through Cycle 2 of 
decitabine. An indication for hydrea following 2 cycles of decitabine is evidence 
that the patient is not responding and decitabine should be discontinued. Other 
than decitabine, patients cannot receive chemotherapy during this trial.  
 
5.4.2 Growth Factors 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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Erythroid growth factors should not be routinely administered to patients during 
the study. 
 
Thrombopoietin receptor agonists should not be routinely administered to patients 
during the study. 
 
Filgrastim (G-CSF) or sargramostim (GM-CSF) may be administered to patients 
with septic shock or febrile neutropenia at the discretion of the treating physician. 
 
5.4.3 Transfusions 
 
 Packed red blood cells (RBCs) transfusion:   
RBC transfusions will be administered at the discretion of the treating 
physician.  
 
 Platelet transfusion:   
Platelet transfusions will be administered at the discretion of the treating 
physician  
5.4.4 Prophylactic Antimicrobial Agents  
 
Antimicrobial agents will be administered at the discretion of the treating 
physician. 
 
5.4.5 Radiotherapy 
 
Patients cannot receive radiotherapy within 14 days prior to study enrollment or 
while on study. 
 


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6.0 
DOSE MODIFICATIONS 
 
A criterion for discontinuation of the study is a delay of > 8 weeks between any two 
cycles.  
 
6.1 
Delay for Cytopenia or Infection 
 
As cytopenias are a defining feature of AML and MDS, their presence alone does not 
generally warrant dose modifications or delays. Patients will receive four cycles of 
therapy without adjustment for any cytopenia. Following Cycle 4, dose modifications or 
delays for cytopenias will be made at the discretion of the investigator.  
 
Patients with febrile neutropenia (temperature ≥ 38.5° with ANC < 1,000/l) or systemic 
infections should have treatment held until clinical resolution of the infection.  
 
6.2 
Delay for Organ Dysfunction 
 
If renal dysfunction (creatinine ≥ 3 X institutional upper limit of normal) or hepatic 
dysfunction (bilirubin ≥ 2.5 mg/dl or ALT/AST ≥ 3 X institutional upper limit of normal) 
occurs, therapy may not resume until the abnormalities have resolved to below the limits 
stated above. If renal or hepatic dysfunction occurs during a treatment course, the next 
dose of decitabine should be held, and serum chemistries should be checked twice per 
week.  Decitabine can be resumed at full dose when creatinine, bilirubin, ALT and AST 
are below the limits listed above.  
 
For grade 3-4 non-hematologic toxicities related to decitabine, decitabine treatment 
should be delayed until recovery to baseline or to grade ≤ 1. Decitabine may be 
reintroduced with a 50% dose reduction, or at full dose at the discretion of the 
investigator. 
 
6.3 
Modification in Dose or Schedule for Treatment Response 
 
Once AML patients achieve morphologic complete remission with incomplete blood 
count recovery (CRi) or MDS patients achieve a marrow complete response (Marrow 
CR) as defined in Section 9.0, decitabine administration may be decreased to 5 
consecutive days to limit myelosuppressive toxicity at the discretion of the treating 
physician. After two cycles of five consecutive days of treatment, if the patient meets the 
definition of CR with complete cytogenetic response as defined in Section 9.0, 
administration may further be decreased to three  consecutive days per cycle at the 
discretion of the treating physician. 
 


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7.0 
PHARMACEUTICAL INFORMATION 
 
7.1 
Decitabine 
 
Decitabine is an antimetabolite that can replace cytosine in DNA but, unlike cytosine, it 
cannot be methylated. The exact mechanism of decitabine activity has not been 
determined and may involve multiple pathways. Decitabine is thought primarily to act by 
correcting DNA methylation, a major mechanism for gene expression. In some cancer 
cells, hypermethylation blocks the activity of tumor suppressor genes, which regulate cell 
division and differentiation to prevent malignant transformation. When suppressor gene 
activity is blocked, cell division becomes unregulated, leading to the formation of 
neoplastic cells. Decitabine regulates DNA methylation (specifically targets the 
methyltransferase, DNMT1), effectively demethylating and reactivating different tumor 
suppressor genes. Because hypermethylation occurs early in the malignant transformation 
of cells, decitabine may have activity in chemoprevention. Other possible mechanisms 
for decitabine activity include cytotoxicity due to incorporation into DNA similar to other 
antimetabolites, methylation-independent induction of gene expression, and degradation 
of DNA methyltransferase 1. 
 
7.1.1 Study Drug Preparation  
 
OSHA Guidelines for handling cytotoxic drugs outlined in the American Journal 
of Hospital Pharmacy must be followed. 
 
Decitabine (5-aza-2’-deoxycytidine) is supplied as a lyophilized powder for 
injection, 50 mg in 20-ml vials. When reconstituted with 10 ml of sterile water for 
injection, each ml will contain 5 mg of decitabine and 6.8 mg of KH2PO4. The 
reconstituted solution should be diluted to 0.1-1.0 mg/mL with pre-chilled 0.9% 
sodium chloride intravenous infusion, which has been cooled to 2-8oC. 
Reconstitution of the powder results in a rapidly decomposing solution. Unless 
used within 15 minutes of reconstitution, the diluted solution must be prepared 
using cold (2—8 degrees C) infusion fluids and stored at 2—8 degrees C (36—46 
degrees F) for a maximum of 7 hours. 
Decitabine will be administered per dosing schedule every 28 ± 4 days.  
 
Decitabine may be administered either as an inpatient or an outpatient.  
 
7.1.2 Supplier 
 
Decitabine is available commercially. 
 
7.1.3 Toxicities 
 
Likely (>15%) 
Cytopenias: 
neutropenia, 
thrombocytopenia, 
anemia, 
leukopenia, 
febrile 
neutropenia.  


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Bleeding: petechiae, ecchymosis. 
Infections: pneumonia. 
Constitutional: fatigue, pallor, fever, rigors, arthralgia, cough. 
GI: nausea/vomiting, constipation, diarrhea, decreased appetite. 
Electrolyte abnormalities: hyperglycemia, hypoalbuminemia, hypomagnesemia, 
hypokalemia, hyponatremia.  
Cardiac: cardiac murmur. 
Neurologic: headache, insomnia, dizziness. 
Dermatologic: rash. 
 
Less likely (1-15%)  
Hematologic: thrombocythemia, hematoma. 
Other infections: cellulitis, candidal infection, urinary tract infection, sinusitis. 
GI: abdominal 
pain, hyperbilirubinemia, 
stomatitis, 
dyspepsia, ascites, 
hemorrhoids, oral ulceration, dysphagia, abdominal distention, gastro-esophageal 
reflux disease/pyrosis (heartburn), and glossodynia. 
Electrolyte abnormalities: hyperkalemia, and dehydration. 
Urologic: dysuria, increased urinary frequency.  
Dermatologic: pruritus, alopecia, urticaria, edema. 
 
Rare (<1%) 
Cardiac: arrest/respiratory arrest, atrial fibrillation, and tachycardia. 
 
  
 
  
 


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8.0 
CORRELATIVE STUDIES SAMPLE COLLECTIONS 
 
Bone marrow samples will be collected pre-study, on Cycle 1 Day 10 ± 1, and on Day 28 ± 4 of 
Cycle 1, 2, 4, and 6 for correlative studies. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. The 
bone marrow samples will be collected under HRPO# 201011766 (“Tissue Acquisition for 
Analysis of Genetic Progression Factors in Hematologic Diseases”) that all patients will be 
concurrently enrolled in.  
 
Skin biopsy sample will be collected as part of the patient’s participation in HRPO# 201011766 
(“Tissue Acquisition for Analysis of Genetic Progression Factors in Hematologic 
Malignancies”). There is no required time frame for this sample, and it may have been collected 
months or even years prior to the first dose of decitabine.  
 
Peripheral blood sample (3 mL) will be collected on Cycle 1 Day 4 ± 1.  Sample must be 
obtained 45 minutes ± 15 minutes after beginning the infusion, before the decitabine infusion is 
complete, and should be obtained from the opposite arm as the infusion.  The sample should be 
collected in a 3 mL K3EDTA tube pre-loaded with 8 mcL of tetrahydrouridine (THU 500 
mcg/mL), an inhibitor of cytidine deaminase.  The sample should be processed by the Tissue 
Procurement Core Facility at the Siteman Cancer Center.  Plasma should be separated and stored 
per protocols. 
 
 


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9.0 
EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
 
A secondary endpoint of this study is to estimate morphologic complete remission rate. 
  
9.1 
Response Criteria - AML 
 
AML Patients will be assessed for response according to the IWG criteria:1  
 
Morphologic complete remission (CR) – Defined as morphologic leukemia-free state, 
defined as less than 5% blasts in aspirate with marrow spicules and a count of ≥200 
nucleated cells and no blasts with Auer rods or any persistence of extramedullary disease. 
When erythroid precursors constitute less than 50% of bone marrow nucleated cells, the 
percentage of blasts is based on all nucleated cells; when there are 50% or more erythroid 
cells, the percentage blasts should be based on the nonerythroid cells. The presence of 
mild megaloblastoid changes may be permitted if they are thought to be consistent with 
treatment effect. However, persistence of pretreatment abnormalities (eg, pseudo-Pelger-
Hüet cells, ringed sideroblasts, dysplastic megakaryocytes) are not consistent with CR.  
In addition, ANC >1000/μl and platelet count of ≥100,000/μl. Patient must be 
independent of transfusions. No duration requirement for this designation. 
 
Cytogenetic complete remission (CCR) – Defined as morphologic complete remission 
plus a reversion to a normal karyotype. For this study, reversion of a normal karyotype is 
defined as no clonal abnormalities detected in a minimum of 20 mitotic cells. 
 
Morphologic complete remission with incomplete blood count recovery (CRi) – Defined 
as morphologic complete recovery with the exception of neutropenia <1000/μl or 
thrombocytopenia <100,000/μl.  
 
Partial remission (PR) – Defined as meeting the above criteria for complete remission, 
with the following exceptions: decrease of ≥50% in the percentage of blast to 5-25% in 
the bone marrow aspirate. A value of 5% blasts in bone marrow with Auer rods present 
may also be considered a partial remission.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, CRi, PR, PD, or 
recurrence/morphologic relapse.   
 
Progressive disease (PD) – Defined as a clear increase in bone marrow blast count, 
typically a greater than 50% increase compared with baseline, or new extramedullary 
disease. Patients with a > 50% increase in peripheral blast count with an increase of the 
total peripheral white blood cell count to > 10,000/l should undergo evaluation by bone 
marrow biopsy to access for progressive disease.  
 
Recurrence/morphologic relapse - Defined as relapse following complete remission is 
defined as reappearance of blasts in the blood or the finding of ≥5% blasts in the bone 
marrow, not attributable to any other cause. New dysplastic changes is considered 


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relapse. If there are no blasts in the peripheral blood and 5-20% blasts in the bone 
marrow, bone marrow biopsy should be repeated in > 1 week to confirm relapse. 
 
9.2 
Response Criteria - MDS 
 
MDS Patients will be assessed for response according to the IWG criteria:69 
 
Complete remission (CR) – Defined as < 5% myeloblasts with normal maturation of all 
cell lines in the bone marrow and peripheral blood values of Hgb > 11g/dL, Platelets > 
100 x 109/L, Neutrophils > 1.0 x 109/L, and 0% blasts. Persistent dysplasia does not 
exclude CR but will be noted.  
 
Marrow Complete Response (Marrow CR) – Defined as < 5% myeloblasts in the bone 
marrow and a decrease by > 50% from pretreatment values, but not meeting the 
definition of CR above.  
 
Partial Remission (PR) – Defined as meeting the definition of CR above with a decrease 
of myeloeblasts in the bone marrow by > 50% from pretreatment values, but absolute 
myeloblasts still >5%.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, Marrow CR, PR, 
SD, PD, or recurrence/morphologic relapse.   
 
Progressive Disease//Relapse (PD) – Defined as ≥ 50% increase in blasts to > 5% blasts 
(for patients with less than 5% blasts at baseline only), ≥ 50% increase to > 10% blasts 
(for patients with 5-10% blasts at baseline only), ≥ 50% increase to > 20% blasts (for 
patients with l0-20% blasts at baseline only), ≥ 50% increase to > 30% blasts (for patients 
with 20-30% blasts at baseline only) or any of the following: At least 50% decrement 
from maximum remission/response in granulocytes or platelets, reduction in Hgb by ≥ 2 
g/dL, or New or worsened transfusion dependence not related to study drug toxicity. Or 
for patients with a CR, Marrow CR, or PR as defined above and subsequently 
development of one of the following: Return to pretreatment bone marrow blast 
percentage, decrement of ≥ 50% from maximum remission/response levels in 
granulocytes or platelets, or reduction in Hgb concentration by ≥ 1.5 g/dL or transfusion 
dependence. 
 
9.2.1 Hematologic Improvement (HI) 
 
 
 
Progressive disease as defined above nullifies hematologic improvement.  
 
Erythroid response requires all of the following (only required if pretreatment 
HgB <11 g/dL): 
 Hgb increase by ≥ 1.5 g/dL 
 Relevant reduction of units of RBC transfusions by an absolute number of at 
least 4 RBC transfusions/8 wk compared with the pretreatment transfusion 


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number in the previous 8 wk. Only RBC transfusions given for a Hgb of ≤ 9.0 
g/dL pretreatment will count in the RBC transfusion response evaluation 
 
Platelet response requires one of the following (only required if pretreatment 
platelets < 100 x 109/L):  
 Absolute increase of ≥ 30 x 109/L (for patients starting with > 20 x 109/L 
platelets) 
 Increase from < 20 x 109/L to > 20 x 109/L and absolute increase > 100% (for 
patients starting with < 20 x 109/L) 
 
Neutrophil response requires the following (only required if pretreatment ANC 
<1.0 x 109/L):  
 At least 100% increase and an absolute increase > 0.5 x 109/L 
 
9.3.1 Cytogenetic Response  
 
Complete Cytogenetic Response – Defined as reversion to a normal karyotype. 
For this study, reversion of a normal karyotype is defined as no clonal 
abnormalities detected in a minimum of 20 mitotic cells. Progressive disease as 
defined above nullifies cytogenetic response.  
 
9.3 
Guidelines for Evaluation of Disease 
 
Bone marrow biopsy should be performed on Day 28 ± 4 of cycles 1, 2, 4 and 6 for 
clinical disease assessment. Following Cycle 6, bone marrow biopsies should be 
performed at the discretion of the treating physician to assess for relapse or progression. 
 
9.4 
Other Secondary Efficacy Measures 
 
9.4.1 Treatment Failure, Overall Survival and Event-free Survival 
 
Treatment failure is defined as failure to achieve a complete or partial remission 
following 4 cycles of therapy.  
 
Overall survival is defined as the date of first dose of study drug to the date of 
death from any cause.  Patients still alive at the time of the final analysis will be 
right-censored. 
 
Event-free survival is defined as the interval from the date of first dose of study 
drug to date of treatment failure, recurrence, death due to any cause, or loss to 
follow up. 
 
9.4.2 Duration of Remission and Relapse-free Survival for Patients Having 
Complete Remission 
 


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Duration of complete remission is defined as the interval from the date that 
complete remission is documented to the date of recurrence, death due to any 
cause, or loss to follow-up.  
 
Relapse-free survival is defined only for those patients achieving a complete 
remission. It is defined as the interval from the date of first documentation of a 
leukemia-free state to the date of recurrence, death due to any cause, or loss to 
follow up.   
  
9.4.3 Safety 
 
Safety evaluation will include assessments of adverse experiences, medical 
history, physical examinations, vital signs, and laboratory assessments at baseline 
and throughout the study period. Local laboratories may be used for this study.  
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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10.0 
ADVERSE EVENT REPORTING  
 
10.1 
Adverse Events (AEs) 
 
Definition:  any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading:  the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for all 
toxicity reporting.  A copy of the CTCAE version 4.0 can be downloaded from the CTEP 
web site:  http://ctep.cancer.gov/reporting/ctc.html. 
 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the terms 
listed that should be used are those provided by the Department of Health and Human 
Services’ Office for Human Research Protections (OHRP).  A copy of this guidance can 
be found on OHRP’s website: (http://www.hhs.gov/ohrp/policy/AdvEvntGuid.htm). 
 
10.2 
Unanticipated Problems 
 
Definition: 
 
 unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied; 
 related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the 
incident, experience, or outcome may have been caused by the procedures 
involved in the research); and 
 suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
 
10.3 
Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that govern 
human 
subjects 
research 
or 
failure 
to 
follow 
the 
determinations 
of 
the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate choice to 
ignore regulations, institutional policies, or determinations of the IRB. 
 
10.4 
Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial harm 
to subjects or others, or that materially compromises the rights or welfare of participants. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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10.5 
Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under the research 
team’s control. Exceptions apply only to a single participant or a singular situation. 
 
Pre-approval of all protocol exceptions must be obtained prior to the event. 
 
10.6 
Reporting to the Human Research Protection Office (HRPO) and the Quality 
Assurance and Safety Monitoring Committee (QASMC) at Washington 
University: 
 
The PI is required to promptly notify the IRB of the following events: 
 
 Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct 
of the study. 
 Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 
 Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence of 
the event or notification to the PI of the event.  The death of a research participant that 
qualifies as a reportable event should be reported within 1 working day of the occurrence 
of the event or notification to the PI of the event. 
 
10.7 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER 
FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the investigator to report any unanticipated problem to the FDA as 
follows: 
 
 Report any unexpected fatal or life-threatening adverse experiences associated 
with use of the drug (i.e., there is a reasonable possibility that the experience may 
have been caused by the drug) by telephone or fax no later than 7 calendar days 
after initial receipt of the information.  A life-threatening adverse experience is 
defined as any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it 
does not include a reaction that, had it occurred in a more severe form, might have 
caused death. 
 Report any serious, unexpected adverse experiences, as well as results from 
animal studies that suggest significant clinical risk within 15 calendar days after 
initial receipt of this information.  A serious adverse drug experience is defined as 


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any adverse drug experience occurring at any dose that results in any of the 
following outcomes: 
o Death 
o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial 
disruption of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 
o Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical 
intervention to prevent one of the outcomes listed above 
An unexpected adverse drug experience is defined as any adverse drug 
experience, the specificity or severity of which is not consistent with the current 
investigator brochure (or risk information, if an IB is not required or available). 
 
All MedWatch forms will be sent by the investigator or investigator’s team to the FDA at 
the following address or by fax: 
 
Food and Drug Administration 
Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
10.8 
Timeframe for Reporting Required Events:  
 
Reportable adverse events will be tracked for 30 days following the last day of study 
treatment, or until the start of a new chemotherapy/treatment, whichever comes first. 
 
Deaths 
Any reportable death while on study or within 
30 days of study 
Immediately, within 24 hours, to PI 
and the IRB 
Any reportable death while off study  
Immediately, within 24 hours, to PI 
and the IRB  
Adverse Events/Unanticipated Problems 
Any reportable adverse events as described in 
Sections 10.1 and 10.2 (other than death) and 
10.7 
Immediately, within 24 hours to PI 
and within 10 working days to the 
IRB, and within 7 or 15 days to the 
FDA  
All adverse events regardless of grade and 
attribution should be submitted cumulatively  
Include in DSM report 
Noncompliance and Serious Noncompliance 
All noncompliance and serious noncompliance 
as described in Sections 10.3 and 10.4 
Immediately, within 24 hours, to PI 
and within 10 working days to the IRB 


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11.0 
DATA SAFETY MONITORING PLAN 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, 
the Principal Investigator will provide a Data and Safety Monitoring (DSM) report to the 
Washington University Quality Assurance and Safety Monitoring Committee (QASMC) semi-
annually beginning six months after accrual has opened (if at least five patients have been 
enrolled) or one year after accrual has opened (if fewer than five patients have been enrolled at 
the six-month mark). 
 
The Principal Investigator will review all patient data at least every six months, and provide a 
semi-annual report to the QASMC. This report will include: 
 HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
 Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of 
study 
 History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol 
exceptions, error, or breach of confidentiality including start/stop dates and reason 
 Study-wide target accrual and study-wide actual accrual 
 Protocol activation date 
 Average rate of accrual observed in year 1, year 2, and subsequent years 
 Expected accrual end date 
 Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
 Measures of efficacy 
 Early stopping rules with supporting data and list the number of participants who 
have met the early stopping rules 
 Summary of toxicities 
 Abstract submissions/publications 
 Summary of any recent literature that may affect the safety or ethics of the study  
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC 
according to institutional guidelines. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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12.0 
STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
 
12.1 
Analysis Populations 
 
Intent-to-Treat - The intent-to-treat (ITT) population will be defined as all patients who 
receive at least one dose of decitabine.  
 
Efficacy-Evaluable - The efficacy-evaluable (EE) population will be defined as all 
patients who receive at least one cycle of treatment.   
 
Safety - The safety population will be defined as all patients with at least one dose of 
decitabine. 
 
12.2 
Statistical Analysis 
 
12.2.1 Descriptive Analyses 
 
Patient Disposition: The number of patients discontinued, the reasons for 
discontinuation, and the number of cycles administered will be summarized. 
 
Demographics and Baseline Characteristics: Descriptive summary statistics will 
be provided for demographic and important baseline characteristics.  For 
continuous variables, the number of patients, mean, standard deviation, median, 
minimum and maximum will be provided.  For categorical variables, the number 
and percent of patients in each demographic/characteristic category will be 
summarized. 
 
12.2.2 Primary Endpoint Analysis 
 
The primary end-point of this phase II study is the correlation biomarkers with 
overall response in AML/MDS patients to decitabine. 
 
We will correlate the presence of patient specific mutations (especially mutations 
in DNMT3A, IDH1, IDH2, and TET2) with overall response rates. Mutation rates 
in these genes and in other patient-specific genes will be correlated with overall 
response and their respective 95% confidence intervals will be provided. 
 
Statistical considerations. The relationship between AML/MDS-associated 
mutations and the responsiveness to decitabine will be described by contingency 
tables and assessed using Chi-square test or Fisher’s exact test as appropriate. The 
differentially expressed genes between responders and non-responders will be 
determined using the significance analysis of microarray (SAM). Multivariate 
logistic regression will also be fit to adjust the potential confounding effects of 
other demographic and clinical characteristics. The resultant critical P values will 
be corrected for multiple testing.  Since the Bonferroni correction is known to be 
too conservative, a permutation analysis (e.g., 10,000 random assignments for 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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responsive status) will be used to derive an empirical critical P value.  The new 
critical p-value is the number of permutation tests which yield a lower p-value 
than the original test divided by the total number of permutation tests.[61]   
 
A power analysis was performed using Chi-square test for the association 
between mutation and response to decitabine. The frequency of DNMT3A, 
IDH1/2, and TET2 mutations in AML and MDS patients are each between 15% 
and 30% in multiple cohorts.[29-37] Table 3 shows the minimal detectable 
differences of response rates between mutant and wild-type cases, assuming an 
expected overall 45% CR/CRi rate and 80% power. For an average of 20% 
frequency of a mutated gene, for example, 100 patients will provide 80% power 
to detect 30% difference at a 2-sided 0.1 alpha error. The purpose of this study is 
to determine whether any of 
these mutations result in a 
clinically 
significant 
association 
that 
would 
warrant 
pre-treatment 
testing. We believe that a 
reasonable cut off would be 
if at least 30% of clinical 
response could be attributed 
to the presence of a specific 
mutation. 
However, 
we 
anticipate that more power 
could be achieved as we 
assess patient groups with 
combined 
characteristics 
(e.g. DNMT3A mutation or 
loss 
of 
DNMT3A 
expression). In addition, we 
expect that the majority of the enrolled 125 patients can be used in the analysis 
(since all of our molecular information will be acquired within 30 days of 
enrollment, we anticipate a drop-out rate of 20% or less). 
 
12.2.3 Secondary Endpoint Analyses 
 
The overall response rate (CR/CRi/PR) and complete response rate (CR/CRi) will 
be compared with historical controls[2], and the 95% confidence intervals will be 
provided. 
 
The rate of patient specific mutation clearance after 10, 28, and 56 days of therapy 
will be determined using an Illumina based platform. We will compare the rate of 
mutation clearance between the patients who achieve a CR/CRi after 4 cycles and 
those who do not. We anticipate that we will identify between 6 and 25 exonic 
SNVs per case (the range obtained with >200 AML samples sequenced to date 
with whole genome or exome sequencing), and that the expected 95% confidence 
Table 3. Detectable mutation associated response rates (80% power, α = 0.05) 
 
Total 
N 
% of patients 
with a given 
mutation  
Detectable 
response 
difference 
Sensitivity of mutation to 
predict response at minimum 
detectable response difference* 
100 
15%  
20%  
30% 
40% 
50% 
0.40 
0.35 
0.30 
0.27 
0.26 
80% 
75% 
67% 
61% 
58% 
* Assuming the overall response  rate (CR/Cri) of 45%   


Genomic Predictors of Decitabine Response in AML/MDS 
 
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interval of the mean variant allele frequency will be ±2.56% and ±1.25%, 
respectively, if we assume an anticipated standard deviation of 3.2% within the 
same clone. The change of tumor burden over time and its association with 
clinical response will  be assessed using 2-way ANOVA for repeated 
measurement data, followed by the ad-hoc multiple comparisons for between-
group differences (e.g., responders vs. non-responders) at given time points as 
well as the between-time differences within each group. The resultant critical P 
values will also be corrected using permutation analysis for multiple testing. 
 
The steady-state plasma decitabine concentration on day 4 ± 1 will be measured 
and correlated with clinical overall response. 
 
The percent decrease in bone marrow methylcytosine will be calculated on day 
10±1. We will compare this reduction with previously published data and between 
the patients who achieve CR/CRi and those who do not. The correlation of peak 
plasma levels of decitabine with response will be assessed using 2-sample t-test or 
Mann-Whitney non-parametric rank-sum test as appropriate. The change of total 
bone marrow DNA methylcytosine from baseline and its association with both 
steady-state plasma drug levels and response will be assessed using 2-way 
ANOVA for repeated measurement data, followed by the ad-hoc multiple 
comparisons for between-group differences (e.g., responders vs. non-responders) 
and between-time differences. Similar analyses will be performed for the 
correlation of expression profiles and SNP analysis with pharmacokinetic data.   
 
Statistical power justification.  A power analysis was performed using two-
sample t-test for between-group difference and paired t-test for the change over 
time. For tumor clearance, this will provide > 99% power to detect a 10% 
difference in the means at two separate time points for clones with 6 or more 
variants, and > 98% power to detect a 15% difference if there are 3 or more 
variants per clone with α = 0.05 (power will decrease to 91% and 82%, 
respectively, in cases where the standard deviation is 4.7%, the maximum we 
have observed thus far). For pharmacologic correlations, we anticipate 85% 
power to determine a 25 ng/ml difference between the average Cmax of 45 
evaluable responders and 55 evaluable non-responders with an anticipated 
average Cmax of 70 ng/ml and a standard deviation of 40 ng/ml within the entire 
cohort, and with α = 0.05.[22] We believe a less significant difference would not 
be sufficient to warrant application in clinical decision making (i.e. whether to 
continue therapy for more than 1 cycle). We anticipate 99% power to determine a 
difference of 20% in the reduction of bone marrow methylcytosine between 45 
evaluable responders and 55 evaluable non-responders with an expected standard 
deviation of 15% and α = 0.05.[28, 66-68] The difference in methylcytosine 
reduction previously observed was 30% between patients achieving CR/PR and 
those with only hematologic improvement.[28]  
 
Time to relapse and time to death will also be listed for each patient. 
 


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12.2.4 Safety Analysis 
 
Extent of Exposure – Duration (number of cycles/infusions) of decitabine doses 
(cumulative and intensity) will be summarized. 
 
Adverse Events - All adverse events will be coded using NCI CTCAE v. 4.0.  The 
incidence of treatment-emergent adverse events (number and percent of patients 
reporting the adverse event at least once during the study) will be summarized.  In 
addition, adverse events will be summarized by investigator attribution of 
relationship to study medication and by grade.  Similar summaries will be 
provided by treatment cycle. 
 
Clinical Laboratory Evaluation – Applicable laboratory parameters will be 
graded according to NCI CTCAE version 4.0. The incidence of maximum grade 
(number and percent of patients experiencing the maximum grade during the 
study) will be summarized for each laboratory parameter.  Similar summaries will 
be provided by treatment cycle.  Graphical displays of select hematological 
parameters will also be provided. 
 
Vital Signs, Physical Findings, and Other Safety Observations – Vital signs, 
findings from physical examinations, ECOG scores and weights will be presented 
in listing format. 
 
12.2.5 Description of Planned Subgroup Analyses 
 
The following subgroups (if sufficient numbers of patients) will be explored with 
respect to the primary and overall survival endpoints. 
 Age (60-69 years old vs ≥ 70 years old) 
 Cytogenetics 
 Patients with AML-associated mutations in DNMT3A, TET2, IDH1, or 
IDH2 vs those who do not have somatic variants in these genes. 
 
12.3 
Sample Size 
 
We anticipate initially enrolling 25 patients at Washington University. Once we establish 
a routine exome analysis with validation and calculation of the rate of patient-specific 
mutation clearance, we will enroll an additional 100 patients from at least 2 additional 
centers.  
 


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13.0 
DATA SUBMISSION SCHEDULE 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to registration 
Patient Registration Form 
Disease Diagnosis Form 
Inclusion Criteria Worksheet 
Exclusion Criteria Worksheet 
 
Prior to starting treatment 
Treatment Record Form 
To be maintained throughout a patient’s treatment 
cycle.  The complete record is due at completion of 
treatment. 
Bone Marrow Sample Collection 
Form 
To be maintained throughout the course of the 
study at designated bone marrow sample 
collections time points (See Appendix 2). 
Pathology: Bone Marrow Biopsy 
To be completed upon the return of pathology 
report for each bone marrow biopsy. 
Study Calendar Form 
To be completed with the date of each time point 
upon occurrence. 
Labs Form 
To be completed upon the return of laboratory 
reports for each study time point (See Appendix 2) 
Dose Modifications 
To be maintained throughout the course of the 
study if dose modifications are performed. 
Therapy Response Form 
To be filled out upon remission (prior to, and/or on 
therapy) 
End of Study Form 
 
Due at the completion of the EOS visit, at the time 
of death, and/or at the time that the patient is lost to 
follow-up 
Follow Up Form 
 30 days post date of last decitabine dose. 
 Every 6 months for 2 years after the last dose of 
study drug. 
Record of Adverse Events 
At the time of any AE 
 
 


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Appendix 1 
ECOG Performance Scale 
Activity Status 
Description 
0 
Asymptomatic, fully active, and able to carry on all predisease 
performance without restrictions. 
 
1 
Symptomatic, fully ambulatory but restricted in physically strenuous 
activity and able to carry out performance of a light or sedentary 
nature, eg, light housework, office work. 
 
2 
Symptomatic, ambulatory and capable of all self-care but unable to 
carry out any work activities.  Up and about more than 50% of 
waking hours: in bed less than 50% of day. 
 
3 
Symptomatic, capable of only limited self-care, confined to bed or 
chair more than 50% of waking hours, but not bedridden. 
 
4 
Completely disabled.  Cannot carry on any self-care.   Totally 
bedridden. 
 
5 
Dead 
 
 


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Appendix 2 
Study Flow Chart 
Study Flow Chart 
Test 
Pre-
study1 
Base-
line2 
All 
Cycles 
Day 1 
All Cycles 
Days 1-103 
Cycle 1 Day 
4 ± 1 
 
Cycle 1 
Day 10 ± 1 
and 28 ± 4 
Every 
week 
Every 
2 
weeks 
Even 
cycles 
Day 28±4 
End of 
Study 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Medical History 
 
X 
 
 
 
 
 
 
 
 
Physical Exam 
 
Xa 
Xb 
 
 
 
 
 
 
X 
Transfusion 
Requirementsc 
 
Xc 
Ongoing 
Bone Marrow 
Aspirate and Biopsy 
Xd 
 
 
 
 
Xd 
 
 
Xd 
Xd 
Flow Cytometrye 
X 
 
 
 
 
 
 
 
 
 
Peripheral blood for 
correlative studies 
 
 
 
 
X 
 
 
 
 
 
Pregnancy test 
 
X 
 
 
 
 
 
 
 
 
CBC with differential 
and platelets 
 
X 
X 
 
 
 
Xf 
 
 
X 
Serum Chemistriesg 
 
X 
X 
 
 
 
 
Xf 
 
X 
Decitabine 
administrationh 
 
 
 
X 
 
 
 
 
 
 
Adverse Events  
 
 
Ongoing 
Post-study follow-up 
 
 
 
 
 
 
 
 
 
Xi 
1 To be performed within 14 days of the first dose of decitabine.  
2 To be performed within 7 days of the first dose of decitabine.  
3 Decitabine may be reduced to Days 1-5 or Days 1-3. See Section 6.3 for details.  
a To include height, weight, and ECOG. 
b To include weight. 
c Care should be given to record the frequency of transfusions from 8 weeks prior to first dose of study drug through 
the duration of the study. 
d Bone marrow biopsy and aspirate should be performed at baseline, at Cycle 1 Day 10 ± 1, and at  Day 28 ± 4 of 
Cycles 1, 2, 4 and 6 for clinical disease assessment. Following Cycle 6, bone marrow biopsies should be performed at 
the discretion of the treating physician to assess for relapse or progression. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. To include: correlative studies (collected under HRPO# 
201011766), morphologic assessment, cytogenetics and FISH at all time points. A skin biopsy sample (collected under 
HRPO# 201011766) is required at pre-study, but may have been collected months or even years prior to first dose of 
decitabine.  
e On bone marrow or peripheral blood.  
f After Cycle 1, these labs may be obtained every other week (CBC) or once a month (Chemistries) at the discretion of 
the treating physician. 
g Serum chemistries include bicarbonate, calcium, glucose, total protein, albumin, BUN, potassium, chloride, sodium, 
creatinine, total bilirubin, AST, ALT, alkaline phosphatase, and LDH. 
h Decitabine treatment will be 20 mg/m2 over a 1-hour IV infusion on consecutive Days 1-10 every 28-day cycle.   The 
dose will be calculated based on actual patient weight at the beginning of each cycle. Patients may be premedicated 
with lorazepam, prochlorperazine, palonosetron, or ondansetron at the treating physician’s discretion. 
i Patients will be followed for  30 days following the date of the last decitabine dose to monitor for toxicity, survival 
and response. Thereafter, the investigator or designee will record information on subsequent therapy, disease 
progression, and death, every 6 months for 2 years after first dose of study drug or until the patient is lost to follow-up.


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
14.0 
REFERENCES 
 
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Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
16. 
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treatment of older AML patients judged unfit for induction chemotherapy. 
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Ley, T.J., et al., DNMT3A mutations in acute myeloid leukemia. N Engl J Med, 
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31. 
Mardis, E.R., et al., Recurring mutations found by sequencing an acute myeloid 
leukemia genome. N Engl J Med, 2009. 361(11): p. 1058-66. 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
32. 
Abdel-Wahab, O., J. Patel, and R.L. Levine, Clinical Implications of Novel 
Mutations in Epigenetic Modifiers in AML. Hematol Oncol Clin North Am, 2011. 
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33. 
Reuther, G.W., Recurring mutations in myeloproliferative neoplasms alter 
epigenetic regulation of gene expression. Am J Cancer Res, 2011. 1(6): p. 752-62. 
34. 
Paschka, P., et al., IDH1 and IDH2 mutations are frequent genetic alterations in 
acute myeloid leukemia and confer adverse prognosis in cytogenetically normal 
acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem 
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35. 
Shen, Y., et al., Gene mutation patterns and their prognostic impact in a cohort of 
1185 patients with acute myeloid leukemia. Blood, 2011. 118(20): p. 5593-603. 
36. 
Tefferi, A., et al., Detection of mutant TET2 in myeloid malignancies other than 
myeloproliferative neoplasms: CMML, MDS, MDS/MPN and AML. Leukemia, 
2009. 23(7): p. 1343-5. 
37. 
Walter, M.J., et al., Recurrent DNMT3A mutations in patients with 
myelodysplastic syndromes. Leukemia, 2011. 25(7): p. 1153-8. 
38. 
Lin, J., et al., Recurrent DNMT3A R882 Mutations in Chinese Patients with Acute 
Myeloid Leukemia and Myelodysplastic Syndrome. PLoS One, 2011. 6(10): p. 
e26906. 
39. 
Bejar, R., et al., Clinical effect of point mutations in myelodysplastic syndromes. 
N Engl J Med, 2011. 364(26): p. 2496-506. 
40. 
Walter, M.J., et al., Clonal architecture of secondary acute myeloid leukemia. N 
Engl J Med, 2012. 366(12): p. 1090-8. 
41. 
Kantarjian, H.M., et al., Survival advantage with decitabine versus intensive 
chemotherapy in patients with higher risk myelodysplastic syndrome: comparison 
with historical experience. Cancer, 2007. 109(6): p. 1133-7. 
42. 
Lubbert, M., et al., Continued Low-Dose Decitabine (DAC) Is an Active First-
Line Treatment in All Cytogenetic Subgroups of Older AML Patients: Results of 
the FR00331 Multicenter Phase II Study. ASH Annual Meeting Abstracts, 2007. 
110(11): p. 300-. 
43. 
Blum, W., et al., Phase I Study of Decitabine Alone or in Combination With 
Valproic Acid in Acute Myeloid Leukemia. J Clin Oncol, 2007. 25(25): p. 3884-
3891. 
44. 
Issa, J.-P.J., et al., Phase 1 study of low-dose prolonged exposure schedules of the 
hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in hematopoietic 
malignancies. Blood, 2004. 103(5): p. 1635-1640. 
45. 
Garcia-Manero, G., et al., Phase 1/2 study of the combination of 5-aza-2'-
deoxycytidine with valproic acid in patients with leukemia. Blood, 2006. 108(10): 
p. 3271-3279. 
46. 
Issa, J.P., S.B. Baylin, and J.G. Herman, DNA methylation changes in 
hematologic malignancies: biologic and clinical implications. Leukemia, 1997. 
11 Suppl 1: p. S7-11. 
47. 
Ganetzky, R.D., et al., Differences Between Normal and Leukemic Stem Cell-
Specific Methylome Indicates Aberrantly Silenced Genes Involved in the 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 2 Version date: 03/10/14 
 
Pathogenesis of Malignant Evolution. ASH Annual Meeting Abstracts, 2008. 
112(11): p. 599-. 
48. 
Christman, J.K., 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA 
methylation: mechanistic studies and their implications for cancer therapy. 
Oncogene, 2002. 21(35): p. 5483-95. 
49. 
Lubbert, M., DNA methylation inhibitors in the treatment of leukemias, 
myelodysplastic syndromes and hemoglobinopathies: clinical results and possible 
mechanisms of action. Curr Top Microbiol Immunol, 2000. 249: p. 135-64. 
50. 
Kantarjian, H., et al., Results of a randomized study of 3 schedules of low-dose 
decitabine in higher-risk myelodysplastic syndrome and chronic myelomonocytic 
leukemia. Blood, 2007. 109(1): p. 52-57. 
51. 
Steensma, D.P., et al., Multicenter Study of Decitabine Administered Daily for 5 
Days Every 4 Weeks to Adults With Myelodysplastic Syndromes: The Alternative 
Dosing for Outpatient Treatment (ADOPT) Trial. J Clin Oncol, 2009: p. 
JCO.2008.19.6550. 
52. 
Cashen, A.F., et al., Preliminary Results of a Multicenter Phase II Trial of 5-Day 
Decitabine as Front-Line Therapy for Elderly Patients with Acute Myeloid 
Leukemia (AML). ASH Annual Meeting Abstracts, 2008. 112(11): p. 560-. 
53. 
Jabbour, E., et al., Evolution of decitabine development: accomplishments, 
ongoing investigations, and future strategies. Cancer, 2008. 112(11): p. 2341-51. 
54. 
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decitabine in higher-risk myelodysplastic syndrome and chronic myelomonocytic 
leukemia. Blood, 2007. 109(1): p. 52-7. 
55. 
Blum, W.G., Klisovic, R., Liu, S., Kefauver, C., Grever, M. R., Schaaf, L., Chan, 
K., Byrd, J. C., Villalona-Calero, M., Marcucci,  G. Efficacy of a novel schedule 
of decitabine in previously untreated AML, age 60 or older. in ASCO Annual 
Meeting. 2009. 
56. 
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deoxycytidine (NSC 127716) in cancer patients. Cancer Res, 1986. 46(9): p. 
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58. 
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determination of 5-aza-2'-deoxycytidine in plasma by high-performance liquid 
chromatography. J Chromatogr, 1985. 345(1): p. 162-7. 
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deoxycytidine in patients with acute leukemia. Pharmacol Ther, 1985. 30(3): p. 
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aza-2'-deoxycytidine in mice. Coll Czech Chem Commun, 1965. 30: p. 3001-
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Momparler, R.L., et al., Pilot phase I-II study on 5-aza-2'-deoxycytidine 
(Decitabine) in patients with metastatic lung cancer. Anticancer Drugs, 1997. 
8(4): p. 358-68. 


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62. 
Aparicio, A., et al., Phase I trial of continuous infusion 5-aza-2'-deoxycytidine. 
Cancer Chemother Pharmacol, 2003. 51(3): p. 231-9. 
63. 
Bryan, J., et al., Pharmacokinetic evaluation of decitabine for the treatment of 
leukemia. Expert Opin Drug Metab Toxicol, 2011. 7(5): p. 661-72. 
64. 
Scandura, J.M., et al., Phase 1 study of epigenetic priming with decitabine prior 
to standard induction chemotherapy for patients with AML. Blood, 2011. 118(6): 
p. 1472-80. 
65. 
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methods for p-value adjustment. 1993, New York: John Wiley & Sons, Inc. 
66. 
Samlowski, W.E., et al., Evaluation of a 7-day continuous intravenous infusion of 
decitabine: inhibition of promoter-specific and global genomic DNA methylation. 
J Clin Oncol, 2005. 23(17): p. 3897-905. 
67. 
Blum, W., et al., Phase I study of decitabine alone or in combination with 
valproic acid in acute myeloid leukemia. J Clin Oncol, 2007. 25(25): p. 3884-91. 
68. 
Yang, A.S., et al., DNA methylation changes after 5-aza-2'-deoxycytidine therapy 
in patients with leukemia. Cancer Res, 2006. 66(10): p. 5495-503. 
69.       Cheson, B.D., et al., Clinical application and proposal for modification of the 
International Working Group (IWG) response criteria in myelodysplasia. Blood, 
2006. 108(2): p. 419-25. 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
 
NCT# NCT01687400 
 
 
Document Version Date:  10/29/2013 
 
IRB Approval Date:  12/13/2013 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 1 Version date: 10/29/2013 
 
 
 
Genomic Predictors of Decitabine Response in AML/MDS 
 
Washington University School of Medicine 
Division of Oncology 
660 South Euclid Avenue, Box 8056 
St. Louis, MO 63110 
 
Protocol #: 201210102 
Protocol Version Date: 10/29/13 
 
Principal Investigator 
John Welch, M.D./Ph.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-2626 
Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Sub-Investigator 
Timothy Ley, M.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-8832 
 
Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Pathology 
Jeffery M. Klco, M.D., Ph.D. 
Department of Anatomic and Molecular Pathology 
Telephone: (314) 362-8832 
Email: klcoj@wustl.edu 
 
Statistician 
Feng Gao, Ph.D. 
Division of Biostatistics 
Telephone: (314) 362-3682 
E-mail: feng@wubios.wustl.edu 
 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to obtain 
informed consent, may not be disclosed to another party unless law or regulations require such disclosure.  Persons 
to whom the information is disclosed must be informed that the information is confidential and may not be further 
disclosed by them. 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 1 Version date: 10/29/2013 
 
Study Schema: 
 
 
Patients with progressive disease after two cycles (defined as a clear increase in bone marrow blast count, typically a 
> 50% increase in bone marrow blasts over baseline) should be removed from study. 
Patients with stable disease may continue on study and should undergo a diagnostic bone marrow biopsy at the end 
of even numbered cycles. 
Once AML patients achieve morphologic complete remission with incomplete blood count recovery (CRi) or MDS 
patients achieve a marrow complete response (Marrow CR) as defined in Section 9.0, decitabine 
administration may be decreased to five consecutive days to limit myelosuppressive toxicityat the 
discretion of the treating physician. After two cycles of five consecutive days of treatment, if the patient 
meets the definition of CR with complete cytogenetic response as defined in Section 9.0, administration 
may further be decreased to three consecutive days per cycle at the discretion of the treating physician. 
Sample collection for correlative studies: 
Bone marrow biopsies should be performed at baseline, at Cycle 1 Day 10 ± 1, at Cycle 1Day 28 ± 4, and 
at the end of even numbered cycles. 
Peripheral blood samples should be collected on Day 4± 1 of Cycle 1. Collection should be obtained 45 ± 
15 minutes after decitabine infusion is initiated.
20 
mg/m
2/day
Cycle 1 
20 
mg/m
2/day
Cycle 2+ 
Day 1 
Day 28 
BM Bx 
PB 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 1 Version date: 10/29/2013 
3 
Genomic Predictors of Decitabine Response in AML/MDS 
Table of Contents 
Section 
Page 
1.0 INTRODUCTION 
5 
1.1 
Rationale 
5 
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
7 
1.2.1 
Pharmacokinetics and Metabolism of Decitabine 
9 
1.2.2 
Experience with extended dosing schedules 
10 
1.3 
Correlative Studies 
11 
2.0 OBJECTIVES 
13 
2.1 
Primary Objective 
13 
2.2 
Secondary Objectives 
13 
3.0 PATIENT SELECTION 
14 
3.1 
Inclusion Criteria 
14 
3.2 
Exclusion Criteria 
14 
3.3 
Inclusion of Women and Minorities 
15 
4.0 REGISTRATION PROCEDURES 
16 
4.1 
Confirmation of Patient Eligibility 
16 
4.2 
Patient Registration in the Siteman Cancer Center Database 
16 
4.3 
Assignment of UPN 
16 
5.0 INVESTIGATIONAL PLAN 
17 
5.1 
Summary of Study Design 
17 
5.2 
Study Procedures 
17 
5.2.1 
Pre-study Procedures 
17 
5.2.2 
Baseline Evaluation 
18 
5.2.3 
Day 1 of Each Cycle 
18 
5.2.4 
Cycle 1 Days 1-10 
18 
5.2.5 
Cycle 1 Day 4 ± 1 
18 
5.2.6 
Cycle 1 Day 10 ± 1 
19 
5.2.7 
Weekly 
19 
5.2.8 
Every Other Week 
19 
5.2.9 
Cycle 1 and Even Cycles Day 28 ± 4 days 
19 
5.2.10 
End-of-Study Procedures 
19 
5.2.11 
Post-study Follow-up 
19 
5.3 
Duration of Therapy 
20 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
20 
5.4.1 
Chemotherapy 
20 
5.4.2 
Growth Factors 
20 
5.4.3 
Transfusions 
21 
5.4.4 
Prophylactic Antimicrobial Agents 
21 
5.4.5 
Radiotherapy 
21 
6.0 DOSE MODIFICATIONS 
22 
6.1 
Delay for Cytopenia or Infection 
22 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 1 Version date: 10/29/2013 
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6.2 
Delay for Organ Dysfunction 
22 
6.3 
Modification in Dose or Schedule 
22 
7.0 PHARMACEUTICAL INFORMATION 
23 
7.1 
Decitabine 
23 
7.1.1 
Study Drug Preparation 
23 
7.1.2 
Supplier 
23 
7.1.3 
Toxicities 
23 
8.0 CORRELATIVE STUDIES SAMPLE COLLECTIONS 
25 
9.0 EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
26 
9.1 
Response Criteria 
26 
9.2 
Guidelines for Evaluation of Disease 
28 
9.3 
Progressive Disease 
Error! Bookmark not defined. 
9.4 
Other Secondary Efficacy Measures 
28 
9.4.1 
Overall Survival and Event-free Survival 
28 
9.4.2 
Duration of Remission and Relapse-free Survival for Patients Having Complete 
Remission 
28 
9.4.3 
Safety 
29 
10.0 ADVERSE EVENT REPORTING 
30 
10.1 Adverse Events (AEs) 
30 
10.2 Unanticipated Problems 
30 
10.3 Noncompliance 
30 
10.4 Serious Noncompliance 
30 
10.5 Protocol Exceptions 
31 
10.6 Reporting to the Human Research Protection Office (HRPO) and the Quality Assurance 
and Safety Monitoring Committee (QASMC) at Washington University: 
31 
10.7 Timeframe for Reporting Required Events: 
32 
11.0 DATA SAFETY MONITORING PLAN 
33 
12.0 STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
34 
12.1 Analysis Populations 
34 
12.2 Statistical Analysis 
34 
12.2.1 
Descriptive Analyses 
34 
12.2.2 
Primary Endpoint Analysis 
34 
12.2.3 
Secondary Endpoint Analyses 
35 
12.2.4 
Safety Analysis 
37 
12.2.5 
Description of Planned Subgroup Analyses 
37 
12.3 Sample Size 
37 
13.0 DATA SUBMISSION SCHEDULE 
38 
  


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 1 Version date: 10/29/2013 
5 
1.0 
INTRODUCTION 
 
1.1 
Rationale   
 
Personalized therapy for cancer requires that 1) patient-specific factors (e.g. patient-
specific mutations, expression profiles, or pharmacogenomics) influence treatment 
algorithms, and 2) once therapy is initiated, in vivo responsiveness is used to guide 
further therapy. The modern era of genomics has provided significant insights into cancer 
pathogenesis by describing patterns of germline and somatic variants, as well as defining 
disease-specific patterns of gene expression. However, the application and integration of 
this data with therapeutics remains a difficult task. 
 
Decitabine is a commonly used single-agent therapy for patients with acute myeloid 
leukemia (AML) and/or myelodysplastic syndrome (MDS). Historically, it has yielded 
clinical complete responses in approximately 25% of patients across diverse clinical 
trials, it is well tolerated, and it can be given as in the outpatient setting. However, the 
majority of patients do not respond to decitabine, the molecular etiology of decitabine 
sensitivity is unknown, and there are no algorithms to predict patient responsiveness a 
priori or even within one month of starting therapy.  
 
AML and MDS are two closely related hematological disorders characterized by 
ineffective hematopoiesis and malignant expansion of clonal myeloid cells.  Both AML 
and MDS frequently occur in elderly patients and some cases are secondary and/or 
related to long-term bone marrow damage after radiation or chemotherapy. Current 
therapy for AML consists of cytotoxic chemotherapy followed by consolidation 
chemotherapy or stem cell transplantation. Using these approaches, current cure rates 
may be as high as 60% for patients younger than 60 with good risk cytogenetics (e.g. 
t(15;17), t(8;21), inv(16)).[4]  Outcomes in patients ≥ 60 years old and with relapsed 
disease remain poor with 2 year survival of only ~25%.[5] Historically, the available 
treatment for MDS has been symptomatic and has not been shown to be effective in 
producing sustained improvement in hematopoiesis or in delaying leukemic evolution[6, 
7].  
 
Current AML therapy is poorly tolerated in elderly and pre-treated patients due to 
hematologic and non-hematologic toxicities; following such therapy, patients typically 
require 3-4 weeks of hospitalization for treatment of neutropenic fevers and for regular 
transfusions. Even among the relatively healthy patients enrolled in clinical trials, 
induction mortality in the elderly can be as high as 20-40% following standard induction 
therapy,[8-11] and moderate to severe non-hematologic toxicity occurs in at least an 
additional 30% of patients.[9, 12-14] Consequently, many older AML patients are not 
offered traditional intensive chemotherapy, but receive supportive care alone.[15]  More 
effective and less toxic therapies are needed to provide durable remissions in a significant 
proportion of elderly AML in populations. 
 
Decitabine has been studied in MDS patients and in elderly and/or relapsed AML 
patients. It has been found to be reasonably well tolerated, can be given in the outpatient 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 1 Version date: 10/29/2013 
6 
setting, and has overall response rates of 20-45%.[2, 3, 16-20] Our own experience has 
been of CR/PR rates of 25%, [2] and this is typical of many other groups for both MDS 
and AML patients (reviewed in [21]  Decitabine is an S-phase depended drug.  [22-24] 
However, decitabine is usually administered in one hour daily infusions during 3 to 5 
consecutive days (too short to expose decitabine to most AML blasts during at least 2 
cycles of replication, which divide every 2 to 7 days).[25-27]  Recent experience with 
increasing the number of infusions per cycle from 5 consecutive days to 10 consecutive 
days has improved overall response rates to 67%.[3] Response to decitabine can be slow, 
sometimes requiring 3 to 5 cycles before maximum response is observed.[2] As a 
consequence, many patients are exposed to prolonged therapy, gaining little benefit, 
before determining resistance. There are currently no biomarkers that predict response to 
decitabine. 
 
Decitabine is a cytosine analogue that cannot be methylated. Incorporation of decitabine 
into DNA prevents DNA methylation at cytosine residues and derepresses epigenetically 
silenced gene expression. Therefore, the reduction in total genomic DNA methylcytosine 
content acts as a marker of effective decitabine dosing. Indeed, two groups have recently 
correlated effective methylcytosine reduction and gene expression derepression to 
response (Figure 1.1).[3, 28] 
 
 
 
   
Figure 1.1. From Shen et al JCO (2010) 28:611 and Blum et al JCO (2007) 25: 3884. 
 
We and others have recently used whole genome sequencing to identify recurrent, 
somatic mutations in AML and MDS patients in four genes that are involved in 
methylcytosine regulation: DNMT3A, IDH1, IDH2, and TET2.[29-37] Collectively, 
nearly half of all AML/MDS patients have a mutation in one of these four genes.  
DNMT3A mutations are associated with an adverse outcome in AML patients treated with 
cytotoxic chemotherapy.[35, 38] However, it is not yet clear whether the presence of 
mutations in one or more of these genes is associated with response or resistance to 
decitabine. 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 1 Version date: 10/29/2013 
7 
In this study, we will treat patients with the current state-of-the-art decitabine 
schedule.[3] We will perform exome sequencing on all patients and correlate response 
with mutation status, rate of mutation clearance, expression profile, plasma decitabine 
levels, and DNA methylcytosine content reduction. This approach will comprehensively 
assess genomic and pharmacologic parameters that could clinically predict patient 
response to decitabine.  
 
In this study, we will combine analysis of patients with AML and MDS. Recent genomic 
analysis has shown that AML and MDS have shared mutations in many of the same 
genes, shared patterns of mutation acquisition, and shared patterns of mutation 
evolution.[39, 40] Furthermore, MDS, like AML, presents with clonal hematopoiesis, 
even when the morphologic blast count is zero.[36]Because this study will identify and 
track the rate of clearance of patient specific mutations, and because decitabine has been 
shown to have efficacy in both AML and MDS patients, [2, 3, 16-19, 41-45] combining 
analysis of AML and MDS patients is reasonable.  Asymptomatic MDS patients are 
excluded because these patients are typically followed with watchful-waiting rather than 
with chemotherapy. 
  
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
 
Methylation of cytosine in the CpG dinucleotide by DNA methyltransferase leads to 
transcriptional silencing of genes during normal development and has emerged as a 
significant mechanism for the loss of tumor suppressor gene expression in human cancer, 
including AML and MDS.[46, 47] Like azacitidine, decitabine (5-aza-2’-deoxycitidine) is 
incorporated into DNA during S phase and forms covalent adducts with DNA 
methyltransferase. The irreversible inhibition of this enzyme results in loss of 
methylation and is associated with differentiation.[48]  Decitabine is an approximately 5-
fold more potent inhibitor of DNA methyltransferase than azacitidine, which might 
explain why decitabine can induce differentiation of myeloid progenitors in vitro at lower 
doses than azacitidine. In addition to their differentiation inducing activity at low doses, 
both agents have a direct cytotoxic effect at higher doses (1-2 µM).[49]  
 
Decitabine has been investigated in hematologic malignancies using a range of doses and 
schedules.  It is currently FDA approved for the treatment of adults with MDS at a dose 
of 15 mg/m2 every 8 hours for 3 days, based on a Phase III study that demonstrated 
improved outcomes in the treatment arm compared to patients treated with supportive 
care alone.[15]  However, the overall response rate in this trial was modest (17%). Of 
note, 18% of the patients in this trial had 20-30% bone marrow blasts, meeting the 
current WHO criteria for AML.  Subsequent phase I and II studies in AML, using a 
variety of low-dose decitabine regimens, have reported overall response rates of 22-
49%.[2, 3, 16-19, 41-45] Comparisons of multiple low dose schedules have noted 
improved efficacy in the MDS population using a dose of 20 mg/m2 on Days 1-5 of a 28-
day cycle.[50, 51] Because of these results, we have recently explored this dosing 
schedule in the treatment of elderly patients with AML.[52] In this study, we observed an 
overall response rate of 24%, and an additional 44% of patients achieved stable disease 
during a median of 5 monthly cycles.[48] In this study, we noted that decitabine does not 


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act quickly and the median time to response was 3 months. These results are similar to 
the recently published experience in Germany treating 227 patients with 15 mg/m2 over 3 
hours TID for 3 days every 6 weeks, which resulted in a CR/PR rate of 26%.[16]  
 
A recent study using a prolonged administration schedule (20 mg/m2 IV over 1 hour on 
consecutive days 1-10) showed improved response (64% CR/PR) and improved time to 
respond (50% of patients had bone marrow blasts < 5% after 1 cycle of therapy).[3]  
These results are an improvement over previous experience by other groups using 
decitabine to treat elderly AML patients or patients with relapsed AML, and this 
experience is summarized below: 
Trials of decitabine in AML 
Status 
Other Therapy 
DAC Dose 
(mg x days) 
Evaluable 
CR 
de novo 
daunomycin1 
90 mg/m2 x 5 
6 
100% 
Salvage 
None4 
0.75-80 mg/kg 
16 
  
None5 
37-81 mg/kg 
6 
33% 
None7.  Ara-C 
Refractory 
Various 
4 
0 
AMSA7 
250-500 mg/m2 x 6 
10 
80% 
idarubicin or 
AMSA3 
250 mg/m2 x 6 
56 
39% 
Elderly 
None6 
270-360 mg/m2 x 3 
10 
30% 
None2 
45 mg/m2 x 3 
4 
50% 
None8 
20 mg/m2day 1-5,8-12 
50 
23% 
None9 
20 mg/m2 x 5 
55 
25% 
 
None10 
50 mg/m2 CIVI x 3 
29 
48% 
 
None11 
20 mg/m2 x 10 
53 
64% 
 
ATRA cycle 212 
15 mg/m2 TID x 3 
227 
26% 
1 Schwartsmann G.  Leukemia 1997; 11(Suppl 1):S28-31. 
2 Lubbert M. ASH 2002 Abstract #4601. 
3 Willemze R.  Leukemia.  1997; 11(Suppl 1):S24-27 
4 Rivard GE.  Leukemia Research 1981; 5:453-462. 
5 Momparler RL.  Pharmac Therapy 1985; 30:277-286.  
6 Petti MC.  Leukemia.  1993; 7(Suppl 1):36-41.  
7 Richel DJ.  Br. J. Cancer 1991; 64:144-148 
8 Issa, JP. Blood 2004. 103: 1635. 
9 Cashen AF. ASH Annual Meeting. 2008 abstract 560. 
10 Wijermans PW. Leukemia 1997. 11: Suppl 1, 19-23. 
11 Blum, W. PNAS 2010; 107:7473. 
12 Lubbert. Haematologica 2011. ePub 11/04/2011. 
 
In general, decitabine is well tolerated. Unlike conventional cytotoxic chemotherapy, 
decitabine can be administered in the outpatient setting. However, AML and MDS are 
aggressive hematologic malignancies. Cytopenias commonly result from AML/MDS 
inhibition of normal hematopoiesis, and often do not resolve until a complete response is 
achieved. Thus, neutropenic sepsis and thrombocytopenic bleeding remain significant 
problems regardless of therapy, especially during the first 4-8 weeks. In our experience 
treating elderly AML patients, neutropenia occurred in 24%, sepsis in 9%, and 
bacteremia in 7%.  Similar results have been observed in MDS patients.[20, 53, 54] As a 


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result, death is not uncommon in the first 8 weeks of therapy, occurring in 15-17% of 
patients, and most commonly resulting from infection and cytopenias related to disease 
and therapy.[16, 52, 55] While unsettling, these numbers are better than many historical 
patients treated with cytotoxic chemotherapy, and suggest a need for continued 
improvements in therapy.[9, 12]  
 
1.2.1 Pharmacokinetics and Metabolism of Decitabine 
 
In human subjects, after IV administration, decitabine displayed a distribution 
phase with a half-life of 7 minutes and a terminal phase elimination half-life of 10-
35 minutes as measured by bioassay.[56, 57]   The short plasma half-life is due to 
rapid inactivation of decitabine by deamination by liver cytidine deaminase.[23] 
Total body clearance (Clp) in humans was high with a mean value of 126 
mL/min/kg.[58]  
 
Among five adult patients with advanced solid tumors administered seven courses 
of 100 mg/m2 of decitabine by 1-hour infusions,[56] maximum plasma 
concentration ranged from 1 to 4 µM. Disappearance from plasma was biphasic 
with a rapid initial decline to less than 0.1 µM during the first hour post infusion 
and a slower decline over the next two hours to 6 nM, the level of detectability. 
Plasma half-life (meanSE) was estimated to be approximately 71 minutes in 
the initial phase (t½α) and 355 minutes in the second phase (t½β). The area under 
the plasma concentration-time curve was 40888 ng.hour/mL for these patients, 
with a peak plasma concentration of 2.010.44 µM (0.440.1 µg/mL), while mean 
volume of distribution (Vdss) and CLp was 4.591.42 L/kg and 12621 
mL/min/kg, respectively. The time course of DAC concentrations was similar in 1 
of 2 patients administered 75 mg/m2, while DAC was usually undetectable in the 
plasma of patients administered 25 to 60 mg/m2. 
 
Using a longer infusion time (up to 40 hours), Rivard et al.[57] reported a plasma 
concentration of 0.1 to 0.4 g/mL. With infusion times of 40-60 hours, at an 
infusion rate of 1 mg/kg/h, plasma concentrations of 0.43-0.76 µg/mL were 
achieved.[59]  The steady-state plasma concentration at an infusion rate of 
1 mg/kg/h was estimated to be 0.2-0.5 µg/mL. The half-life after discontinuing the 
infusion was 12-20 min.[57]  
 
Urinary excretion of unchanged decitabine was low, ranging from less than 0.01% 
to 0.9% of the total dose, and there was no relationship between excretion and dose 
or plasma drug levels. High clearance values and a total urinary excretion of <1% 
of the administered dose suggest that decitabine is eliminated rapidly and largely by 
metabolic processes, including extrahepatic metabolism.[60] 
 
The steady state plasma concentrations (Css) ranged from 0.31 to 0.39 µg/mL 
(1.27 to 1.60 µM) during the infusion of 100 mg/m² decitabine over a 6-hour 
period.[61] The range of concentrations during a 600 mg/m2 infusion over an 8-
hour period was 0.41-16 g/mL(1.68 to 4.76 µM).[60] These plasma 


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concentrations are in the same range as the in vitro concentrations that activated 
the expression of tumor suppressor gene p16 in three human non-small-cell lung 
cancer (NSCLC) cell lines.[61]  
 
Steady state decitabine levels of 0.1 to 0.2 µM were achieved in solid tumor 
patients dosed with 30 mg/m²/day and 40 mg/m²/day by continuous intravenous 
infusion over 72 hours.[62]  
 
Summary of published steady state serum levels are below and are in proportion to 
infusion rate (R2 = 0.98, p < 0.0001):[63] 
 
Rate 
Serum 
Concentration 
mg/m2/hr 
ng/ml 
nM 
5 
55 
241 
7.5 
80 
351 
15 
180 
789 
21 
182.4 
800 
22.5 
350 
1535 
42 
456 
2000 
84 
1140 
5000 
 
In our experience treating primary patient blasts in vitro, concentrations of 100 – 
500 nM are required maximize methylcytosine reduction without causing cellular 
toxicity. Because decitabine incorporation into DNA is S-phase dependent, 
exposure of cells during cell division is necessary for efficacy. 
  
 
 
 
1.2.2 Experience with extended dosing schedules 
 
Blum et al recently published their experience treating elderly AML patients with 
a 10-day course of decitabine 20 mg/m2/day as a 1-hour infusion.[3] This 
 


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prolonged exposure course was designed to increase the number of AML blast 
divisions exposed to decitabine per cycle. Toxicities were very similar to a similar 
regiment given as a 3 and 5 day course,[2, 16] but the overall response (CR/PR) 
was much improved (64% vs 25%). In all three studies, the toxicities were 
primarily myeloid suppression and the resultant infectious and bleeding 
complications associated with leukemic and pharmacologic hematopoietic 
suppression. Furthermore, Blum et al noted that the percent methylcytosine 
reduction was improved (22% vs 11%) as was the time to blast clearance (50% of 
patients who ultimately achieved a CR had ≤ 5% bone marrow blasts after 1 cycle 
vs 0% of patients after 2 cycles). Finally, like Shen et al, they observed a 
correlation between the extent of methylcytosine reduction and response.[28]  
This study suggests that improvement in response rates and time to response can 
be achieved by prolonged administration of decitabine. Further, that DNA 
methylcytosine reduction can be achieved with prolonged exposure to decitabine 
and that this might be a patient-specific biological marker of adequate dosing. 
  
Scandura et al. recently published a study of infusional decitabine prior to 
cytarabine + daunorubicin induction therapy for AML.[64] They escalated the 
duration of infusional therapy and reached their planned goal of 20 mg/m2/day x 7 
days of continuous therapy given prior to cytarabine and daunorubicin. They 
noted similar toxicities (primarily hematopoietic and the associated neutropenic 
infections), but that these were not different from their experience giving 
cytarabine + daunorubicin alone. 
 
1.3 
Correlative Studies 
 
With exception of the peripheral blood sample collected on Cycle 1 Day 4, all correlative 
studies will be collected under HRPO# 201011766 (“Tissue Acquisition for Analysis of 
Genetic Progression Factors in Hematologic Diseases”) that all patients will be 
concurrently enrolled in. The goals of the correlative studies include: 
 
1. To correlate patient-specific mutations with response to decitabine. 
We will perform exome sequencing of each patient’s pre-study bone marrow sample 
and skin sample. We will determine AML/MDS-associated variants found in the bone 
marrow, but not skin sample. We will correlate the presence of mutations with 
response (especially mutations in DNMT3A, IDH1, IDH2, and TET2). This study is 
powered to identify mutations with >80% sensitivity to predict decitabine response if 
used as a clinical assay. 
 
2. To correlate the velocity and depth of response with response to decitabine. 
We will assess tumor burden by allele frequency of patient-specific mutations in bone 
marrow samples collected pre study, Cycle 1 Day 10±1, Cycle 1 Day 28±4, and 
Cycle 2 Day 28±4. We will assess mutations in at least 24 recurrently mutated genes 
(including, but not limited to: FLT3, DNMT3A, NPM1, TET2, WT1, IDH1, IDH2, 
RUNX1, NRAS, TP53, U2AF1, KRAS, PHF6, MSTP9, PTPN11, KIT, SMC3, 
PRUNE2, ETV6, CEBPA, SMC1A, ASXL1, STAG2, RAD21) as well as all patient-


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specific mutations discovered during exome sequencing. The leukemic allele 
frequency will be measured using the Illumnia MySeq or HiSeq platform using DNA 
capture by gene specific biotinylated-oligonucleotides. Patient bone marrow samples 
will be assessed pre-study, Cycle 1 Day 10 ± 1, Cycle 1 Day 28 ± 4, and Cycle 2 Day 
28 ± 4 and compared with patient specific skin samples (normal control). The 
velocity of response will be calculated as the natural log rate of patient-specific 
mutation allele frequency clearance. Depth of response will be defined as the lowest 
patient-specific mutation allele frequency in a bone marrow sample. The velocity and 
depth of tumor response will be correlated to: 
 Achievement of clinical CR, 
 Time to best response, 
 Mutational status, 
 Time to disease progression. 
Because all patient-specific exome-discovered mutations will be assessed a second 
time in the primary bone marrow sample as well as the  Cycle 1 Day 10 ± 1, Cycle 1 
Day 28 ± 4, and Cycle 2 Day 28 ± 4 bone marrow samples, this study will also serve 
to validate all exome-discovered mutations. This is important, because exome 
sequencing is associated with a 20-50% false-discovery rate depending on type I error 
tolerated (mutation-calling algorithms are typically set for greater sensitivity than 
specificity).  Thus, many mutations discovered during exome sequencing are 
incorrect.  This second analysis of these mutations will server to exclude these false 
positive calls. 
 
3. To correlate the bone marrow expression profile from pre-study and Cycle 1 Day 10 
± 1 with response to decitabine. 
We will correlate expression signatures, and changes in expression signatures, with: 
 Achievement of clinical CR, 
 Mutation status. 
 
4. To correlate steady-state plasma decitabine concentration on Cycle 1 Day 4 ± 1 with: 
 
Achievement of CR. 
 
5. To correlate the extent of bone marrow DNA hypomethylation on Cycle 1 Day 10 ± 1 
with response to decitabine. 
We will assess the percent reduction in bone marrow methylcytosine content using 
LC-MS. Patients will be evaluated pre-study and Cycle 1 Day 10 ± 1. The change in 
DNA methylcytosine content will be correlated to: 
 Achievement of clinical CR, 
 Change in expression signature. 
This will determine whether inadequate blast exposure to decitabine correlates with 
drug resistance. 
 


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2.0 
OBJECTIVES 
 
2.1 
Primary Objective 
 
To determine whether patient-specific mutations correlate with clinical overall response 
to decitabine in patients with AML or MDS. 
 
2.2 
Secondary Objectives 
 
- 
To determine whether 10 consecutive days per cycle of decitabine improves 
outcomes compared with historical controls treated with 5 consecutive days per cycle 
of decitabine. 
 
- 
To determine whether other biomarkers correlate with clinical overall response to 
decitabine in patients with AML or MDS. These will include:  
 
1. The velocity and depth of patient-specific mutation clearance; 
2. The bone marrow expression profile and change in expression profile during 
decitabine treatment; 
3. The steady-state plasma decitabine concentrations; 
4. The decrease in bone marrow methylcytosine content in response to decitabine.  
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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3.0 
PATIENT SELECTION 
 
3.1 
Inclusion Criteria 
 
One of the following: 
 Patient must have non-M3 AML and be > 60 years of age OR 
 non-M3 AML with relapsed disease OR 
 Symptomatic MDS with one of the following: 
□ Symptomatic anemia with either hemoglobin < 10.0 g/dL or requiring RBC 
transfusion 
□ Thrombocytopenia with a history of two or more platelet counts < 
50,000/mcL or a significant hemorrhage requiring platelet transfusions 
□ Neutropenia with two or more ANC < 1,000/mcL 
 
All of the following: 
1. Patient must have an ECOG performance status ≤ 2. 
2. Patient must have >10% disease burden measured by cytomorphology, flow 
cytometry, or cytogenetics. 
3. Patient must have peripheral white blood cell count < 50,000/mcl. 
4. Patient must have adequate organ function, defined as: 
a. 
Total bilirubin < 1.5 x ULN 
b. 
AST/ALT < 2.5 x ULN 
c. 
Serum creatinine < 2.0 x ULN  
5. Patient must have undergone ≤ 2 cycles of prior hypomethylating agent 
(decitabine or azacitidine). 
6. Patient must be enrolled in HRPO# 201011766 (“Tissue Acquisition for Analysis 
of Genetic Progression Factors in Hematologic Diseases”). 
7. Patient must be > 18 years of age. 
8. Patient must be able to understand and willing to sign an IRB-approved written 
informed consent document. 
 
3.2 
Exclusion Criteria 
 
1. Patient must not be pregnant or nursing. 
2. Patient must not have known CNS leukemia.  
3. Patient must not have a history of positive HIV serology.  
4. Patient must not have a history of positive Hepatitis C serology. 
5. Patient must not have undergone a prior allogeneic stem cell transplant. 
6. Patient must not have any uncontrolled intercurrent illness including, but not 
limited to, ongoing or active infection, ongoing or active GVHD, congestive heart 
failure of NYHA class 3 or 4, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situation that would limit compliance with study 
requirements. 
7. Patient must not have had radiation therapy within 14 days of enrollment. 
8. Patient must not have received any chemotherapy within 21 days of enrollment, 
and any acute treatment-related toxicities must have returned to baseline. Patients 


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may be receiving hydrea at time of enrollment.    
 
3.3 
Inclusion of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible for this 
trial. 


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4.0 
REGISTRATION PROCEDURES 
 
Patients must not start any protocol intervention prior to registration through the Siteman 
Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility 
2. Registration of patient in the Siteman Cancer Center database 
3. Assignment of unique patient number (UPN) 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility collecting the information listed below  
1. The registering MD’s name 
2. Patient’s race, sex, and DOB 
3. Three letters (or two letters and a dash) for the patient’s initials 
4. Copy of signed consent form 
5. Completed eligibility checklist, signed and dated by a member of the study team 
6. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center Database 
 
All patients must be registered through the Siteman Cancer Center database. 
 
4.3 
Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  
Patients will also be identified by first, middle, and last initials.  If the patient has no 
middle initial, a dash will be used on the case report forms (CRFs).  All data will be 
recorded with this identification number on the appropriate CRFs. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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5.0 
INVESTIGATIONAL PLAN 
 
5.1 
Summary of Study Design 
 
This is an open-label, single-arm, Phase II, biomarker study of patients with AML or 
MDS who are treated with 10 consecutive days of decitabine. FAB-M3 AML is excluded 
from this trial, because other well-tolerated options exist for the treatment of FAB-M3 
acute promyelocytic leukemia. AML patients who are < 60 years old and have not 
received cytotoxic induction chemotherapy are also excluded, because cytotoxic therapy 
offers a curative possibility for these patients.  MDS patients without cytopenias are also 
excluded because these patients can be followed with watchful-waiting. 
 
All patients will undergo bone marrow biopsies pre-study, on Cycle 1 Day 10 ± 1, and on 
Day 28 ± 4 of Cycle 1 and all even number cycles. Patients will provide a skin biopsy 
sample pre-study. Patients will also provide peripheral blood samples on Cycle 1 Day 4 ± 
1 for pharmacokinetic studies. See Section 8.0 for details on correlative studies.  
 
Patients will receive decitabine 20 mg/m2/day as a 1-hour infusion on consecutive Days 
1-10 in 28-day cycles.  The dose will be calculated based on actual patient weight at the 
beginning of each cycle. 
 
Once AML patients achieve morphologic complete remission with incomplete blood 
count recovery (CRi) or MDS patients achieve a marrow complete response (Marrow 
CR) as defined in Section 9.0, decitabine administration may be decreased to five 
consecutive days to limit myelosuppressive toxicity at the discretion of the treating 
physician. After two cycles of five consecutive days of treatment, if the patient meets the 
definition of CR with complete cytogenetic response as defined in Section 9.0, 
administration may further be decreased to three consecutive days per cycle at the 
discretion of the treating physician. 
 
It will be a logistical goal of this trial consistently to return preliminary exome results to 
the treating physician by the end of Cycle 1 and validated results by the end of Cycle 2. 
However, this logistical goal is not a primary or secondary aim of this study. Given the 
limited data regarding mutation presence and drug response, we do not anticipate that the 
results will directly influence therapeutic decisions made by the treating physician. 
However, the development of comprehensive genomic pipelines and integration of this 
data into a clinical trial setting will be required before personalized medicine can be 
realized. 
 
5.2 
Study Procedures 
 
5.2.1 Pre-study Procedures 
 
Pre-study procedures must take place no more than 14 days prior to the first dose 
of decitabine except where noted below. 
 


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1. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments 
performed on bone marrow will include correlative studies (collected under 
HRPO# 201011766), morphologic assessment, cytogenetics, and FISH.  
2. Bone marrow or peripheral blood for flow cytometry 
3. Skin biopsy sample (collected under HRPO# 201011766). There is no 
required time frame for this sample; it may have been collected months or 
even years prior to the first dose of decitabine.  
 
5.2.2 Baseline Evaluation 
 
Baseline evaluations must take place no more than 7 days prior to the first dose of 
decitabine.  
 
1. Medical history 
2. Physical exam including height, weight, and ECOG performance score. 
3. Record transfusion requirements for 8 weeks before first dose of study drug 
4. CBC with differential and platelets 
5. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
6. Urine βHCG pregnancy test if appropriate 
 
5.2.3 Day 1 of Each Cycle 
 
1. Physical exam, including weight  
2. CBC with differential and platelets 
3. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
 
5.2.4 Cycle 1 Days 1-10 
 
Decitabine administration, 20 mg/m2 per day given as a 1-hour continuous 
intravenous infusion.  The dose will be calculated based on actual patient weight 
at the beginning of each cycle.  Patients may be premedicated with lorazepam, 
prochlorperazine, palonosetron, or ondansetron at the treating physician’s 
discretion. 
 
5.2.5 Cycle 1 Day 4 ± 1 
 
Obtain 3 mL peripheral blood for correlative studies. Sample must be obtained 45 
minutes ± 15 minutes after beginning the infusion, before the decitabine infusion 
is complete, and should be obtained from the opposite arm as the infusion.  The 
sample should be collected in a 3 mL K3EDTA tube pre-loaded with 8 mcL of 
tetrahydrouridine (THU 500 mcg/mL), an inhibitor of cytidine deaminase.  The 


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sample should be processed by the Tissue Procurement Core Facility at the 
Siteman Cancer Center.  Plasma should be separated and stored per protocols. 
  
5.2.6 Cycle 1 Day 10 ± 1 
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH. 
 
5.2.7 Weekly 
 
CBC with differential and platelets. After the first cycle, this may be changed to 
every other week at the discretion of the treating physician. 
 
5.2.8 Every Other Week 
 
Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH. After the first cycle, this may be changed to 
every month at the discretion of the treating physician. 
 
5.2.9 Cycle 1 and Even Cycles Day 28 ± 4 days  
 
Bone marrow aspirate and biopsy. Standard bone marrow procurement procedures 
will be followed for collection of the tissue. Assessments performed on bone 
marrow will include: correlative studies (collected under HRPO# 201011766), 
morphologic assessment, cytogenetics, and FISH 
 
5.2.10 End-of-Study Procedures  
 
1. Physical exam 
2. CBC with differential and platelets.  
3. Serum chemistries including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, and LDH.  
4. Bone marrow aspirate and biopsy. Standard bone marrow procurement 
procedures will be followed for collection of the tissue. Assessments 
performed on bone marrow will include morphologic assessment, 
cytogenetics, and FISH.  
 
5.2.11 Post-study Follow-up 
 
Patients are to be followed according to standard patient care procedures for 30 
days following the date of the last decitabine dose to monitor for toxicity, 
survival, and response. Thereafter, the investigator or designee will record 


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information on subsequent therapy, disease progression, and/or death every 6 
months for 2 years after last dose of study drug or until the patient is lost to 
follow-up or dies. 
 
5.3 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to the 
patient’s health and/or the patient no longer wishes to continue protocol therapy, the 
protocol therapy should be discontinued and the reason(s) for discontinuation 
documented in the case report forms. 
 
In the absence of treatment delays due to adverse events, treatment may continue 
indefinitely or until one of the following criteria applies: 
 
 Documented and confirmed disease progression 
 Death 
 Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
 General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
 Suspected pregnancy 
 Serious non-compliance with the study protocol 
 Lost to follow-up 
 Patient withdraws consent 
 Investigator removes the patient from study 
 The Siteman Cancer Center decides to close the study 
 
Patients who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar. 
 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
 
5.4.1 Chemotherapy 
 
Patients cannot receive any chemotherapy within 21 days of study enrollment and 
must have recovered from any treatment-related toxicities. However, patients may 
be receiving hydrea at enrollment and may continue on hydrea through Cycle 2 of 
decitabine. An indication for hydrea following 2 cycles of decitabine is evidence 
that the patient is not responding and decitabine should be discontinued. Other 
than decitabine, patients cannot receive chemotherapy during this trial.  
 
5.4.2 Growth Factors 
 
Erythroid growth factors should not be routinely administered to patients during 
the study. 
 


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Thrombopoietin receptor agonists should not be routinely administered to patients 
during the study. 
 
Filgrastim (G-CSF) or sargramostim (GM-CSF) may be administered to patients 
with septic shock or febrile neutropenia at the discretion of the treating physician. 
 
5.4.3 Transfusions 
 
 Packed red blood cells (RBCs) transfusion:   
RBC transfusions will be administered at the discretion of the treating 
physician.  
 
 Platelet transfusion:   
Platelet transfusions will be administered at the discretion of the treating 
physician  
5.4.4 Prophylactic Antimicrobial Agents  
 
Antimicrobial agents will be administered at the discretion of the treating 
physician. 
 
5.4.5 Radiotherapy 
 
Patients cannot receive radiotherapy within 14 days prior to study enrollment or 
while on study. 
 


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6.0 
DOSE MODIFICATIONS 
 
A criterion for discontinuation of the study is a delay of > 8 weeks between any two 
cycles.  
 
6.1 
Delay for Cytopenia or Infection 
 
As cytopenias are a defining feature of AML and MDS, their presence alone does not 
generally warrant dose modifications or delays. Patients will receive four cycles of 
therapy without adjustment for any cytopenia. Following Cycle 4, dose modifications or 
delays for cytopenias will be made at the discretion of the investigator.  
 
Patients with febrile neutropenia (temperature ≥ 38.5° with ANC < 1,000/l) or systemic 
infections should have treatment held until clinical resolution of the infection.  
 
6.2 
Delay for Organ Dysfunction 
 
If renal dysfunction (creatinine ≥ 3 X institutional upper limit of normal) or hepatic 
dysfunction (bilirubin ≥ 2.5 mg/dl or ALT/AST ≥ 3 X institutional upper limit of normal) 
occurs, therapy may not resume until the abnormalities have resolved to below the limits 
stated above. If renal or hepatic dysfunction occurs during a treatment course, the next 
dose of decitabine should be held, and serum chemistries should be checked twice per 
week.  Decitabine can be resumed at full dose when creatinine, bilirubin, ALT and AST 
are below the limits listed above.  
 
For grade 3-4 non-hematologic toxicities related to decitabine, decitabine treatment 
should be delayed until recovery to baseline or to grade ≤ 1. Decitabine may be 
reintroduced with a 50% dose reduction, or at full dose at the discretion of the 
investigator. 
 
6.3 
Modification in Dose or Schedule for Treatment Response 
 
Once AML patients achieve morphologic complete remission with incomplete blood 
count recovery (CRi) or MDS patients achieve a marrow complete response (Marrow 
CR) as defined in Section 9.0, decitabine administration may be decreased to 5 
consecutive days to limit myelosuppressive toxicity at the discretion of the treating 
physician. After two cycles of five consecutive days of treatment, if the patient meets the 
definition of CR with complete cytogenetic response as defined in Section 9.0, 
administration may further be decreased to three  consecutive days per cycle at the 
discretion of the treating physician. 
 


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7.0 
PHARMACEUTICAL INFORMATION 
 
7.1 
Decitabine 
 
Decitabine is an antimetabolite that can replace cytosine in DNA but, unlike cytosine, it 
cannot be methylated. The exact mechanism of decitabine activity has not been 
determined and may involve multiple pathways. Decitabine is thought primarily to act by 
correcting DNA methylation, a major mechanism for gene expression. In some cancer 
cells, hypermethylation blocks the activity of tumor suppressor genes, which regulate cell 
division and differentiation to prevent malignant transformation. When suppressor gene 
activity is blocked, cell division becomes unregulated, leading to the formation of 
neoplastic cells. Decitabine regulates DNA methylation (specifically targets the 
methyltransferase, DNMT1), effectively demethylating and reactivating different tumor 
suppressor genes. Because hypermethylation occurs early in the malignant transformation 
of cells, decitabine may have activity in chemoprevention. Other possible mechanisms 
for decitabine activity include cytotoxicity due to incorporation into DNA similar to other 
antimetabolites, methylation-independent induction of gene expression, and degradation 
of DNA methyltransferase 1. 
 
7.1.1 Study Drug Preparation  
 
OSHA Guidelines for handling cytotoxic drugs outlined in the American Journal 
of Hospital Pharmacy must be followed. 
 
Decitabine (5-aza-2’-deoxycytidine) is supplied as a lyophilized powder for 
injection, 50 mg in 20-ml vials. When reconstituted with 10 ml of sterile water for 
injection, each ml will contain 5 mg of decitabine and 6.8 mg of KH2PO4. The 
reconstituted solution should be diluted to 0.1-1.0 mg/mL with pre-chilled 0.9% 
sodium chloride intravenous infusion, which has been cooled to 2-8oC. 
Reconstitution of the powder results in a rapidly decomposing solution. Unless 
used within 15 minutes of reconstitution, the diluted solution must be prepared 
using cold (2—8 degrees C) infusion fluids and stored at 2—8 degrees C (36—46 
degrees F) for a maximum of 7 hours. 
Decitabine will be administered per dosing schedule every 28 ± 4 days.  
 
Decitabine may be administered either as an inpatient or an outpatient.  
 
7.1.2 Supplier 
 
Decitabine is available commercially. 
 
7.1.3 Toxicities 
 
Likely (>15%) 
Cytopenias: 
neutropenia, 
thrombocytopenia, 
anemia, 
leukopenia, 
febrile 
neutropenia.  


Genomic Predictors of Decitabine Response in AML/MDS 
 
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Bleeding: petechiae, ecchymosis. 
Infections: pneumonia. 
Constitutional: fatigue, pallor, fever, rigors, arthralgia, cough. 
GI: nausea/vomiting, constipation, diarrhea, decreased appetite. 
Electrolyte abnormalities: hyperglycemia, hypoalbuminemia, hypomagnesemia, 
hypokalemia, hyponatremia.  
Cardiac: cardiac murmur. 
Neurologic: headache, insomnia, dizziness. 
Dermatologic: rash. 
 
Less likely (1-15%)  
Hematologic: thrombocythemia, hematoma. 
Other infections: cellulitis, candidal infection, urinary tract infection, sinusitis. 
GI: abdominal 
pain, hyperbilirubinemia, 
stomatitis, 
dyspepsia, ascites, 
hemorrhoids, oral ulceration, dysphagia, abdominal distention, gastro-esophageal 
reflux disease/pyrosis (heartburn), and glossodynia. 
Electrolyte abnormalities: hyperkalemia, and dehydration. 
Urologic: dysuria, increased urinary frequency.  
Dermatologic: pruritus, alopecia, urticaria, edema. 
 
Rare (<1%) 
Cardiac: arrest/respiratory arrest, atrial fibrillation, and tachycardia. 
 
  
 
  
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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8.0 
CORRELATIVE STUDIES SAMPLE COLLECTIONS 
 
Bone marrow samples will be collected pre-study, on Cycle 1 Day 10 ± 1, and on Day 28 ± 4 of 
Cycle 1 and of even-numbered cycles for correlative studies. The bone marrow samples will be 
collected under HRPO# 201011766 (“Tissue Acquisition for Analysis of Genetic Progression 
Factors in Hematologic Diseases”) that all patients will be concurrently enrolled in.  
 
Skin biopsy sample will be collected as part of the patient’s participation in HRPO# 201011766 
(“Tissue Acquisition for Analysis of Genetic Progression Factors in Hematologic 
Malignancies”). There is no required time frame for this sample, and it may have been collected 
months or even years prior to the first dose of decitabine.  
 
Peripheral blood sample (3 mL) will be collected on Cycle 1 Day 4 ± 1.  Sample must be 
obtained 45 minutes ± 15 minutes after beginning the infusion, before the decitabine infusion is 
complete, and should be obtained from the opposite arm as the infusion.  The sample should be 
collected in a 3 mL K3EDTA tube pre-loaded with 8 mcL of tetrahydrouridine (THU 500 
mcg/mL), an inhibitor of cytidine deaminase.  The sample should be processed by the Tissue 
Procurement Core Facility at the Siteman Cancer Center.  Plasma should be separated and stored 
per protocols. 
 
 


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9.0 
EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
 
A secondary endpoint of this study is to estimate morphologic complete remission rate. 
  
9.1 
Response Criteria - AML 
 
AML Patients will be assessed for response according to the IWG criteria:1  
 
Morphologic complete remission (CR) – Defined as morphologic leukemia-free state, 
defined as less than 5% blasts in aspirate with marrow spicules and a count of ≥200 
nucleated cells and no blasts with Auer rods or any persistence of extramedullary disease. 
When erythroid precursors constitute less than 50% of bone marrow nucleated cells, the 
percentage of blasts is based on all nucleated cells; when there are 50% or more erythroid 
cells, the percentage blasts should be based on the nonerythroid cells. The presence of 
mild megaloblastoid changes may be permitted if they are thought to be consistent with 
treatment effect. However, persistence of pretreatment abnormalities (eg, pseudo-Pelger-
Hüet cells, ringed sideroblasts, dysplastic megakaryocytes) are not consistent with CR.  
In addition, ANC >1000/μl and platelet count of ≥100,000/μl. Patient must be 
independent of transfusions. No duration requirement for this designation. 
 
Cytogenetic complete remission (CCR) – Defined as morphologic complete remission 
plus a reversion to a normal karyotype. For this study, reversion of a normal karyotype is 
defined as no clonal abnormalities detected in a minimum of 20 mitotic cells. 
 
Morphologic complete remission with incomplete blood count recovery (CRi) – Defined 
as morphologic complete recovery with the exception of neutropenia <1000/μl or 
thrombocytopenia <100,000/μl.  
 
Partial remission (PR) – Defined as meeting the above criteria for complete remission, 
with the following exceptions: decrease of ≥50% in the percentage of blast to 5-25% in 
the bone marrow aspirate. A value of 5% blasts in bone marrow with Auer rods present 
may also be considered a partial remission.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, CRi, PR, PD, or 
recurrence/morphologic relapse.   
 
Progressive disease (PD) – Defined as a clear increase in bone marrow blast count, 
typically a greater than 50% increase compared with baseline, or new extramedullary 
disease. Patients with a > 50% increase in peripheral blast count with an increase of the 
total peripheral white blood cell count to > 10,000/l should undergo evaluation by bone 
marrow biopsy to access for progressive disease.  
 
Recurrence/morphologic relapse - Defined as relapse following complete remission is 
defined as reappearance of blasts in the blood or the finding of ≥5% blasts in the bone 
marrow, not attributable to any other cause. New dysplastic changes is considered 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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relapse. If there are no blasts in the peripheral blood and 5-20% blasts in the bone 
marrow, bone marrow biopsy should be repeated in > 1 week to confirm relapse. 
 
9.2 
Response Criteria - MDS 
 
MDS Patients will be assessed for response according to the IWG criteria:69 
 
Complete remission (CR) – Defined as < 5% myeloblasts with normal maturation of all 
cell lines in the bone marrow and peripheral blood values of Hgb > 11g/dL, Platelets > 
100 x 109/L, Neutrophils > 1.0 x 109/L, and 0% blasts. Persistent dysplasia does not 
exclude CR but will be noted.  
 
Marrow Complete Response (Marrow CR) – Defined as < 5% myeloblasts in the bone 
marrow and a decrease by > 50% from pretreatment values, but not meeting the 
definition of CR above.  
 
Partial Remission (PR) – Defined as meeting the definition of CR above with a decrease 
of myeloeblasts in the bone marrow by > 50% from pretreatment values, but absolute 
myeloblasts still >5%.  
 
Stable Disease (SD) – Defined as not meeting the definitions of CR, Marrow CR, PR, 
SD, PD, or recurrence/morphologic relapse.   
 
Progressive Disease//Relapse (PD) – Defined as ≥ 50% increase in blasts to > 5% blasts 
(for patients with less than 5% blasts at baseline only), ≥ 50% increase to > 10% blasts 
(for patients with 5-10% blasts at baseline only), ≥ 50% increase to > 20% blasts (for 
patients with l0-20% blasts at baseline only), ≥ 50% increase to > 30% blasts (for patients 
with 20-30% blasts at baseline only) or any of the following: At least 50% decrement 
from maximum remission/response in granulocytes or platelets, reduction in Hgb by ≥ 2 
g/dL, or New or worsened transfusion dependence not related to study drug toxicity. Or 
for patients with a CR, Marrow CR, or PR as defined above and subsequently 
development of one of the following: Return to pretreatment bone marrow blast 
percentage, decrement of ≥ 50% from maximum remission/response levels in 
granulocytes or platelets, or reduction in Hgb concentration by ≥ 1.5 g/dL or transfusion 
dependence. 
 
9.2.1 Hematologic Improvement (HI) 
 
 
 
Progressive disease as defined above nullifies hematologic improvement.  
 
Erythroid response requires all of the following (only required if pretreatment 
HgB <11 g/dL): 
 Hgb increase by ≥ 1.5 g/dL 
 Relevant reduction of units of RBC transfusions by an absolute number of at 
least 4 RBC transfusions/8 wk compared with the pretreatment transfusion 


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number in the previous 8 wk. Only RBC transfusions given for a Hgb of ≤ 9.0 
g/dL pretreatment will count in the RBC transfusion response evaluation 
 
Platelet response requires one of the following (only required if pretreatment 
platelets < 100 x 109/L):  
 Absolute increase of ≥ 30 x 109/L (for patients starting with > 20 x 109/L 
platelets) 
 Increase from < 20 x 109/L to > 20 x 109/L and absolute increase > 100% (for 
patients starting with < 20 x 109/L) 
 
Neutrophil response requires the following (only required if pretreatment ANC 
<1.0 x 109/L):  
 At least 100% increase and an absolute increase > 0.5 x 109/L 
 
9.3.1 Cytogenetic Response  
 
Complete Cytogenetic Response – Defined as reversion to a normal karyotype. 
For this study, reversion of a normal karyotype is defined as no clonal 
abnormalities detected in a minimum of 20 mitotic cells. Progressive disease as 
defined above nullifies cytogenetic response.  
 
9.3 
Guidelines for Evaluation of Disease 
 
Bone marrow biopsy should be performed on Day 28 ± 4 of cycle 1 and on Day 28 ± 4 of 
each even cycle for clinical disease assessment. 
 
9.4 
Other Secondary Efficacy Measures 
 
9.4.1 Treatment Failure, Overall Survival and Event-free Survival 
 
Treatment failure is defined as failure to achieve a complete or partial remission 
following 4 cycles of therapy.  
 
Overall survival is defined as the date of first dose of study drug to the date of 
death from any cause.  Patients still alive at the time of the final analysis will be 
right-censored. 
 
Event-free survival is defined as the interval from the date of first dose of study 
drug to date of treatment failure, recurrence, death due to any cause, or loss to 
follow up. 
 
9.4.2 Duration of Remission and Relapse-free Survival for Patients Having 
Complete Remission 
 
Duration of complete remission is defined as the interval from the date that 


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complete remission is documented to the date of recurrence, death due to any 
cause, or loss to follow-up.  
 
Relapse-free survival is defined only for those patients achieving a complete 
remission. It is defined as the interval from the date of first documentation of a 
leukemia-free state to the date of recurrence, death due to any cause, or loss to 
follow up.   
  
9.4.3 Safety 
 
Safety evaluation will include assessments of adverse experiences, medical 
history, physical examinations, vital signs, and laboratory assessments at baseline 
and throughout the study period. Local laboratories may be used for this study.  
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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10.0 
ADVERSE EVENT REPORTING  
 
10.1 
Adverse Events (AEs) 
 
Definition:  any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading:  the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for all 
toxicity reporting.  A copy of the CTCAE version 4.0 can be downloaded from the CTEP 
web site:  http://ctep.cancer.gov/reporting/ctc.html. 
 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the terms 
listed that should be used are those provided by the Department of Health and Human 
Services’ Office for Human Research Protections (OHRP).  A copy of this guidance can 
be found on OHRP’s website: (http://www.hhs.gov/ohrp/policy/AdvEvntGuid.htm). 
 
10.2 
Unanticipated Problems 
 
Definition: 
 
 unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied; 
 related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the 
incident, experience, or outcome may have been caused by the procedures 
involved in the research); and 
 suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
 
10.3 
Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that govern 
human 
subjects 
research 
or 
failure 
to 
follow 
the 
determinations 
of 
the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate choice to 
ignore regulations, institutional policies, or determinations of the IRB. 
 
10.4 
Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial harm 
to subjects or others, or that materially compromises the rights or welfare of participants. 
 


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10.5 
Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under the research 
team’s control. Exceptions apply only to a single participant or a singular situation. 
 
Pre-approval of all protocol exceptions must be obtained prior to the event. 
 
10.6 
Reporting to the Human Research Protection Office (HRPO) and the Quality 
Assurance and Safety Monitoring Committee (QASMC) at Washington 
University: 
 
The PI is required to promptly notify the IRB of the following events: 
 
 Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct 
of the study. 
 Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 
 Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence of 
the event or notification to the PI of the event.  The death of a research participant that 
qualifies as a reportable event should be reported within 1 working day of the occurrence 
of the event or notification to the PI of the event. 
 
10.7 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER 
FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the investigator to report any unanticipated problem to the FDA as 
follows: 
 
 Report any unexpected fatal or life-threatening adverse experiences associated 
with use of the drug (i.e., there is a reasonable possibility that the experience may 
have been caused by the drug) by telephone or fax no later than 7 calendar days 
after initial receipt of the information.  A life-threatening adverse experience is 
defined as any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it 
does not include a reaction that, had it occurred in a more severe form, might have 
caused death. 
 Report any serious, unexpected adverse experiences, as well as results from 
animal studies that suggest significant clinical risk within 15 calendar days after 
initial receipt of this information.  A serious adverse drug experience is defined as 


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any adverse drug experience occurring at any dose that results in any of the 
following outcomes: 
o Death 
o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial 
disruption of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 
o Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical 
intervention to prevent one of the outcomes listed above 
An unexpected adverse drug experience is defined as any adverse drug 
experience, the specificity or severity of which is not consistent with the current 
investigator brochure (or risk information, if an IB is not required or available). 
 
All MedWatch forms will be sent by the investigator or investigator’s team to the FDA at 
the following address or by fax: 
 
Food and Drug Administration 
Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
10.8 
Timeframe for Reporting Required Events:  
 
Reportable adverse events will be tracked for 30 days following the last day of study 
treatment, or until the start of a new chemotherapy/treatment, whichever comes first. 
 
Deaths 
Any reportable death while on study or within 
30 days of study 
Immediately, within 24 hours, to PI 
and the IRB 
Any reportable death while off study  
Immediately, within 24 hours, to PI 
and the IRB  
Adverse Events/Unanticipated Problems 
Any reportable adverse events as described in 
Sections 10.1 and 10.2 (other than death) and 
10.7 
Immediately, within 24 hours to PI 
and within 10 working days to the 
IRB, and within 7 or 15 days to the 
FDA  
All adverse events regardless of grade and 
attribution should be submitted cumulatively  
Include in DSM report 
Noncompliance and Serious Noncompliance 
All noncompliance and serious noncompliance 
as described in Sections 10.3 and 10.4 
Immediately, within 24 hours, to PI 
and within 10 working days to the IRB 


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11.0 
DATA SAFETY MONITORING PLAN 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, 
the Principal Investigator will provide a Data and Safety Monitoring (DSM) report to the 
Washington University Quality Assurance and Safety Monitoring Committee (QASMC) semi-
annually beginning six months after accrual has opened (if at least five patients have been 
enrolled) or one year after accrual has opened (if fewer than five patients have been enrolled at 
the six-month mark). 
 
The Principal Investigator will review all patient data at least every six months, and provide a 
semi-annual report to the QASMC. This report will include: 
 HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
 Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of 
study 
 History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol 
exceptions, error, or breach of confidentiality including start/stop dates and reason 
 Study-wide target accrual and study-wide actual accrual 
 Protocol activation date 
 Average rate of accrual observed in year 1, year 2, and subsequent years 
 Expected accrual end date 
 Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
 Measures of efficacy 
 Early stopping rules with supporting data and list the number of participants who 
have met the early stopping rules 
 Summary of toxicities 
 Abstract submissions/publications 
 Summary of any recent literature that may affect the safety or ethics of the study  
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC 
according to institutional guidelines. 
 


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12.0 
STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
 
12.1 
Analysis Populations 
 
Intent-to-Treat - The intent-to-treat (ITT) population will be defined as all patients who 
receive at least one dose of decitabine.  
 
Efficacy-Evaluable - The efficacy-evaluable (EE) population will be defined as all 
patients who receive at least one cycle of treatment.   
 
Safety - The safety population will be defined as all patients with at least one dose of 
decitabine. 
 
12.2 
Statistical Analysis 
 
12.2.1 Descriptive Analyses 
 
Patient Disposition: The number of patients discontinued, the reasons for 
discontinuation, and the number of cycles administered will be summarized. 
 
Demographics and Baseline Characteristics: Descriptive summary statistics will 
be provided for demographic and important baseline characteristics.  For 
continuous variables, the number of patients, mean, standard deviation, median, 
minimum and maximum will be provided.  For categorical variables, the number 
and percent of patients in each demographic/characteristic category will be 
summarized. 
 
12.2.2 Primary Endpoint Analysis 
 
The primary end-point of this phase II study is the correlation biomarkers with 
overall response in AML/MDS patients to decitabine. 
 
We will correlate the presence of patient specific mutations (especially mutations 
in DNMT3A, IDH1, IDH2, and TET2) with overall response rates. Mutation rates 
in these genes and in other patient-specific genes will be correlated with overall 
response and their respective 95% confidence intervals will be provided. 
 
Statistical considerations. The relationship between AML/MDS-associated 
mutations and the responsiveness to decitabine will be described by contingency 
tables and assessed using Chi-square test or Fisher’s exact test as appropriate. The 
differentially expressed genes between responders and non-responders will be 
determined using the significance analysis of microarray (SAM). Multivariate 
logistic regression will also be fit to adjust the potential confounding effects of 
other demographic and clinical characteristics. The resultant critical P values will 
be corrected for multiple testing.  Since the Bonferroni correction is known to be 
too conservative, a permutation analysis (e.g., 10,000 random assignments for 


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responsive status) will be used to derive an empirical critical P value.  The new 
critical p-value is the number of permutation tests which yield a lower p-value 
than the original test divided by the total number of permutation tests.[61]   
 
A power analysis was performed using Chi-square test for the association 
between mutation and response to decitabine. The frequency of DNMT3A, 
IDH1/2, and TET2 mutations in AML and MDS patients are each between 15% 
and 30% in multiple cohorts.[29-37] Table 3 shows the minimal detectable 
differences of response rates between mutant and wild-type cases, assuming an 
expected overall 45% CR/CRi rate and 80% power. For an average of 20% 
frequency of a mutated gene, for example, 100 patients will provide 80% power 
to detect 30% difference at a 2-sided 0.1 alpha error. The purpose of this study is 
to determine whether any of 
these mutations result in a 
clinically 
significant 
association 
that 
would 
warrant 
pre-treatment 
testing. We believe that a 
reasonable cut off would be 
if at least 30% of clinical 
response could be attributed 
to the presence of a specific 
mutation. 
However, 
we 
anticipate that more power 
could be achieved as we 
assess patient groups with 
combined 
characteristics 
(e.g. DNMT3A mutation or 
loss 
of 
DNMT3A 
expression). In addition, we 
expect that the majority of the enrolled 125 patients can be used in the analysis 
(since all of our molecular information will be acquired within 30 days of 
enrollment, we anticipate a drop-out rate of 20% or less). 
 
12.2.3 Secondary Endpoint Analyses 
 
The overall response rate (CR/CRi/PR) and complete response rate (CR/CRi) will 
be compared with historical controls[2], and the 95% confidence intervals will be 
provided. 
 
The rate of patient specific mutation clearance after 10, 28, and 56 days of therapy 
will be determined using an Illumina based platform. We will compare the rate of 
mutation clearance between the patients who achieve a CR/CRi after 4 cycles and 
those who do not. We anticipate that we will identify between 6 and 25 exonic 
SNVs per case (the range obtained with >200 AML samples sequenced to date 
with whole genome or exome sequencing), and that the expected 95% confidence 
Table 3. Detectable mutation associated response rates (80% power, α = 0.05) 
 
Total 
N 
% of patients 
with a given 
mutation  
Detectable 
response 
difference 
Sensitivity of mutation to 
predict response at minimum 
detectable response difference* 
100 
15%  
20%  
30% 
40% 
50% 
0.40 
0.35 
0.30 
0.27 
0.26 
80% 
75% 
67% 
61% 
58% 
* Assuming the overall response  rate (CR/Cri) of 45%   


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interval of the mean variant allele frequency will be ±2.56% and ±1.25%, 
respectively, if we assume an anticipated standard deviation of 3.2% within the 
same clone. The change of tumor burden over time and its association with 
clinical response will  be assessed using 2-way ANOVA for repeated 
measurement data, followed by the ad-hoc multiple comparisons for between-
group differences (e.g., responders vs. non-responders) at given time points as 
well as the between-time differences within each group. The resultant critical P 
values will also be corrected using permutation analysis for multiple testing. 
 
The steady-state plasma decitabine concentration on day 4 ± 1 will be measured 
and correlated with clinical overall response. 
 
The percent decrease in bone marrow methylcytosine will be calculated on day 
10±1. We will compare this reduction with previously published data and between 
the patients who achieve CR/CRi and those who do not. The correlation of peak 
plasma levels of decitabine with response will be assessed using 2-sample t-test or 
Mann-Whitney non-parametric rank-sum test as appropriate. The change of total 
bone marrow DNA methylcytosine from baseline and its association with both 
steady-state plasma drug levels and response will be assessed using 2-way 
ANOVA for repeated measurement data, followed by the ad-hoc multiple 
comparisons for between-group differences (e.g., responders vs. non-responders) 
and between-time differences. Similar analyses will be performed for the 
correlation of expression profiles and SNP analysis with pharmacokinetic data.   
 
Statistical power justification.  A power analysis was performed using two-
sample t-test for between-group difference and paired t-test for the change over 
time. For tumor clearance, this will provide > 99% power to detect a 10% 
difference in the means at two separate time points for clones with 6 or more 
variants, and > 98% power to detect a 15% difference if there are 3 or more 
variants per clone with α = 0.05 (power will decrease to 91% and 82%, 
respectively, in cases where the standard deviation is 4.7%, the maximum we 
have observed thus far). For pharmacologic correlations, we anticipate 85% 
power to determine a 25 ng/ml difference between the average Cmax of 45 
evaluable responders and 55 evaluable non-responders with an anticipated 
average Cmax of 70 ng/ml and a standard deviation of 40 ng/ml within the entire 
cohort, and with α = 0.05.[22] We believe a less significant difference would not 
be sufficient to warrant application in clinical decision making (i.e. whether to 
continue therapy for more than 1 cycle). We anticipate 99% power to determine a 
difference of 20% in the reduction of bone marrow methylcytosine between 45 
evaluable responders and 55 evaluable non-responders with an expected standard 
deviation of 15% and α = 0.05.[28, 66-68] The difference in methylcytosine 
reduction previously observed was 30% between patients achieving CR/PR and 
those with only hematologic improvement.[28]  
 
Time to relapse and time to death will also be listed for each patient. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 1 Version date: 10/29/2013 
37 
12.2.4 Safety Analysis 
 
Extent of Exposure – Duration (number of cycles/infusions) of decitabine doses 
(cumulative and intensity) will be summarized. 
 
Adverse Events - All adverse events will be coded using NCI CTCAE v. 4.0.  The 
incidence of treatment-emergent adverse events (number and percent of patients 
reporting the adverse event at least once during the study) will be summarized.  In 
addition, adverse events will be summarized by investigator attribution of 
relationship to study medication and by grade.  Similar summaries will be 
provided by treatment cycle. 
 
Clinical Laboratory Evaluation – Applicable laboratory parameters will be 
graded according to NCI CTCAE version 4.0. The incidence of maximum grade 
(number and percent of patients experiencing the maximum grade during the 
study) will be summarized for each laboratory parameter.  Similar summaries will 
be provided by treatment cycle.  Graphical displays of select hematological 
parameters will also be provided. 
 
Vital Signs, Physical Findings, and Other Safety Observations – Vital signs, 
findings from physical examinations, ECOG scores and weights will be presented 
in listing format. 
 
12.2.5 Description of Planned Subgroup Analyses 
 
The following subgroups (if sufficient numbers of patients) will be explored with 
respect to the primary and overall survival endpoints. 
 Age (60-69 years old vs ≥ 70 years old) 
 Cytogenetics 
 Patients with AML-associated mutations in DNMT3A, TET2, IDH1, or 
IDH2 vs those who do not have somatic variants in these genes. 
 
12.3 
Sample Size 
 
We anticipate initially enrolling 25 patients at Washington University. Once we establish 
a routine exome analysis with validation and calculation of the rate of patient-specific 
mutation clearance, we will enroll an additional 100 patients from at least 2 additional 
centers.  
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 1 Version date: 10/29/2013 
38 
13.0 
DATA SUBMISSION SCHEDULE 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to registration 
Patient Registration Form 
Disease Diagnosis Form 
Inclusion Criteria Worksheet 
Exclusion Criteria Worksheet 
 
Prior to starting treatment 
Treatment Record Form 
To be maintained throughout a patient’s treatment 
cycle.  The complete record is due at completion of 
treatment. 
Bone Marrow Sample Collection 
Form 
To be maintained throughout the course of the 
study at designated bone marrow sample 
collections time points (See Appendix 2). 
Pathology: Bone Marrow Biopsy 
To be completed upon the return of pathology 
report for each bone marrow biopsy. 
Study Calendar Form 
To be completed with the date of each time point 
upon occurrence. 
Labs Form 
To be completed upon the return of laboratory 
reports for each study time point (See Appendix 2) 
Dose Modifications 
To be maintained throughout the course of the 
study if dose modifications are performed. 
Therapy Response Form 
To be filled out upon remission (prior to, and/or on 
therapy) 
End of Study Form 
 
Due at the completion of the EOS visit, at the time 
of death, and/or at the time that the patient is lost to 
follow-up 
Follow Up Form 
 30 days post date of last decitabine dose. 
 Every 6 months for 2 years after the last dose of 
study drug. 
Record of Adverse Events 
At the time of any AE 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 1 Version date: 10/29/2013 
39 
Appendix 1 
ECOG Performance Scale 
Activity Status 
Description 
0 
Asymptomatic, fully active, and able to carry on all predisease 
performance without restrictions. 
 
1 
Symptomatic, fully ambulatory but restricted in physically strenuous 
activity and able to carry out performance of a light or sedentary 
nature, eg, light housework, office work. 
 
2 
Symptomatic, ambulatory and capable of all self-care but unable to 
carry out any work activities.  Up and about more than 50% of 
waking hours: in bed less than 50% of day. 
 
3 
Symptomatic, capable of only limited self-care, confined to bed or 
chair more than 50% of waking hours, but not bedridden. 
 
4 
Completely disabled.  Cannot carry on any self-care.   Totally 
bedridden. 
 
5 
Dead 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 1 Version date: 10/29/2013 
40 
Appendix 2 
Study Flow Chart 
Study Flow Chart 
Test 
Pre-
study1 
Base-
line2 
All 
Cycles 
Day 1 
All Cycles 
Days 1-103 
Cycle 1 Day 
4 ± 1 
 
Cycle 1 
Day 10 ± 1 
and 28 ± 4 
Every 
week 
Every 
2 
weeks 
Even 
cycles 
Day 28±4 
End of 
Study 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Medical History 
 
X 
 
 
 
 
 
 
 
 
Physical Exam 
 
Xa 
Xb 
 
 
 
 
 
 
X 
Transfusion 
Requirementsc 
 
Xc 
Ongoing 
Bone Marrow 
Aspirate and Biopsy 
Xd 
 
 
 
 
Xd 
 
 
Xd 
Xd 
Flow Cytometrye 
X 
 
 
 
 
 
 
 
 
 
Peripheral blood for 
correlative studies 
 
 
 
 
X 
 
 
 
 
 
Pregnancy test 
 
X 
 
 
 
 
 
 
 
 
CBC with differential 
and platelets 
 
X 
X 
 
 
 
Xf 
 
 
X 
Serum Chemistriesg 
 
X 
X 
 
 
 
 
Xf 
 
X 
Decitabine 
administrationh 
 
 
 
X 
 
 
 
 
 
 
Adverse Events  
 
 
Ongoing 
Post-study follow-up 
 
 
 
 
 
 
 
 
 
Xi 
1 To be performed within 14 days of the first dose of decitabine.  
2 To be performed within 7 days of the first dose of decitabine.  
3 Decitabine may be reduced to Days 1-5 or Days 1-3. See Section 6.3 for details.  
a To include height, weight, and ECOG. 
b To include weight. 
c Care should be given to record the frequency of transfusions from 8 weeks prior to first dose of study drug through 
the duration of the study. 
d Standard bone marrow procurement procedures will be followed for collection of the tissue. To include: correlative 
studies (collected under HRPO# 201011766), morphologic assessment, cytogenetics and FISH at all time points. A 
skin biopsy sample (collected under HRPO# 201011766) is required at pre-study, but may have been collected months 
or even years prior to first dose of decitabine.  
e On bone marrow or peripheral blood.  
f After Cycle 1, these labs may be obtained every other week (CBC) or once a month (Chemistries) at the discretion of 
the treating physician. 
g Serum chemistries include bicarbonate, calcium, glucose, total protein, albumin, BUN, potassium, chloride, sodium, 
creatinine, total bilirubin, AST, ALT, alkaline phosphatase, and LDH. 
h Decitabine treatment will be 20 mg/m2 over a 1-hour IV infusion on consecutive Days 1-10 every 28-day cycle.   The 
dose will be calculated based on actual patient weight at the beginning of each cycle. Patients may be premedicated 
with lorazepam, prochlorperazine, palonosetron, or ondansetron at the treating physician’s discretion. 
i Patients will be followed for  30 days following the date of the last decitabine dose to monitor for toxicity, survival 
and response. Thereafter, the investigator or designee will record information on subsequent therapy, disease 
progression, and death, every 6 months for 2 years after first dose of study drug or until the patient is lost to follow-up.


Genomic Predictors of Decitabine Response in AML/MDS 
 
Amendment 1 Version date: 10/29/2013 
41 
14.0 
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Genomic Predictors of Decitabine Response in AML/MDS 
 
 
NCT# NCT01687400 
 
 
Document Version Date:  09/12/2012 
 
IRB Approval Date:  12/06/2012 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Version date: 09/12/12 
 
 
 
Genomic Predictors of Decitabine Response in AML/MDS 
 
Washington University School of Medicine 
Division of Oncology 
660 South Euclid Avenue, Box 8056 
St. Louis, MO 63110 
 
Protocol #: (Pending) 
Protocol Version Date: 09/12/12 
 
Principal Investigator 
John Welch, M.D./Ph.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-2626 
Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Sub-Investigator 
Timothy Ley, M.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-8832 
 
Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Pathology 
Jeffery M. Klco, M.D., Ph.D. 
Department of Anatomic and Molecular Pathology 
Telephone: (314) 362-8832 
Email: klcoj@wustl.edu 
 
Statistician 
Feng Gao, Ph.D. 
Division of Biostatistics 
Telephone: (314) 362-3682 
E-mail: feng@wubios.wustl.edu 
 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to obtain 
informed consent, may not be disclosed to another party unless law or regulations require such disclosure.  Persons 
to whom the information is disclosed must be informed that the information is confidential and may not be further 
disclosed by them. 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Version date: 09/12/12 
 
 
Study Synopsis 
Title of study  
 
Genomic Predictors of Decitabine Response in AML 
Primary 
investigator/study center 
location 
John S. Welch, M.D./PhD 
Washington University School of Medicine, Division of Oncology 
St. Louis, MO 
Timothy J. Ley, MD 
Washington University School of Medicine, Division of Oncology 
St. Louis, MO 
Phase of development 
Phase II 
Study objectives 
Primary:  
- To determine whether patient-specific mutations correlate with clinical 
overall response to decitabine in patients with AML or MDS. 
Secondary:   
- To determine whether 10 consecutive days per cycle of decitabine 
improves outcomes compared with historical controls treated with 5 
consecutive days per cycle of decitabine. 
- To determine whether other biomarkers correlate with clinical overall 
response to decitabine in patients with AML or MDS. These will include: 
velocity and depth of patient-specific mutation clearance, bone marrow 
expression profile and change in expression profile during decitabine 
treatment, steady-state serum decitabine concentrations, and decrease in 
bone marrow methylcytosine content in response to decitabine. 
 
 
Study design  
Non-randomized, open-label, Phase II study of decitabine in patients with 
AML or MDS.  
Study criteria 
Inclusion Criteria 
 
One of the following: 
1. Patient must have non-M3 AML and be > 60 years of age OR  
2. non-M3 AML with relapsed disease OR 
3. Symptomatic MDS with one of the following: 
o 
Symptomatic anemia with either hemoglobin <10.0 g/dL 
or requiring RBC transfusion 
o 
Thrombocytopenia with a history of two or more platelet 
counts < 50,000 / µL or a significant hemorrhage 
requiring platelet transfusions, or 
o 
Neutropenia with two or more absolute neutrophil counts 
< 1,000 /µL. 
 
All of the following: 
1. Patient must have an ECOG performance status ≤ 2. 
2. Patient must have >10% disease burden measured by 
cytomorphology, flow cytometry, or cytogenetics. 
3. Patient must have peripheral white blood cell count < 50,000/mcl. 
4. Patient must have adequate organ function, defined as: 
a. 
Total bilirubin < 1.5 x ULN 
b. AST/ALT < 2.5 x ULN 
c. 
Serum creatinine < 2.0 x ULN  
5. Patient must have undergone ≤ 2 cycles of prior hypomethylating 
agent (decitabine or azacitidine). 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Version date: 09/12/12 
 
6. Patient must be able to understand and willing to sign an IRB-
approved written informed consent document. 
7. Patient must be enrolled in HRPO# 201011766 (“Tissue 
Acquisition for Analysis of Genetic Progression Factors in 
Hematologic Malignancies”). 
8. Patient must be > 18 years of age. 
 
 
Exclusion Criteria 
1. Patient must not be pregnant or nursing. 
2. Patient must not have known CNS leukemia.  
3. Patient must not have a history of positive HIV serology.  
4. Patient must not have a history of positive Hepatitis C serology. 
5. Patient must not have undergone prior allogeneic stem cell 
transplant. 
6. Patient must not have any uncontrolled intercurrent illness including, 
but not limited to, ongoing or active infection, ongoing or active 
GVHD, congestive heart failure of NYHA class 3 or 4, unstable 
angina pectoris, cardiac arrhythmia, or psychiatric illness/social 
situation that would limit compliance with study requirements. 
7. Patient must not have had radiation therapy within 14 days of 
enrollment.  
 
Expected number of 
patients 
Total: 125. 
Evaluable for all molecular studies: 100. 
Enrollment time-table: 25 patients will be enrolled initially at Washington 
University. Once the molecular pipeline for routine exome analysis is 
established, at least 2 additional sites will be added.  
Study drug formulation 
and administration  
Decitabine (5-aza-2’-deoxycytidine) is a lyophilized white powder available 
for injection in 20-mL single-use vials containing 50 mg of drug. The 
product should be stored at room temperature. Prior to use, the test product is 
reconstituted in 10 mL of sterile water for injection. The reconstituted 
concentrate is further diluted with cold infusion solution to obtain a final 
concentration of decitabine of between 0.1 to 1.0 mg/mL. 
 
All patients will receive decitabine 20 mg/m2 given as a 1 hour continuous 
IV infusion on consecutive days 1-10 of each cycle; the dose should be 
calculated based on actual body weight at the beginning of each cycle.  
 
Once patients achieve morphologic complete remission with incomplete 
blood count recovery (CRi) (morphologic leukemia-free state, defined as less 
than 5% blasts in aspirate with marrow spicules and a count of  ≥ 200 
nucleated cells and no blasts with Auer rods, no persistence of 
extramedullary disease, and no persistent pre-treatment morphologic 
abnormalities, such as pseudo-Pelger-Hüet cells, ringed sideroblasts, or 
dysplastic megakaryocytes) administration may be decreased to 5 
consecutive days to limit myelosuppressive toxicity, per treating physician. 
After two cycles with bone marrow blast counts < 5% and no evidence of 
disease by cytogenetics or immunophenotype, patients may further decrease 
therapy to 3 consecutive days per cycle, per treating physician. 
Each cycle will be 28 ± 4 days. 
Duration of treatment 
Treatment may continue until one of the following criteria applies: 
 Death, 
 Disease progression (as defined in 3.7.1.3), 
 Intercurrent illness that prevents further administration of treatment, 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Version date: 09/12/12 
 
 Unacceptable adverse event(s), 
 Patient decides to withdraw from the study, or 
 General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the treating 
physician.  
Correlative studies  
Bone marrow samples for correlative studies will be collected prestudy, on 
Cycle 1 day 10 ± 1 and day 28 ± 4, and on day 28 ± 4 of subsequent even 
numbered cycles.  
Peripheral blood samples for correlative studies will be collected on Cycle 1 
day 4 ± 1.   
 
1. Identify Patient-specific mutations 
We will perform exome sequencing of each patient’s day 0 bone 
marrow sample and skin sample. We will determine AML/MDS-
associated variants, defined as variants found in the bone marrow 
sample and not in the skin sample. We will correlate the presence of 
mutations with overall response. 
2. Velocity and depth of response. 
We will assess the rate of tumor clearance by measuring the allele 
frequency of patient-specific mutations in bone marrow samples 
during cycles 1 and 2. We will assess mutations in at least 24 
recurrently mutated genes (including, but not limited to: FLT3, 
DNMT3A, NPM1, TET2, WT1, IDH1, IDH2, RUNX1, NRAS, TP53, 
U2AF1, KRAS, PHF6, MSTP9, PTPN11, KIT, SMC3, PRUNE2, 
ETV6, CEBPA, SMC1A, ASXL1, STAG2, RAD21) as well as all 
patient-specific mutations discovered during exome sequencing. The 
leukemic allele frequency will be measured using the Illumnia 
MySeq or HiSeq platform using DNA capture by gene specific 
biotinylated-oligonucleotides, or by a comparable technique. Patient 
bone marrow samples will be assessed on day 0, day 10 ± 1, and day 
28 ± 4 of cycle 1, and on day 28 ± 4 of cycle 2 and compared with 
patient-specific skin samples (normal control). The velocity of 
response will be calculated as the natural log rate of patient-specific 
mutation allele frequency clearance. Depth of response will be 
defined as the lowest patient-specific mutation allele frequency in a 
bone marrow sample. The velocity and depth of tumor response will 
be correlated to: 
 
Overall response, 
 
Time to best response, 
 
Mutation status, 
 
Time to disease progression. 
 
3. Expression profile. 
Expression array profiling will be performed on bone marrow 
samples from day 0 and day 10 ± 1. We will correlate expression 
signatures, and changes in expression signatures, with: 
 
Overall response, 
 
Mutational status. 
 
4. Steady-state serum decitabine concentration. 
We will assess serum decitabine concentration on day 4 ± 1 and 
correlation with: 
 
Overall response. 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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5. Bone marrow DNA hypomethylation. 
We will assess the percent reduction in bone marrow methylcytosine 
content using LC-MS. This may serve as a better biomarker of 
adequate dosing than serum drug concentration. Patients will be 
evaluated on day 0 and day 10 ± 1 of cycle 1. The change in DNA 
methylcytosine content will be correlated to: 
 
Overall response, 
 
Change in expression signatures. 
 
  
Criteria for evaluation 
Efficacy-  
Bone marrow biopsies should be performed at baseline and at day 10 ± 1 and 
day 28 ± 4 of cycle 1 and at the end of subsequent even numbered 
cycles. Standard bone marrow procurement procedures will be 
followed for collection of the tissue. Jeffrey Klco, MD/PhD will act 
as a central reviewer for all bone marrow biopsies. 
 
Peripheral blood counts will be followed by weekly CBC with differential. 
After cycle 1, the frequency of routine CBC analysis may be 
decreased by the treating physician as clinically indicated.  
 
Patients will be assessed for response according to the IWG criteria:[1]  
Morphologic complete remission (CR)– Defined as morphologic 
leukemia-free state, defined as less than 5% blasts in aspirate with 
marrow spicules and a count of  ≥ 200 nucleated cells and no blasts with 
Auer rods or any persistence of extramedullary disease, and no evidence 
for dysplasia. When erythroid precursors constitute less than 50% of bone 
marrow nucleated cells, the percentage of blasts is based on all nucleated 
cells; when there are 50% or more erythroid cells, the percentage blasts 
should be based on the nonerythroid cells. The presence of mild 
megaloblastoid changes may be permitted if they are thought to be 
consistent with treatment effect. However, persistence of pretreatment 
abnormalities (eg, pseudo-Pelger-Hüet cells, ringed sideroblasts, 
dysplastic megakaryocytes) are not consistent with CR. In addition, ANC 
> 1000/μl and platelet count of ≥ 100,000/μl. Patient must be independent 
of transfusions. No duration requirement for this designation. 
Cytogenetic complete remission (CCR) – Defined as morphologic complete 
remission plus a reversion to a normal karyotype. For this study, reversion 
to a normal karyotype is defined as no clonal abnormalities detected in a 
minimum of 20 mitotic cells. Fluorescent in situ hybridization (FISH) 
may be used as a supplement to follow a specifically defined cytogenetic 
abnormality. 
Morphologic complete remission with incomplete blood count recovery (CRi) 
– Defined as morphologic complete recovery with the exception of 
neutropenia <1000/μl or thrombocytopenia <100,000/μl.  
Partial remission (PR) – Requires that the above criteria for complete 
remission be met, with the following exceptions: decrease of ≥50% in the 
percentage of blasts to 5-25% in the bone marrow aspirate. A value of 
5% blasts in bone marrow with Auer rods present may also be 
considered a partial remission.  


Genomic Predictors of Decitabine Response in AML/MDS 
 
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Treatment failure – Includes all patients who did not achieve a complete or 
partial remission.  
Recurrence/morphologic relapse - Relapse following complete remission is 
defined as reappearance of blasts in the blood or the finding of ≥5% blasts 
in the bone marrow, not attributable to any other cause. New dysplastic 
changes are considered a relapse. If there are no blasts in the peripheral 
blood and 5-20% blasts in the bone marrow, bone marrow biopsy should 
be repeated in > 1 week to confirm relapse. 
Safety-- All patients receiving at least one dose of decitabine will be 
evaluated for safety. 
Statistical considerations 
This trial will be conducted as a phase II biomarker trial. 
 
This trial is designed to detect the most clinically meaningful mutational 
predictors of response. These are mutations that have sufficient positive 
predictive value to be used as a clinical assay. 
 
A power analysis was performed with Fisher’s exact test for mutation status 
and response to decitabine, assuming an expected 45% CR/CRi. With alpha = 
0.05, and a total anticipated enrollment of 100 evaluable patients, we 
anticipate 80% power to detect a 27% and 40% response difference between 
patients with and without a mutation, if the mutation occurs with a respective 
frequency of 30% and 15%. Such mutations would provide 80%  sensitivity 
to predict response to decitabine. These are values that would warrant 
diagnostic clinical testing prior to therapy.  
 
This trial is also designed to detect an improvement in clinical response 
compared to historical controls treated with 5 consecutive days of decitabine 
(20 mg/m2/day every 28 days)[2]. A power analysis was performed with 
Fisher’s exact test for response to decitabine compared to a historical rate of 
24% observed in 55 patients. With 100 evaluable patients enrolled, we 
anticipate 90% power with alpha = 0.1 to detect a 21% improvement in 
response (CR/ PR ≥ 45%). The reported CR/PR rate of the proposed 
treatment schedule (10 consecutive days of decitabine 20 mg/m2/day) is 
64%[3]; the expected CR/PR rate of 45% is a conservative estimation of 
expected outcomes.  
 
 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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Study Schema: 
 
 
Patients with progressive disease after two cycles (defined as a clear increase in bone marrow blast count, typically a 
> 50% increase in bone marrow blasts over baseline) should be removed from study. 
Patients with stable disease may continue on study and should undergo a diagnostic bone marrow biopsy at the end 
of even numbered cycles. 
After 2 cycles, if patients achieve morphologic CRi, administration will be decreased to 5 days to limit 
myelosuppressive toxicity (20 mg/m2 over 1 hour on Days 1-5 every 28 ± 4 days). After two cycles with 
morphologic CRi and no evidence of disease by cytogenetics or immunophenotype, patients may further 
decrease therapy to 3 days per cycle (20 mg/m2 over 1 hour on Days 1-3 every 28 ± 4 days). 
Sample collection for correlative studies: 
Bone marrow biopsies should be performed at baseline and at Day 10 ± 1 and Day 28 ± 4 of Cycle 1 and at 
the end of even numbered cycles. 
 
Peripheral blood samples should be collected on Day 4± 1 of Cycle 1. Collection should be obtained 45 ± 
15 minutes after decitabine infusion is initiated.
20 
mg/m
2/da
Cycle 1 
20 
mg/m
2/da
Cycle 2+ 
Day 1 
Day 28 
BM Bx 
PB 


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8 
Genomic Predictors of Decitabine Response in AML/MDS 
Table of Contents 
Section 
Page 
1.0 INTRODUCTION 
10 
1.1 
Rationale 
10 
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
12 
1.2.1 
Pharmacokinetics and Metabolism of Decitabine 
14 
1.2.2 
Experience with extended dosing schedules 
15 
1.3 
Correlative Studies 
16 
2.0 OBJECTIVES 
18 
2.1 
Primary Objective 
18 
2.2 
Secondary Objectives 
18 
3.0 PATIENT SELECTION 
19 
3.1 
Inclusion Criteria 
19 
3.2 
Exclusion Criteria 
19 
3.3 
Inclusion of Women and Minorities 
20 
4.0 REGISTRATION PROCEDURES 
21 
4.1 
Confirmation of Patient Eligibility 
21 
4.2 
Patient Registration in the Siteman Cancer Center Database 
21 
4.3 
Assignment of UPN 
21 
5.0 INVESTIGATIONAL PLAN 
22 
5.1 
Summary of Study Design 
22 
5.2 
Study Procedures 
22 
5.2.1 
Pre-study Procedures 
22 
5.2.2 
Baseline Evaluation 
23 
5.2.3 
Day 1 of Each Cycle 
23 
5.2.4 
Cycle 1 Days 1-10 
23 
5.2.5 
Cycle 1 Day 4 ± 1 
23 
5.2.6 
Cycle 1 Day 10 ± 1 
24 
5.2.7 
Weekly 
24 
5.2.8 
Every Other Week 
24 
5.2.9 
Cycle 1 and Even Cycles Day 28 ± 4 days 
24 
5.2.10 
End-of-Study Procedures 
24 
5.2.11 
Post-study Follow-up 
24 
5.3 
Duration of Therapy 
25 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
25 
5.4.1 
Chemotherapy 
25 
5.4.2 
Growth Factors 
25 
5.4.3 
Transfusions 
26 
5.4.4 
Prophylactic Antimicrobial Agents 
26 
5.4.5 
Radiotherapy 
26 
6.0 DOSE MODIFICATIONS 
27 
6.1 
Delay for Cytopenia or Infection 
27 


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9 
6.2 
Delay for Organ Dysfunction 
27 
6.3 
Modification in Dose or Schedule 
27 
7.0 PHARMACEUTICAL INFORMATION 
28 
7.1 
Decitabine 
28 
7.1.1 
Study Drug Preparation 
28 
7.1.2 
Supplier 
28 
7.1.3 
Toxicities 
28 
8.0 CORRELATIVE STUDIES SAMPLE COLLECTIONS 
30 
9.0 EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
31 
9.1 
Response Criteria 
31 
9.2 
Guidelines for Evaluation of Disease 
32 
9.3 
Progressive Disease 
32 
9.4 
Other Secondary Efficacy Measures 
32 
9.4.1 
Overall Survival and Event-free Survival 
32 
9.4.2 
Duration of Remission and Relapse-free Survival for Patients Having Complete 
Remission 
32 
9.4.3 
Safety 
33 
10.0 ADVERSE EVENT REPORTING 
34 
10.1 Adverse Events (AEs) 
34 
10.2 Unanticipated Problems 
34 
10.3 Noncompliance 
34 
10.4 Serious Noncompliance 
34 
10.5 Protocol Exceptions 
35 
10.6 Reporting to the Human Research Protection Office (HRPO) and the Quality Assurance 
and Safety Monitoring Committee (QASMC) at Washington University: 
35 
10.7 Timeframe for Reporting Required Events: 
36 
11.0 DATA SAFETY MONITORING PLAN 
37 
12.0 STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
38 
12.1 Analysis Populations 
38 
12.2 Statistical Analysis 
38 
12.2.1 
Descriptive Analyses 
38 
12.2.2 
Primary Endpoint Analysis 
38 
12.2.3 
Secondary Endpoint Analyses 
39 
12.2.4 
Safety Analysis 
41 
12.2.5 
Description of Planned Subgroup Analyses 
41 
12.3 Sample Size 
41 
13.0 DATA SUBMISSION SCHEDULE 
42 
  


Genomic Predictors of Decitabine Response in AML/MDS 
 
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10 
1.0 
INTRODUCTION 
 
1.1 
Rationale   
 
Personalized therapy for cancer requires that 1) patient-specific factors (e.g. patient-
specific mutations, expression profiles, or pharmacogenomics) influence treatment 
algorithms, and 2) once therapy is initiated, in vivo responsiveness is used to guide further 
therapy. The modern era of genomics has provided significant insights into cancer 
pathogenesis by describing patterns of germline and somatic variants, as well as defining 
disease-specific patterns of gene expression. However, the application and integration of 
this data with therapeutics remains a difficult task. 
 
Decitabine is a commonly used single-agent therapy for patients with acute myeloid 
leukemia (AML) and/or myelodysplastic syndrome (MDS). Historically, it has yielded 
clinical complete responses in approximately 25% of patients across diverse clinical trials, 
it is well tolerated, and it can be given as in the outpatient setting. However, the majority 
of patients do not respond to decitabine, the molecular etiology of decitabine sensitivity is 
unknown, and there are no algorithms to predict patient responsiveness a priori or even 
within one month of starting therapy.  
 
AML and MDS are two closely related hematological disorders characterized by 
ineffective hematopoiesis and malignant expansion of clonal myeloid cells.  Both AML 
and MDS frequently occur in elderly patients and some cases are secondary and/or related 
to long-term bone marrow damage after radiation or chemotherapy. Current therapy for 
AML consists of cytotoxic chemotherapy followed by consolidation chemotherapy or stem 
cell transplantation. Using these approaches, current cure rates may be as high as 60% for 
patients younger than 60 with good risk cytogenetics (e.g. t(15;17), t(8;21), inv(16)).[4]  
Outcomes in patients ≥ 60 years old and with relapsed disease remain poor with 2 year 
survival of only ~25%.[5] Historically, the available treatment for MDS has been 
symptomatic and has not been shown to be effective in producing sustained improvement 
in hematopoiesis or in delaying leukemic evolution[6, 7].  
 
Current AML therapy is poorly tolerated in elderly and pre-treated patients due to 
hematologic and non-hematologic toxicities; following such therapy, patients typically 
require 3-4 weeks of hospitalization for treatment of neutropenic fevers and for regular 
transfusions. Even among the relatively healthy patients enrolled in clinical trials, induction 
mortality in the elderly can be as high as 20-40% following standard induction therapy,[8-
11] and moderate to severe non-hematologic toxicity occurs in at least an additional 30% 
of patients.[9, 12-14] Consequently, many older AML patients are not offered traditional 
intensive chemotherapy, but receive supportive care alone.[15]  More effective and less 
toxic therapies are needed to provide durable remissions in a significant proportion of 
elderly AML in populations. 
 
Decitabine has been studied in MDS patients and in elderly and/or relapsed AML patients. 
It has been found to be reasonably well tolerated, can be given in the outpatient setting, 
and has overall response rates of 20-45%.[2, 3, 16-20] Our own experience has been of 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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11 
CR/PR rates of 25%, [2] and this is typical of many other groups for both MDS and AML 
patients (reviewed in [21]  Decitabine is an S-phase depended drug.  [22-24] However, 
decitabine is usually administered in one hour daily infusions during 3 to 5 consecutive 
days (too short to expose decitabine to most AML blasts during at least 2 cycles of 
replication, which divide every 2 to 7 days).[25-27]  Recent experience with increasing the 
number of infusions per cycle from 5 consecutive days to 10 consecutive days has 
improved overall response rates to 67%.[3] Response to decitabine can be slow, sometimes 
requiring 3 to 5 cycles before maximum response is observed.[2] As a consequence, many 
patients are exposed to prolonged therapy, gaining little benefit, before determining 
resistance. There are currently no biomarkers that predict response to decitabine. 
 
Decitabine is a cytosine analogue that cannot be methylated. Incorporation of decitabine 
into DNA prevents DNA methylation at cytosine residues and derepresses epigenetically 
silenced gene expression. Therefore, the reduction in total genomic DNA methylcytosine 
content acts as a marker of effective decitabine dosing. Indeed, two groups have recently 
correlated effective methylcytosine reduction and gene expression derepression to 
response (Figure 1.1).[3, 28] 
 
 
 
   
Figure 1.1. From Shen et al JCO (2010) 28:611 and Blum et al JCO (2007) 25: 3884. 
 
We and others have recently used whole genome sequencing to identify recurrent, somatic 
mutations in AML and MDS patients in four genes that are involved in methylcytosine 
regulation: DNMT3A, IDH1, IDH2, and TET2.[29-37] Collectively, nearly half of all 
AML/MDS patients have a mutation in one of these four genes.  DNMT3A mutations are 
associated with an adverse outcome in AML patients treated with cytotoxic 
chemotherapy.[35, 38] However, it is not yet clear whether the presence of mutations in 
one or more of these genes is associated with response or resistance to decitabine. 
 
In this study, we will treat patients with the current state-of-the-art decitabine schedule.[3] 
We will perform exome sequencing on all patients and correlate response with mutation 
status, rate of mutation clearance, expression profile, serum decitabine levels, and DNA 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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12 
methylcytosine content reduction. This approach will comprehensively assess genomic and 
pharmacologic parameters that could clinically predict patient response to decitabine.  
 
In this study, we will combine analysis of patients with AML and MDS. Recent genomic 
analysis has shown that AML and MDS have shared mutations in many of the same genes, 
shared patterns of mutation acquisition, and shared patterns of mutation evolution.[39, 40] 
Furthermore, MDS, like AML, presents with clonal hematopoiesis, even when the 
morphologic blast count is zero.[36]Because this study will identify and track the rate of 
clearance of patient specific mutations, and because decitabine has been shown to have 
efficacy in both AML and MDS patients, [2, 3, 16-19, 41-45] combining analysis of AML 
and MDS patients is reasonable.  Asymptomatic MDS patients are excluded because these 
patients are typically followed with watchful-waiting rather than with chemotherapy. 
  
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
 
Methylation of cytosine in the CpG dinucleotide by DNA methyltransferase leads to 
transcriptional silencing of genes during normal development and has emerged as a 
significant mechanism for the loss of tumor suppressor gene expression in human cancer, 
including AML and MDS.[46, 47] Like azacitidine, decitabine (5-aza-2’-deoxycitidine) is 
incorporated into DNA during S phase and forms covalent adducts with DNA 
methyltransferase. The irreversible inhibition of this enzyme results in loss of methylation 
and is associated with differentiation.[48]  Decitabine is an approximately 5-fold more 
potent inhibitor of DNA methyltransferase than azacitidine, which might explain why 
decitabine can induce differentiation of myeloid progenitors in vitro at lower doses than 
azacitidine. In addition to their differentiation inducing activity at low doses, both agents 
have a direct cytotoxic effect at higher doses (1-2 µM).[49]  
 
Decitabine has been investigated in hematologic malignancies using a range of doses and 
schedules.  It is currently FDA approved for the treatment of adults with MDS at a dose of 
15 mg/m2 every 8 hours for 3 days, based on a Phase III study that demonstrated improved 
outcomes in the treatment arm compared to patients treated with supportive care alone.[15]  
However, the overall response rate in this trial was modest (17%). Of note, 18% of the 
patients in this trial had 20-30% bone marrow blasts, meeting the current WHO criteria for 
AML.  Subsequent phase I and II studies in AML, using a variety of low-dose decitabine 
regimens, have reported overall response rates of 22-49%.[2, 3, 16-19, 41-45] Comparisons 
of multiple low dose schedules have noted improved efficacy in the MDS population using 
a dose of 20 mg/m2 on Days 1-5 of a 28-day cycle.[50, 51] Because of these results, we 
have recently explored this dosing schedule in the treatment of elderly patients with 
AML.[52] In this study, we observed an overall response rate of 24%, and an additional 
44% of patients achieved stable disease during a median of 5 monthly cycles.[48] In this 
study, we noted that decitabine does not act quickly and the median time to response was 
3 months. These results are similar to the recently published experience in Germany 
treating 227 patients with 15 mg/m2 over 3 hours TID for 3 days every 6 weeks, which 
resulted in a CR/PR rate of 26%.[16]  
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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13 
A recent study using a prolonged administration schedule (20 mg/m2 IV over 1 hour on 
consecutive days 1-10) showed improved response (64% CR/PR) and improved time to 
respond (50% of patients had bone marrow blasts < 5% after 1 cycle of therapy).[3]  These 
results are an improvement over previous experience by other groups using decitabine to 
treat elderly AML patients or patients with relapsed AML, and this experience is 
summarized below: 
Trials of decitabine in AML 
Status 
Other Therapy 
DAC Dose 
(mg x days) 
Evaluable 
CR 
de novo 
daunomycin1 
90 mg/m2 x 5 
6 
100% 
Salvage 
None4 
0.75-80 mg/kg 
16 
  
None5 
37-81 mg/kg 
6 
33% 
None7.  Ara-C 
Refractory 
Various 
4 
0 
AMSA7 
250-500 mg/m2 x 6 
10 
80% 
idarubicin or 
AMSA3 
250 mg/m2 x 6 
56 
39% 
Elderly 
None6 
270-360 mg/m2 x 3 
10 
30% 
None2 
45 mg/m2 x 3 
4 
50% 
None8 
20 mg/m2day 1-5,8-12 
50 
23% 
None9 
20 mg/m2 x 5 
55 
25% 
 
None10 
50 mg/m2 CIVI x 3 
29 
48% 
 
None11 
20 mg/m2 x 10 
53 
64% 
 
ATRA cycle 212 
15 mg/m2 TID x 3 
227 
26% 
1 Schwartsmann G.  Leukemia 1997; 11(Suppl 1):S28-31. 
2 Lubbert M. ASH 2002 Abstract #4601. 
3 Willemze R.  Leukemia.  1997; 11(Suppl 1):S24-27 
4 Rivard GE.  Leukemia Research 1981; 5:453-462. 
5 Momparler RL.  Pharmac Therapy 1985; 30:277-286.  
6 Petti MC.  Leukemia.  1993; 7(Suppl 1):36-41.  
7 Richel DJ.  Br. J. Cancer 1991; 64:144-148 
8 Issa, JP. Blood 2004. 103: 1635. 
9 Cashen AF. ASH Annual Meeting. 2008 abstract 560. 
10 Wijermans PW. Leukemia 1997. 11: Suppl 1, 19-23. 
11 Blum, W. PNAS 2010; 107:7473. 
12 Lubbert. Haematologica 2011. ePub 11/04/2011. 
 
In general, decitabine is well tolerated. Unlike conventional cytotoxic chemotherapy, 
decitabine can be administered in the outpatient setting. However, AML and MDS are 
aggressive hematologic malignancies. Cytopenias commonly result from AML/MDS 
inhibition of normal hematopoiesis, and often do not resolve until a complete response is 
achieved. Thus, neutropenic sepsis and thrombocytopenic bleeding remain significant 
problems regardless of therapy, especially during the first 4-8 weeks. In our experience 
treating elderly AML patients, neutropenia occurred in 24%, sepsis in 9%, and bacteremia 
in 7%.  Similar results have been observed in MDS patients.[20, 53, 54] As a result, death 
is not uncommon in the first 8 weeks of therapy, occurring in 15-17% of patients, and most 
commonly resulting from infection and cytopenias related to disease and therapy.[16, 52, 
55] While unsettling, these numbers are better than many historical patients treated with 
cytotoxic chemotherapy, and suggest a need for continued improvements in therapy.[9, 12]  


Genomic Predictors of Decitabine Response in AML/MDS 
 
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14 
 
1.2.1 Pharmacokinetics and Metabolism of Decitabine 
 
In human subjects, after IV administration, decitabine displayed a distribution phase 
with a half-life of 7 minutes and a terminal phase elimination half-life of 10-35 
minutes as measured by bioassay.[56, 57]   The short plasma half-life is due to rapid 
inactivation of decitabine by deamination by liver cytidine deaminase.[23] Total 
body clearance (Clp) in humans was high with a mean value of 126 mL/min/kg.[58]  
 
Among five adult patients with advanced solid tumors administered seven courses 
of 100 mg/m2 of decitabine by 1-hour infusions,[56] maximum plasma 
concentration ranged from 1 to 4 µM. Disappearance from plasma was biphasic 
with a rapid initial decline to less than 0.1 µM during the first hour post infusion 
and a slower decline over the next two hours to 6 nM, the level of detectability. 
Plasma half-life (meanSE) was estimated to be approximately 71 minutes in the 
initial phase (t½α) and 355 minutes in the second phase (t½β). The area under the 
plasma concentration-time curve was 40888 ng.hour/mL for these patients, with a 
peak plasma concentration of 2.010.44 µM (0.440.1 µg/mL), while mean volume 
of distribution (Vdss) and CLp was 4.591.42 L/kg and 12621 mL/min/kg, 
respectively. The time course of DAC concentrations was similar in 1 of 2 patients 
administered 75 mg/m2, while DAC was usually undetectable in the plasma of 
patients administered 25 to 60 mg/m2. 
 
Using a longer infusion time (up to 40 hours), Rivard et al.[57] reported a plasma 
concentration of 0.1 to 0.4 g/mL. With infusion times of 40-60 hours, at an infusion 
rate of 1 mg/kg/h, plasma concentrations of 0.43-0.76 µg/mL were achieved.[59]  The 
steady-state plasma concentration at an infusion rate of 1 mg/kg/h was estimated to 
be 0.2-0.5 µg/mL. The half-life after discontinuing the infusion was 12-20 min.[57]  
 
Urinary excretion of unchanged decitabine was low, ranging from less than 0.01% to 
0.9% of the total dose, and there was no relationship between excretion and dose or 
plasma drug levels. High clearance values and a total urinary excretion of <1% of the 
administered dose suggest that decitabine is eliminated rapidly and largely by 
metabolic processes, including extrahepatic metabolism.[60] 
 
The steady state plasma concentrations (Css) ranged from 0.31 to 0.39 µg/mL (1.27 
to 1.60 µM) during the infusion of 100 mg/m² decitabine over a 6-hour period.[61] 
The range of concentrations during a 600 mg/m2 infusion over an 8-hour period was 
0.41-16 g/mL(1.68 to 4.76 µM).[60] These plasma concentrations are in the same 
range as the in vitro concentrations that activated the expression of tumor 
suppressor gene p16 in three human non-small-cell lung cancer (NSCLC) cell 
lines.[61]  
 
Steady state decitabine levels of 0.1 to 0.2 µM were achieved in solid tumor patients 
dosed with 30 mg/m²/day and 40 mg/m²/day by continuous intravenous infusion 
over 72 hours.[62]  


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Summary of published steady state serum levels are below and are in proportion to 
infusion rate (R2 = 0.98, p < 0.0001):[63] 
 
Rate 
Serum 
Concentration 
mg/m2/hr 
ng/ml 
nM 
5 
55 
241 
7.5 
80 
351 
15 
180 
789 
21 
182.4 
800 
22.5 
350 
1535 
42 
456 
2000 
84 
1140 
5000 
 
In our experience treating primary patient blasts in vitro, concentrations of 100 – 
500 nM are required maximize methylcytosine reduction without causing cellular 
toxicity. Because decitabine incorporation into DNA is S-phase dependent, 
exposure of cells during cell division is necessary for efficacy. 
  
 
 
 
1.2.2 Experience with extended dosing schedules 
 
Blum et al recently published their experience treating elderly AML patients with 
a 10-day course of decitabine 20 mg/m2/day as a 1-hour infusion.[3] This prolonged 
exposure course was designed to increase the number of AML blast divisions 
exposed to decitabine per cycle. Toxicities were very similar to a similar regiment 
given as a 3 and 5 day course,[2, 16] but the overall response (CR/PR) was much 
improved (64% vs 25%). In all three studies, the toxicities were primarily myeloid 
suppression and the resultant infectious and bleeding complications associated with 
leukemic and pharmacologic hematopoietic suppression. Furthermore, Blum et al 
noted that the percent methylcytosine reduction was improved (22% vs 11%) as 
 


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was the time to blast clearance (50% of patients who ultimately achieved a CR had 
≤ 5% bone marrow blasts after 1 cycle vs 0% of patients after 2 cycles). Finally, 
like Shen et al, they observed a correlation between the extent of methylcytosine 
reduction and response.[28]  This study suggests that improvement in response 
rates and time to response can be achieved by prolonged administration of 
decitabine. Further, that DNA methylcytosine reduction can be achieved with 
prolonged exposure to decitabine and that this might be a patient-specific biological 
marker of adequate dosing. 
  
Scandura et al. recently published a study of infusional decitabine prior to 
cytarabine + daunorubicin induction therapy for AML.[64] They escalated the 
duration of infusional therapy and reached their planned goal of 20 mg/m2/day x 7 
days of continuous therapy given prior to cytarabine and daunorubicin. They noted 
similar toxicities (primarily hematopoietic and the associated neutropenic 
infections), but that these were not different from their experience giving cytarabine 
+ daunorubicin alone. 
 
1.3 
Correlative Studies 
 
The goals of the correlative studies include: 
 
1. To correlate patient-specific mutations with response to decitabine. 
We will perform exome sequencing of each patient’s Day 0 bone marrow sample and 
skin sample. We will determine AML/MDS-associated variants found in the bone 
marrow, but not skin sample. We will correlate the presence of mutations with response 
(especially mutations in DNMT3A, IDH1, IDH2, and TET2). This study is powered to 
identify mutations with >80% sensitivity to predict decitabine response if used as a 
clinical assay. 
 
2. To correlate the velocity and depth of response with response to decitabine. 
We will assess tumor burden by allele frequency of patient-specific mutations in bone 
marrow samples collected on day 0, 10±1, 28±4, and 56±4. We will assess mutations 
in at least 24 recurrently mutated genes (including, but not limited to: FLT3, DNMT3A, 
NPM1, TET2, WT1, IDH1, IDH2, RUNX1, NRAS, TP53, U2AF1, KRAS, PHF6, 
MSTP9, PTPN11, KIT, SMC3, PRUNE2, ETV6, CEBPA, SMC1A, ASXL1, STAG2, 
RAD21) as well as all patient-specific mutations discovered during exome sequencing. 
The leukemic allele frequency will be measured using the Illumnia MySeq or HiSeq 
platform using DNA capture by gene specific biotinylated-oligonucleotides. Patient 
bone marrow samples will be assessed on day 0, day 10 ± 1, and day 28 ± 4 of cycle 1, 
and day 28 ± 4 of cycle 2 and compared with patient specific skin samples (normal 
control). The velocity of response will be calculated as the natural log rate of patient-
specific mutation allele frequency clearance. Depth of response will be defined as the 
lowest patient-specific mutation allele frequency in a bone marrow sample. The 
velocity and depth of tumor response will be correlated to: 
 Achievement of clinical CR, 
 Time to best response, 


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 Mutational status, 
 Time to disease progression. 
Because all patient-specific exome-discovered mutations will be assessed a second 
time in the primary bone marrow sample as well as the Day 10 ± 1, 28 ± 4, and 56 ± 4 
bone marrow samples, this study will also serve to validate all exome-discovered 
mutations. This is important, because exome sequencing is associated with a 20-50% 
false-discovery rate depending on type I error tolerated (mutation-calling algorithms 
are typically set for greater sensitivity than specificity).  Thus, many mutations 
discovered during exome sequencing are incorrect.  This second analysis of these 
mutations will server to exclude these false positive calls. 
 
3. To correlate the bone marrow expression profile on Day 0 and Day 10 ± 1 with response 
to decitabine. 
We will correlate expression signatures, and changes in expression signatures, with: 
 Achievement of clinical CR, 
 Mutation status. 
 
4. To correlate steady-state serum decitabine concentration on Day 4 ± 1 with: 
 
Achievement of CR. 
 
5. To correlate the extent of bone marrow DNA hypomethylation on Day 10 ± 1 with 
response to decitabine. 
We will assess the percent reduction in bone marrow methylcytosine content using LC-
MS. Patients will be evaluated on Day 0 and Day 10 ± 1 of Cycle 1. The change in 
DNA methylcytosine content will be correlated to: 
 Achievement of clinical CR, 
 Change in expression signature. 
This will determine whether inadequate blast exposure to decitabine correlates with 
drug resistance. 
 


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2.0 
OBJECTIVES 
 
2.1 
Primary Objective 
 
To determine whether patient-specific mutations correlate with clinical overall response to 
decitabine in patients with AML or MDS. 
 
2.2 
Secondary Objectives 
 
- 
To determine whether 10 consecutive days per cycle of decitabine improves outcomes 
compared with historical controls treated with 5 consecutive days per cycle of 
decitabine. 
 
- 
To determine whether other biomarkers correlate with clinical overall response to 
decitabine in patients with AML or MDS. These will include:  
 
1. The velocity and depth of patient-specific mutation clearance; 
2. The bone marrow expression profile and change in expression profile during 
decitabine treatment; 
3. The steady-state serum decitabine concentrations; 
4. The decrease in bone marrow methylcytosine content in response to decitabine.  
 
 


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3.0 
PATIENT SELECTION 
 
3.1 
Inclusion Criteria 
 
One of the following: 
 Patient must have non-M3 AML and be > 60 years of age OR 
 non-M3 AML with relapsed disease OR 
 Symptomatic MDS with one of the following: 
□ Symptomatic anemia with either hemoglobin < 10.0 g/dL or requiring RBC 
transfusion 
□ Thrombocytopenia with a history of two or more platelet counts < 50,000/mcL 
or a significant hemorrhage requiring platelet transfusions 
□ Neutropenia with two or more ANC < 1,000/mcL 
 
All of the following: 
1. Patient must have an ECOG performance status ≤ 2. 
2. Patient must have >10% disease burden measured by cytomorphology, flow 
cytometry, or cytogenetics. 
3. Patient must have peripheral white blood cell count < 50,000/mcl. 
4. Patient must have adequate organ function, defined as: 
a. 
Total bilirubin < 1.5 x ULN 
b. 
AST/ALT < 2.5 x ULN 
c. 
Serum creatinine < 2.0 x ULN  
5. Patient must have undergone ≤ 2 cycles of prior hypomethylating agent (decitabine 
or azacitidine). 
6. Patient must be enrolled in HRPO# 201011766 (“Tissue Acquisition for Analysis 
of Genetic Progression Factors in Hematologic Malignancies”). 
7. Patient must be > 18 years of age. 
8. Patient must be able to understand and willing to sign an IRB-approved written 
informed consent document. 
 
3.2 
Exclusion Criteria 
 
1. Patient must not be pregnant or nursing. 
2. Patient must not have known CNS leukemia.  
3. Patient must not have a history of positive HIV serology.  
4. Patient must not have a history of positive Hepatitis C serology. 
5. Patient must not have undergone a prior allogeneic stem cell transplant. 
6. Patient must not have any uncontrolled intercurrent illness including, but not 
limited to, ongoing or active infection, ongoing or active GVHD, congestive heart 
failure of NYHA class 3 or 4, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situation that would limit compliance with study 
requirements. 
7. Patient must not have had radiation therapy within 14 days of enrollment.  
 
 


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3.3 
Inclusion of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible for this trial. 
 


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4.0 
REGISTRATION PROCEDURES 
 
Patients must not start any protocol intervention prior to registration through the Siteman 
Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility 
2. Registration of patient in the Siteman Cancer Center database 
3. Assignment of unique patient number (UPN) 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility collecting the information listed below  
1. The registering MD’s name 
2. Patient’s race, sex, and DOB 
3. Three letters (or two letters and a dash) for the patient’s initials 
4. Copy of signed consent form 
5. Completed eligibility checklist, signed and dated by a member of the study team 
6. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center Database 
 
All patients must be registered through the Siteman Cancer Center database. 
 
4.3 
Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  Patients 
will also be identified by first, middle, and last initials.  If the patient has no middle initial, 
a dash will be used on the case report forms (CRFs).  All data will be recorded with this 
identification number on the appropriate CRFs. 
 


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5.0 
INVESTIGATIONAL PLAN 
 
5.1 
Summary of Study Design 
 
This is an open-label, single-arm, Phase II, biomarker study of patients with AML or MDS 
who are treated with 10 consecutive days of decitabine. FAB-M3 AML is excluded from 
this trial, because other well-tolerated options exist for the treatment of FAB-M3 acute 
promyelocytic leukemia. AML patients who are < 60 years old and have not received 
cytotoxic induction chemotherapy are also excluded, because cytotoxic therapy offers a 
curative possibility for these patients.  MDS patients without cytopenias are also excluded 
because these patients can be followed with watchful-waiting. 
 
All patients will undergo bone marrow biopsies prior to therapy, on Day 10 ± 1, and on 
Day 28 ± 4 of Cycles 1 and 2 for comprehensive genomic correlative studies. Patients will 
also provide peripheral blood samples on Day 4 ± 1 for pharmacokinetic studies. 
 
Patients will receive decitabine 20 mg/m2/day as a 1-hour infusion on consecutive Days 1-
10 in 28-day cycles.  The dose will be calculated based on actual patient weight at the 
beginning of each cycle. 
 
After 2 cycles, if patients achieve CRi, the administration of decitabine will be decreased 
to 5 days. After two cycles with CRi and no evidence of disease by cytogenetics or 
immunophenotype, patients will further decrease therapy 3 consecutive days.  The dose of 
decitabine will remain 20 mg/m2/day over 1 hour regardless of decreases in the number of 
days per cycle of administration. 
 
Patients who achieve stable disease, partial response, or complete response will continue 
on decitabine therapy (receiving cycles of 3-10 days every 28 days (i.e., Days 1-10, Days 
1-5, or Days 1-3), depending on response) until time of progression, unacceptable toxicity, 
or patient decision to discontinue study drugs. 
 
It will be a logistical goal of this trial consistently to return preliminary exome results to 
the treating physician by the end of Cycle 1 and validated results by the end of Cycle 2. 
However, this logistical goal is not a primary or secondary aim of this study. Given the 
limited data regarding mutation presence and drug response, we do not anticipate that the 
results will directly influence therapeutic decisions made by the treating physician. 
However, the development of comprehensive genomic pipelines and integration of this 
data into a clinical trial setting will be required before personalized medicine can be 
realized. 
 
5.2 
Study Procedures 
 
5.2.1 Pre-study Procedures 
 
Pre-study procedures must take place no more than 14 days prior to the first dose 
of decitabine. 


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 Bone marrow morphologic assessmenta 
 Bone marrow or peripheral blood for flow cytometry 
 Bone marrow for cytogenetics.  
a Standard bone marrow procurement procedures will be followed for collection of the tissue.  
 
5.2.2 Baseline Evaluation 
 
Baseline evaluations must take place no more than 7 days prior to the first dose of 
decitabine and include: 
 
1. Medical history 
2. Physical exam including height, weight, vital signs, and ECOG performance 
score. 
3. Concomitant medications 
4. Record transfusion requirements for 8 weeks before first dose of study drug 
5. CBC with differential and platelets 
6. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
7. Urine βHCG pregnancy test if appropriate 
 
5.2.3 Day 1 of Each Cycle 
 
1. Record adverse events  
2. Physical exam, including weight and ECOG performance score 
3. Vital signs, including blood pressure, pulse and temperature 
4. CBC with differential and platelets 
5. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
 
5.2.4 Cycle 1 Days 1-10 
 
Decitabine administration, 20 mg/m2 per day given as a 1-hour continuous 
intravenous infusion.  The dose will be calculated based on actual patient weight at 
the beginning of each cycle.  Patients may be premedicated with lorazepam, 
prochlorperazine, palonosetron, or ondansetron at the treating physician’s 
discretion. 
 
5.2.5 Cycle 1 Day 4 ± 1 
 
Obtain 3 mL peripheral blood for correlative studies. Sample must be obtained 45 
minutes ± 15 minutes after beginning the infusion, before the decitabine infusion is 
complete, and should be obtained from the opposite arm as the infusion.  The 
sample should be collected in a 3 mL K3EDTA tube pre-loaded with 8 mcL of 
tetrahydrouridine (THU 500 mcg/mL), an inhibitor of cytidine deaminase.  The 


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sample should be processed by the Tissue Procurement Core Facility at the Siteman 
Cancer Center.  Serum should be separated and stored per protocols. 
  
5.2.6 Cycle 1 Day 10 ± 1 
 
Obtain bone marrow biopsy for response and correlative studies. a 
a Standard bone marrow procurement procedures will be followed for collection of the tissue.  
 
5.2.7 Weekly 
 
1. CBC with differential and platelets. After the first cycle, this may be changed 
to every other week at the discretion of the treating physician. 
2. Record adverse events. 
 
5.2.8 Every Other Week 
 
Serum chemistries, including bicarbonate, calcium, glucose, total protein, albumin, 
BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, ALT, alkaline 
phosphatase, LDH. After the first cycle, this may be changed to every month at the 
discretion of the treating physician. 
 
5.2.9 Cycle 1 and Even Cycles Day 28 ± 4 days  
 
1. Bone marrow biopsy and aspirate collection.a    
2. Clinical samples for response determination should be obtained at the end of 
Cycles 1, 2 and 4. Patients who receive > 4 cycles should continue to undergo 
bone marrow biopsies on even cycles for clinical evaluation (or as clinically 
indicated). 
a Standard bone marrow procurement procedures will be followed for collection of the tissue.  
 
5.2.10 End-of-Study Procedures  
 
1. CBC with differential and platelets.  
2. Record adverse events.  
3. Physical examination including weight, vital signs, and ECOG performance 
score.  
4. Serum chemistries including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, and LDH.  
5. Bone marrow collection, with samples sent for routine pathology.a 
a Standard bone marrow procurement procedures will be followed for collection of the tissue.  
 
5.2.11 Post-study Follow-up 
 
Patients are to be followed according to standard patient care procedures for 6 
weeks following the date of the last decitabine dose to monitor for toxicity, survival, 


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and response. Thereafter, the investigator or designee will record information on 
subsequent therapy, disease progression, and/or death every 2 months for 2 years 
after first dose of study drug or until the patient is lost to follow-up or dies. 
 
5.3 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to the 
patient’s health and/or the patient no longer wishes to continue protocol therapy, the 
protocol therapy should be discontinued and the reason(s) for discontinuation documented 
in the case report forms. 
 
In the absence of treatment delays due to adverse events, treatment may continue 
indefinitely or until one of the following criteria applies: 
 
 Documented and confirmed disease progression 
 Death 
 Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
 General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
 Suspected pregnancy 
 Serious non-compliance with the study protocol 
 Lost to follow-up 
 Patient withdraws consent 
 Investigator removes the patient from study 
 The Siteman Cancer Center decides to close the study 
 
Patients who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar. 
 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
 
5.4.1 Chemotherapy 
 
Patients cannot receive any chemotherapy within 21 days of study enrollment and 
must have recovered from any treatment-related toxicities. Patients may have 
received hydrea prior to enrollment for a WBC > 50,000/l but must stop hydrea at 
least one day prior to initiating decitabine. Other than decitabine, patients cannot 
receive chemotherapy during this trial.  
 
5.4.2 Growth Factors 
 
Erythroid growth factors should not be routinely administered to patients during the 
study. 
 


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Thrombopoietin receptor agonists should not be routinely administered to patients 
during the study. 
 
Filgrastim (G-CSF) or sargramostim (GM-CSF) may be administered to patients 
with septic shock or febrile neutropenia at the discretion of the treating physician. 
 
5.4.3 Transfusions 
 
 Packed red blood cells transfusion:   
Two units of packed RBCs are to be administered when the hemoglobin drops 
under 8 g/dL. Transfusion administration when the patient’s hemoglobin is 8-
10 g/dL may be done according to the treating physician’s clinical judgment.  
 
 Platelet transfusion:   
One unit of single-donor platelets will be administered when platelets are < 
20,000/L. 
 
5.4.4 Prophylactic Antimicrobial Agents  
 
Antimicrobial agents will be administered at the discretion of the treating physician. 
 
5.4.5 Radiotherapy 
 
Patients cannot receive radiotherapy within 14 days prior to study enrollment or 
while on study. 
 


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6.0 
DOSE MODIFICATIONS 
 
6.1 
Delay for Cytopenia or Infection 
 
As cytopenias are a defining feature of AML and MDS, their presence alone does not 
generally warrant dose modifications or delays. Patients will receive four cycles of therapy 
without adjustment for any cytopenia. 
 
Patients with febrile neutropenia (temperature ≥ 38.5° with ANC < 1,000/l) or systemic 
infections should have treatment held until clinical resolution of the infection. A patient 
will meet criterion for discontinuation of the study if the patient is observed to have a delay 
of > 8 weeks between any two cycles.  
 
6.2 
Delay for Organ Dysfunction 
 
If renal dysfunction (creatinine ≥ 3 X institutional upper limit of normal) or hepatic 
dysfunction (bilirubin ≥ 2.5 mg/dl or ALT/AST ≥ 3 X institutional upper limit of normal) 
occurs, therapy may not resume until the abnormalities have resolved to below the limits 
stated above. If renal or hepatic dysfunction occurs during a treatment course, the next dose 
of decitabine should be held, and serum chemistries should be checked twice per week.  
Decitabine can be resumed when creatinine, bilirubin, ALT and AST are below the limits 
listed above.  
 
For grade 3-4 non-hematologic toxicities, decitabine treatment should be delayed until 
recovery to baseline or to grade ≤ 1. For toxicities felt to be related to decitabine, decitabine 
may be reintroduced with a 50% dose reduction, or at the discretion of the investigator. 
 
6.3 
Modification in Dose or Schedule 
 
Once patients achieve morphologic complete remission with incomplete blood count 
recovery (CRi) (morphologic leukemia-free state, defined as less than 5% blasts in aspirate 
with marrow spicules and a count of  ≥ 200 nucleated cells and no blasts with Auer rods, 
any persistence of extramedullary disease, or persistent pre-treatment morphologic 
abnormalities, such as pseudo-Pelger-Hüet cells, ringed sideroblasts, or dysplastic 
megakaryocytes), administration may be decreased to 5 consecutive days to limit 
myelosuppressive toxicity, per treating physician. After two cycles with bone marrow blast 
counts < 5% and no evidence of disease by cytogenetics or immunophenotype, patients 
may further decrease therapy to 3 consecutive days per cycle, per treating physician. 
 


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7.0 
PHARMACEUTICAL INFORMATION 
 
7.1 
Decitabine 
 
Decitabine is an antimetabolite that can replace cytosine in DNA but, unlike cytosine, it 
cannot be methylated. The exact mechanism of decitabine activity has not been determined 
and may involve multiple pathways. Decitabine is thought primarily to act by correcting 
DNA methylation, a major mechanism for gene expression. In some cancer cells, 
hypermethylation blocks the activity of tumor suppressor genes, which regulate cell 
division and differentiation to prevent malignant transformation. When suppressor gene 
activity is blocked, cell division becomes unregulated, leading to the formation of 
neoplastic cells. Decitabine regulates DNA methylation (specifically targets the 
methyltransferase, DNMT1), effectively demethylating and reactivating different tumor 
suppressor genes. Because hypermethylation occurs early in the malignant transformation 
of cells, decitabine may have activity in chemoprevention. Other possible mechanisms for 
decitabine activity include cytotoxicity due to incorporation into DNA similar to other 
antimetabolites, methylation-independent induction of gene expression, and degradation of 
DNA methyltransferase 1. 
 
7.1.1 Study Drug Preparation  
 
OSHA Guidelines for handling cytotoxic drugs outlined in the American Journal 
of Hospital Pharmacy must be followed. 
 
Decitabine (5-aza-2’-deoxycytidine) is supplied as a lyophilized powder for 
injection, 50 mg in 20-ml vials. When reconstituted with 10 ml of sterile water for 
injection, each ml will contain 5 mg of decitabine and 6.8 mg of KH2PO4. The 
reconstituted solution should be diluted to 0.1-1.0 mg/mL with pre-chilled 0.9% 
sodium chloride intravenous infusion, which has been cooled to 2-8oC. 
Reconstitution of the powder results in a rapidly decomposing solution. Unless used 
within 15 minutes of reconstitution, the diluted solution must be prepared using 
cold (2—8 degrees C) infusion fluids and stored at 2—8 degrees C (36—46 degrees 
F) for a maximum of 7 hours. 
Decitabine will be administered per dosing schedule every 28 ± 4 days.  
 
Decitabine may be administered either as an inpatient or an outpatient.  
 
7.1.2 Supplier 
 
Decitabine is available commercially. 
 
7.1.3 Toxicities 
 
Likely (>15%) 
Cytopenias: 
neutropenia, 
thrombocytopenia, 
anemia, 
leukopenia, 
febrile 
neutropenia.  


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Bleeding: petechiae, ecchymosis. 
Infections: pneumonia. 
Constitutional: fatigue, pallor, fever, rigors, arthralgia, cough. 
GI: nausea/vomiting, constipation, diarrhea, decreased appetite. 
Electrolyte abnormalities: hyperglycemia, hypoalbuminemia, hypomagnesemia, 
hypokalemia, hyponatremia.  
Cardiac: cardiac murmur. 
Neurologic: headache, insomnia, dizziness. 
Dermatologic: rash. 
 
Less likely (1-15%)  
Hematologic: thrombocythemia, hematoma. 
Other infections: cellulitis, candidal infection, urinary tract infection, sinusitis. 
GI: abdominal 
pain, hyperbilirubinemia, 
stomatitis, 
dyspepsia, ascites, 
hemorrhoids, oral ulceration, dysphagia, abdominal distention, gastro-esophageal 
reflux disease/pyrosis (heartburn), and glossodynia. 
Electrolyte abnormalities: hyperkalemia, and dehydration. 
Urologic: dysuria, increased urinary frequency.  
Dermatologic: pruritis, alopecia, urticaria, edema. 
 
Rare (<1%) 
Cardiac: arrest/respiratory arrest, atrial fibrillation, and tachycardia. 
 
  
 
  
 


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8.0 
CORRELATIVE STUDIES SAMPLE COLLECTIONS 
 
Bone marrow samples will be collected at Baseline, on Cycle 1 Day 10 ± 1, and on Day 28 ± 4 of 
Cycle 1 and of even number cycles for correlative studies. Bone marrow aspirate (6-10 ml) will be 
collected and divided between two EDTA (lavender top) tubes. The bone marrow samples and 
peripheral blood samples should be delivered by hand to Sandra McDonald, M.D. PhD, director 
of the Tissue Procurement Core Facility at the Siteman Cancer Center.  At the Tissue Procurement 
Core Facility, patient identification will be removed from the samples, and the cells will be stored 
as frozen pellets in DMSO at 107 per tube. Subsequently, the samples will be thawed, and cells 
will be assayed. 
 
Peripheral blood sample (3 mL) will be collected on Cycle 1 Day 4 ± 1.  Sample must be obtained 
45 minutes ± 15 minutes after beginning the infusion, before the decitabine infusion is complete, 
and should be obtained from the opposite arm as the infusion.  The sample should be collected in 
a 3 mL K3EDTA tube pre-loaded with 8 mcL of tetrahydrouridine (THU 500 mcg/mL), an 
inhibitor of cytidine deaminase.  The sample should be processed by the Tissue Procurement Core 
Facility at the Siteman Cancer Center.  Serum should be separated and stored per protocols. 
 
 


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9.0 
EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
 
A secondary endpoint of this study is to estimate morphologic complete remission rate. 
  
9.1 
Response Criteria 
 
Patients will be assessed for response according to the IWG criteria:1  
 
Morphologic complete remission (CR) – Defined as morphologic leukemia-free state, 
defined as less than 5% blasts in aspirate with marrow spicules and a count of ≥200 
nucleated cells and no blasts with Auer rods or any persistence of extramedullary disease. 
When erythroid precursors constitute less than 50% of bone marrow nucleated cells, the 
percentage of blasts is based on all nucleated cells; when there are 50% or more erythroid 
cells, the percentage blasts should be based on the nonerythroid cells. The presence of mild 
megaloblastoid changes may be permitted if they are thought to be consistent with 
treatment effect. However, persistence of pretreatment abnormalities (eg, pseudo-Pelger-
Hüet cells, ringed sideroblasts, dysplastic megakaryocytes) are not consistent with CR.  In 
addition, ANC >1000/μl and platelet count of ≥100,000/μl. Patient must be independent of 
transfusions. No duration requirement for this designation. 
 
Cytogenetic complete remission (CCR) – Defined as morphologic complete remission plus 
a reversion to a normal karyotype. For this study, reversion of a normal karyotype is 
defined as no clonal abnormalities detected in a minimum of 20 mitotic cells. 
 
Morphologic complete remission with incomplete blood count recovery (CRi) – Defined as 
morphologic complete recovery with the exception of neutropenia <1000/μl or 
thrombocytopenia <100,000/μl.  
 
Partial remission (PR) – Requires that the above criteria for complete remission be met, 
with the following exceptions: decrease of ≥50% in the percentage of blast to 5-25% in the 
bone marrow aspirate. A value of 5% blasts in bone marrow with Auer rods present may 
also be considered a partial remission.  
 
Treatment failure – Includes all patients who did not achieve a complete or partial 
remission following 4 cycles of therapy.  
 
Recurrence/morphologic relapse - Relapse following complete remission is defined as 
reappearance of blasts in the blood or the finding of ≥5% blasts in the bone marrow, not 
attributable to any other cause. New dysplastic changes are considered a relapse. If there 
are no blasts in the peripheral blood and 5-20% blasts in the bone marrow, bone marrow 
biopsy should be repeated in > 1 week to confirm relapse. 
 
 


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9.2 
Guidelines for Evaluation of Disease 
 
Bone marrow biopsy should be performed at baseline for both the correlative study and 
clinical evaluation. Bone marrow biopsy should be performed on Day 28 ± 4 of cycle 1 
and on Day 28 ± 4 of each even cycle for clinical disease assessment. 
 
Peripheral blood counts will be followed by weekly CBC with differential and platelets.  
After the first cycle, this may be changed to every other week or monthly at the discretion 
of the treating physician. 
 
9.3 
Progressive Disease 
 
Progressive disease is defined as a clear increase in bone marrow blast count, typically a 
greater than 50% increase compared with baseline. Patients with progressive disease will 
be removed from the study. 
 
Patients may also be removed from the study for progressive disease based on evidence of 
new extramedullary disease or based on the clinical judgment of the treating physician. 
 
Patients with a > 50% increase in peripheral blast count with an increase of the total 
peripheral white blood cell count to > 10,000/l should undergo evaluation by bone 
marrow biopsy regardless of the cycle number. 
 
9.4 
Other Secondary Efficacy Measures 
 
9.4.1 Overall Survival and Event-free Survival 
 
Overall survival is defined as the date of first dose of study drug to the date of death 
from any cause.  Patients still alive at the time of the final analysis will be right-
censored. 
 
Event-free survival is defined as the interval from the date of first dose of study 
drug to date of treatment failure, recurrence, death due to any cause, or loss to 
follow up. 
 
9.4.2 Duration of Remission and Relapse-free Survival for Patients Having 
Complete Remission 
 
Duration of complete remission is defined as the interval from the date that 
complete remission is documented to the date of recurrence, death due to any cause, 
or loss to follow-up.  
 
Relapse-free survival is defined only for those patients achieving a complete 
remission. It is defined as the interval from the date of first documentation of a 


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33 
leukemia-free state to the date of recurrence, death due to any cause, or loss to 
follow up.   
  
9.4.3 Safety 
 
Safety evaluation will include assessments of adverse experiences, medical history, 
physical examinations, vital signs, and laboratory assessments at baseline and 
throughout the study period. Local laboratories may be used for this study.  
 


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10.0 
ADVERSE EVENT REPORTING  
 
10.1 
Adverse Events (AEs) 
 
Definition:  any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading:  the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for all 
toxicity reporting.  A copy of the CTCAE version 4.0 can be downloaded from the CTEP 
web site:  http://ctep.cancer.gov/reporting/ctc.html. 
 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the terms 
listed that should be used are those provided by the Department of Health and Human 
Services’ Office for Human Research Protections (OHRP).  A copy of this guidance can 
be found on OHRP’s website: (http://www.hhs.gov/ohrp/policy/AdvEvntGuid.htm). 
 
10.2 
Unanticipated Problems 
 
Definition: 
 
 unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied; 
 related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the incident, 
experience, or outcome may have been caused by the procedures involved in the 
research); and 
 suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
 
10.3 
Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that govern 
human 
subjects 
research 
or 
failure 
to 
follow 
the 
determinations 
of 
the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate choice to 
ignore regulations, institutional policies, or determinations of the IRB. 
 
10.4 
Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial harm 
to subjects or others, or that materially compromises the rights or welfare of participants. 
 


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10.5 
Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under the research 
team’s control. Exceptions apply only to a single participant or a singular situation. 
 
Pre-approval of all protocol exceptions must be obtained prior to the event. 
 
10.6 
Reporting to the Human Research Protection Office (HRPO) and the Quality 
Assurance and Safety Monitoring Committee (QASMC) at Washington 
University: 
 
The PI is required to promptly notify the IRB of the following events: 
 
 Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct of 
the study. 
 Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 
 Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence of the 
event or notification to the PI of the event.  The death of a research participant that qualifies 
as a reportable event should be reported within 1 working day of the occurrence of the 
event or notification to the PI of the event. 
 
10.7 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER 
FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the investigator to report any unanticipated problem to the FDA as 
follows: 
 
 Report any unexpected fatal or life-threatening adverse experiences associated with 
use of the drug (i.e., there is a reasonable possibility that the experience may have 
been caused by the drug) by telephone or fax no later than 7 calendar days after 
initial receipt of the information.  A life-threatening adverse experience is defined 
as any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that, had it occurred in a more severe form, might have caused 
death. 
 Report any serious, unexpected adverse experiences, as well as results from animal 
studies that suggest significant clinical risk within 15 calendar days after initial 
receipt of this information.  A serious adverse drug experience is defined as any 


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adverse drug experience occurring at any dose that results in any of the following 
outcomes: 
o Death 
o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial disruption 
of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 
o Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed above 
An unexpected adverse drug experience is defined as any adverse drug experience, 
the specificity or severity of which is not consistent with the current investigator 
brochure (or risk information, if an IB is not required or available). 
 
All MedWatch forms will be sent by the investigator or investigator’s team to the FDA at 
the following address or by fax: 
 
Food and Drug Administration 
Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
10.8 
Timeframe for Reporting Required Events:  
 
Reportable adverse events will be tracked for 30 days following the last day of study 
treatment. 
 
Deaths 
Any reportable death while on study or within 
30 days of study 
Immediately, within 24 hours, to PI 
and the IRB 
Any reportable death while off study  
Immediately, within 24 hours, to PI 
and the IRB  
Adverse Events/Unanticipated Problems 
Any reportable adverse events as described in 
Sections 10.1 and 10.2 (other than death) and 
10.7 
Immediately, within 24 hours to PI 
and within 10 working days to the 
IRB, and within 7 or 15 days to the 
FDA  
All adverse events regardless of grade and 
attribution should be submitted cumulatively  
Include in DSM report 
Noncompliance and Serious Noncompliance 
All noncompliance and serious noncompliance 
as described in Sections 10.3 and 10.4 
Immediately, within 24 hours, to PI 
and within 10 working days to the IRB 


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11.0 
DATA SAFETY MONITORING PLAN 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, the 
Principal Investigator will provide a Data and Safety Monitoring (DSM) report to the Washington 
University Quality Assurance and Safety Monitoring Committee (QASMC) semi-annually 
beginning six months after accrual has opened (if at least five patients have been enrolled) or one 
year after accrual has opened (if fewer than five patients have been enrolled at the six-month mark). 
 
The Principal Investigator will review all patient data at least every six months, and provide a 
semi-annual report to the QASMC. This report will include: 
 HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
 Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of study 
 History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol exceptions, 
error, or breach of confidentiality including start/stop dates and reason 
 Study-wide target accrual and study-wide actual accrual 
 Protocol activation date 
 Average rate of accrual observed in year 1, year 2, and subsequent years 
 Expected accrual end date 
 Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
 Measures of efficacy 
 Early stopping rules with supporting data and list the number of participants who have 
met the early stopping rules 
 Summary of toxicities 
 Abstract submissions/publications 
 Summary of any recent literature that may affect the safety or ethics of the study  
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC according 
to institutional guidelines. 
 


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12.0 
STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
 
12.1 
Analysis Populations 
 
Intent-to-Treat - The intent-to-treat (ITT) population will be defined as all patients who 
receive at least one dose of decitabine.  
 
Efficacy-Evaluable - The efficacy-evaluable (EE) population will be defined as all patients 
who receive at least one cycle of treatment.   
 
Safety - The safety population will be defined as all patients with at least one dose of 
decitabine. 
 
12.2 
Statistical Analysis 
 
12.2.1 Descriptive Analyses 
 
Patient Disposition: The number of patients discontinued, the reasons for 
discontinuation, and the number of cycles administered will be summarized. 
 
Demographics and Baseline Characteristics: Descriptive summary statistics will 
be provided for demographic and important baseline characteristics.  For 
continuous variables, the number of patients, mean, standard deviation, median, 
minimum and maximum will be provided.  For categorical variables, the number 
and percent of patients in each demographic/characteristic category will be 
summarized. 
 
12.2.2 Primary Endpoint Analysis 
 
The primary end-point of this phase II study is the correlation biomarkers with 
overall response in AML/MDS patients to decitabine. 
 
We will correlate the presence of patient specific mutations (especially mutations 
in DNMT3A, IDH1, IDH2, and TET2) with overall response rates. Mutation rates 
in these genes and in other patient-specific genes will be correlated with overall 
response and their respective 95% confidence intervals will be provided. 
 
Statistical considerations. The relationship between AML/MDS-associated 
mutations and the responsiveness to decitabine will be described by contingency 
tables and assessed using Chi-square test or Fisher’s exact test as appropriate. The 
differentially expressed genes between responders and non-responders will be 
determined using the significance analysis of microarray (SAM). Multivariate 
logistic regression will also be fit to adjust the potential confounding effects of other 
demographic and clinical characteristics. The resultant critical P values will be 
corrected for multiple testing.  Since the Bonferroni correction is known to be too 
conservative, a permutation analysis (e.g., 10,000 random assignments for 


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39 
responsive status) will be used to derive an empirical critical P value.  The new 
critical p-value is the number of permutation tests which yield a lower p-value than 
the original test divided by the total number of permutation tests.[61]   
 
A power analysis was performed using Chi-square test for the association between 
mutation and response to decitabine. The frequency of DNMT3A, IDH1/2, and 
TET2 mutations in AML and MDS patients are each between 15% and 30% in 
multiple cohorts.[29-37] Table 3 shows the minimal detectable differences of 
response rates between mutant and wild-type cases, assuming an expected overall 
45% CR/CRi rate and 80% power. For an average of 20% frequency of a mutated 
gene, for example, 100 patients will provide 80% power to detect 30% difference 
at a 2-sided 0.1 alpha error. The purpose of this study is to determine whether any 
of these mutations result in a 
clinically 
significant 
association 
that 
would 
warrant pre-treatment testing. 
We believe that a reasonable 
cut off would be if at least 
30% of clinical response 
could be attributed to the 
presence 
of 
a 
specific 
mutation. 
However, 
we 
anticipate that more power 
could be achieved as we 
assess patient groups with 
combined 
characteristics 
(e.g. DNMT3A mutation or 
loss of DNMT3A expression). 
In addition, we expect that 
the majority of the enrolled 
125 patients can be used in the analysis (since all of our molecular information will 
be acquired within 30 days of enrollment, we anticipate a drop-out rate of 20% or 
less). 
 
12.2.3 Secondary Endpoint Analyses 
 
The overall response rate (CR/CRi/PR) and complete response rate (CR/CRi) will 
be compared with historical controls[2], and the 95% confidence intervals will be 
provided. 
 
The rate of patient specific mutation clearance after 10, 28, and 56 days of therapy 
will be determined using an Illumina based platform. We will compare the rate of 
mutation clearance between the patients who achieve a CR/CRi after 4 cycles and 
those who do not. We anticipate that we will identify between 6 and 25 exonic 
SNVs per case (the range obtained with >200 AML samples sequenced to date with 
whole genome or exome sequencing), and that the expected 95% confidence 
Table 3. Detectable mutation associated response rates (80% power, α = 0.05) 
 
Total 
N 
% of patients 
with a given 
mutation  
Detectable 
response 
difference 
Sensitivity of mutation to 
predict response at minimum 
detectable response difference* 
100 
15%  
20%  
30% 
40% 
50% 
0.40 
0.35 
0.30 
0.27 
0.26 
80% 
75% 
67% 
61% 
58% 
* Assuming the overall response  rate (CR/Cri) of 45%   


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interval of the mean variant allele frequency will be ±2.56% and ±1.25%, 
respectively, if we assume an anticipated standard deviation of 3.2% within the 
same clone. The change of tumor burden over time and its association with clinical 
response will  be assessed using 2-way ANOVA for repeated measurement data, 
followed by the ad-hoc multiple comparisons for between-group differences (e.g., 
responders vs. non-responders) at given time points as well as the between-time 
differences within each group. The resultant critical P values will also be corrected 
using permutation analysis for multiple testing. 
 
The steady-state serum decitabine concentration on day 4 ± 1 will be measured and 
correlated with clinical overall response. 
 
The percent decrease in bone marrow methylcytosine will be calculated on day 
10±1. We will compare this reduction with previously published data and between 
the patients who achieve CR/CRi and those who do not. The correlation of peak 
serum levels of decitabine with response will be assessed using 2-sample t-test or 
Mann-Whitney non-parametric rank-sum test as appropriate. The change of total 
bone marrow DNA methylcytosine from baseline and its association with both 
steady-state serum drug levels and response will be assessed using 2-way ANOVA 
for repeated measurement data, followed by the ad-hoc multiple comparisons for 
between-group differences (e.g., responders vs. non-responders) and between-time 
differences. Similar analyses will be performed for the correlation of expression 
profiles and SNP analysis with pharmacokinetic data.   
 
Statistical power justification.  A power analysis was performed using two-sample 
t-test for between-group difference and paired t-test for the change over time. For 
tumor clearance, this will provide > 99% power to detect a 10% difference in the 
means at two separate time points for clones with 6 or more variants, and > 98% 
power to detect a 15% difference if there are 3 or more variants per clone with α = 
0.05 (power will decrease to 91% and 82%, respectively, in cases where the 
standard deviation is 4.7%, the maximum we have observed thus far). For 
pharmacologic correlations, we anticipate 85% power to determine a 25 ng/ml 
difference between the average Cmax of 45 evaluable responders and 55 evaluable 
non-responders with an anticipated average Cmax of 70 ng/ml and a standard 
deviation of 40 ng/ml within the entire cohort, and with α = 0.05.[22] We believe a 
less significant difference would not be sufficient to warrant application in clinical 
decision making (i.e. whether to continue therapy for more than 1 cycle). We 
anticipate 99% power to determine a difference of 20% in the reduction of bone 
marrow methylcytosine between 45 evaluable responders and 55 evaluable non-
responders with an expected standard deviation of 15% and α = 0.05.[28, 66-68] 
The difference in methylcytosine reduction previously observed was 30% between 
patients achieving CR/PR and those with only hematologic improvement.[28]  
 
Time to relapse and time to death will also be listed for each patient. 
 


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12.2.4 Safety Analysis 
 
Extent of Exposure – Duration (number of cycles/infusions) of decitabine doses 
(cumulative and intensity) will be summarized. 
 
Adverse Events - All adverse events will be coded using NCI CTCAE v. 4.0.  The 
incidence of treatment-emergent adverse events (number and percent of patients 
reporting the adverse event at least once during the study) will be summarized.  In 
addition, adverse events will be summarized by investigator attribution of 
relationship to study medication and by grade.  Similar summaries will be provided 
by treatment cycle. 
 
Clinical Laboratory Evaluation – Applicable laboratory parameters will be graded 
according to NCI CTCAE version 4.0. The incidence of maximum grade (number 
and percent of patients experiencing the maximum grade during the study) will be 
summarized for each laboratory parameter.  Similar summaries will be provided by 
treatment cycle.  Graphical displays of select hematological parameters will also be 
provided. 
 
Vital Signs, Physical Findings, and Other Safety Observations – Vital signs, 
findings from physical examinations, ECOG scores and weights will be presented 
in listing format. 
 
12.2.5 Description of Planned Subgroup Analyses 
 
The following subgroups (if sufficient numbers of patients) will be explored with 
respect to the primary and overall survival endpoints. 
 Age (60-69 years old vs ≥ 70 years old) 
 Cytogenetics 
 Patients with AML-associated mutations in DNMT3A, TET2, IDH1, or 
IDH2 vs those who do not have somatic variants in these genes. 
 
12.3 
Sample Size 
 
We anticipate initially enrolling 25 patients at Washington University. Once we establish 
a routine exome analysis with validation and calculation of the rate of patient-specific 
mutation clearance, we will enroll an additional 100 patients from at least 2 additional 
centers.  
 


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13.0 
DATA SUBMISSION SCHEDULE 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to registration 
Patient Registration Form 
Disease Diagnosis Form 
Inclusion Criteria Worksheet 
Exclusion Criteria Worksheet 
 
Prior to starting treatment 
Treatment Record Form 
To be maintained throughout a patient’s treatment 
cycle.  The complete record is due at completion of 
treatment. 
Bone Marrow Sample Collection 
Form 
To be maintained throughout the course of the 
study at designated bone marrow sample 
collections time points (See Appendix 2). 
Pathology: Bone Marrow Biopsy 
To be completed upon the return of pathology 
report for each bone marrow biopsy. 
Study Calendar Form 
To be completed with the date of each time point 
upon occurrence. 
Labs Form 
To be completed upon the return of laboratory 
reports for each study time point (See Appendix 2) 
Dose Modifications 
To be maintained throughout the course of the 
study if dose modifications are performed. 
Therapy Response Form 
To be filled out upon remission (prior to, and/or on 
therapy) 
End of Study Form 
 
Due at the completion of the EOS visit, at the time 
of death, and/or at the time that the patient is lost to 
follow-up 
Follow Up Form 
 6 weeks post date of last decitabine dose. 
 Every 2 months for 2 years after the first dose of 
study drug. 
Record of Adverse Events 
At the time of any AE 
 
 


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Appendix 1 
ECOG Performance Scale 
Activity Status 
Description 
0 
Asymptomatic, fully active, and able to carry on all predisease 
performance without restrictions. 
 
1 
Symptomatic, fully ambulatory but restricted in physically strenuous 
activity and able to carry out performance of a light or sedentary 
nature, eg, light housework, office work. 
 
2 
Symptomatic, ambulatory and capable of all self-care but unable to 
carry out any work activities.  Up and about more than 50% of 
waking hours: in bed less than 50% of day. 
 
3 
Symptomatic, capable of only limited self-care, confined to bed or 
chair more than 50% of waking hours, but not bedridden. 
 
4 
Completely disabled.  Cannot carry on any self-care.   Totally 
bedridden. 
 
5 
Dead 
 
 


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Appendix 2 
Study Flow Chart 
Study Flow Chart 
Test 
Pre-
study 
Base-
line 
All 
Cycles 
Day 1 
All Cycles 
Cohort A 
Days 1-10a, b 
Cycle 1 day 
4 ± 1 
 
Cycle 1 
Day 10 ± 1 
and 28 ± 4 
Every 
week 
Every 
2 
weeks 
Even 
cycles 
Day 28±4 
End of 
Study 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Medical History 
 
X 
X 
 
 
 
 
 
 
 
Physical Exam 
 
X 
X 
 
 
 
 
 
 
X 
Concomitant 
Medications 
 
X 
 
 
 
 
 
 
 
 
Transfusion 
Requirementsc 
 
Xc 
 
 
Bone Marrow 
Aspirate and Biopsy 
Xd,e 
 
 
 
 
Xd 
 
 
Xd 
Xd 
Peripheral blood for 
correlative studies 
 
 
 
 
X 
 
 
 
 
 
Pregnancy test 
 
X 
 
 
 
 
 
 
 
 
CBC with differential 
and platelets 
 
X 
X 
 
 
 
Xf 
 
 
X 
Serum Chemistriesg 
 
X 
X 
 
 
 
 
Xf 
 
X 
Decitabine 
administrationa,b,h 
 
 
 
X 
 
 
 
 
 
 
Adverse Events  
 
 
 
Ongoing 
Post-study follow-up 
 
 
 
 
 
 
 
 
 
Xi 
a Once bone marrow blast count is ≤ 5% (measured by cytomorphology, immunophenotype, and cytogenetics), the 
number of days per cycle of decitabine may be decreased to 5 per treating physician discretion. 
b Once bone marrow blast count is ≤ 5% for two or more cycles and the peripheral blood counts have normalized, the 
number of days per cycle of decitabine may be decreased to 3 per treating physician discretion. 
c Care should be given to record the frequency of transfusions from 8 weeks prior to first dose of study drug. 
d Standard bone marrow procurement procedures will be followed for collection of the tissue. Prestudy samples should 
be collected within 14 days of first dose of study drug.  
e The bone marrow biopsy for correlative studies may be collected with the prestudy evaluation. If not, it should be 
done as part of the base-line evaluation 
f Once stable, these labs may be obtained every other week or once a month at the discretion of the treating physician. 
g Serum chemistries include bicarbonate, calcium, glucose, total protein, albumin, BUN, potassium, chloride, sodium, 
creatinine, total bilirubin, AST, ALT, alkaline phosphatase, and LDH. 
h Decitabine treatment will be 20 mg/m2 over a 1-hour IV infusion on consecutive Days 1-10 every 28-day cycle.   The 
dose will be calculated based on actual patient weight at the beginning of each cycle. 
i Patients will be followed for 6 weeks following the date of the last decitabine dose to monitor for toxicity, survival 
and response. Thereafter, the investigator or designee will record information on subsequent therapy, disease 
progression, and death, every 2 months for 2 years after first dose of study drug or until the patient is lost to follow-up.


Genomic Predictors of Decitabine Response in AML/MDS 
 
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45 
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Cheson, B.D., et al., Revised recommendations of the International Working 
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Genomic Predictors of Decitabine Response in AML/MDS 
 
 
NCT# NCT01687400 
 
 
Document Version Date:  08/01/2012 
 
IRB Approval Date:  11/28/2012 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Version date: 08/01/12 
 
 
 
Genomic Predictors of Decitabine Response in AML/MDS 
 
Washington University School of Medicine 
Division of Oncology 
660 South Euclid Avenue, Box 8056 
St. Louis, MO 63110 
 
Protocol #: (Pending) 
Protocol Version Date: 08/01/12 
 
Principal Investigator 
John Welch, M.D./Ph.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-2626 
Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Sub-Investigator 
Timothy Ley, M.D. 
Section of Stem Cell Biology, Department of Oncology 
Washington University School of Medicine in St. Louis 
660 S. Euclid Ave., Box 8007, St. Louis, MO 63110 
Telephone Number: (314) 362-8832 
 
Fax Number: (314) 362-9333 
Email: jwelch@dom.wustl.edu 
 
Pathology 
Jeffery M. Klco, M.D., Ph.D. 
Department of Anatomic and Molecular Pathology 
Telephone: (314) 362-8832 
Email: klcoj@wustl.edu 
 
Statistician 
Feng Gao, Ph.D. 
Division of Biostatistics 
Telephone: (314) 362-3682 
E-mail: feng@wubios.wustl.edu 
 
CONFIDENTIAL 
The information contained in this document is regarded as confidential and, except to the extent necessary to obtain 
informed consent, may not be disclosed to another party unless law or regulations require such disclosure.  Persons 
to whom the information is disclosed must be informed that the information is confidential and may not be further 
disclosed by them. 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Version date: 08/01/12 
 
 
Study Synopsis 
Title of study  
 
Genomic Predictors of Decitabine Response in AML 
Primary 
investigator/study center 
location 
John S. Welch, M.D./PhD 
Washington University School of Medicine, Division of Oncology 
St. Louis, MO 
Timothy J. Ley, MD 
Washington University School of Medicine, Division of Oncology 
St. Louis, MO 
Phase of development 
Phase II 
Study objectives 
Primary:  
- To determine whether patient-specific mutations correlate with clinical 
overall response to decitabine in patients with AML or MDS. 
Secondary:   
- To determine whether 10 consecutive days per cycle of decitabine 
improves outcomes compared with historical controls treated with 5 
consecutive days per cycle of decitabine. 
- To determine whether other biomarkers correlate with clinical overall 
response to decitabine in patients with AML or MDS. These will include: 
velocity and depth of patient-specific mutation clearance, bone marrow 
expression profile and change in expression profile during decitabine 
treatment, steady-state serum decitabine concentrations, and decrease in 
bone marrow methylcytosine content in response to decitabine. 
 
 
Study design  
Non-randomized, open-label, Phase II study of decitabine in patients with 
AML or MDS.  
Study criteria 
Inclusion Criteria 
 
One of the following: 
1. Patient must have non-M3 AML and be > 60 years of age OR  
2. non-M3 AML with relapsed disease OR 
3. Symptomatic MDS with one of the following: 
o 
Symptomatic anemia with either hemoglobin <10.0 g/dL 
or requiring RBC transfusion 
o 
Thrombocytopenia with a history of two or more platelet 
counts < 50,000 / µL or a significant hemorrhage 
requiring platelet transfusions, or 
o 
Neutropenia with two or more absolute neutrophil counts 
< 1,000 /µL. 
 
All of the following: 
1. Patient must have an ECOG performance status ≤ 2. 
2. Patient must have >10% disease burden measured by 
cytomorphology, flow cytometry, or cytogenetics. 
3. Patient must have peripheral white blood cell count < 50,000/mcl. 
4. Patient must have adequate organ function, defined as: 
a. 
Total bilirubin < 1.5 x ULN 
b. AST/ALT < 2.5 x ULN 
c. 
Serum creatinine < 2.0 x ULN  
5. Patient must have undergone ≤ 2 cycles of prior hypomethylating 
agent (decitabine or azacitidine). 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Version date: 08/01/12 
 
6. Patient must be able to understand and willing to sign an IRB-
approved written informed consent document. 
7. Patient must be enrolled in HRPO# 201011766 (“Tissue 
Acquisition for Analysis of Genetic Progression Factors in 
Hematologic Malignancies”). 
8. Patient must be > 18 years of age. 
 
 
Exclusion Criteria 
1. Patient must not be pregnant or nursing. 
2. Patient must not have known CNS leukemia.  
3. Patient must not have a history of positive HIV serology.  
4. Patient must not have a history of positive Hepatitis C serology. 
5. Patient must not have any uncontrolled intercurrent illness including, 
but not limited to, ongoing or active infection, ongoing or active 
GVHD, congestive heart failure of NYHA class 3 or 4, unstable 
angina pectoris, cardiac arrhythmia, or psychiatric illness/social 
situation that would limit compliance with study requirements. 
6. Patient must not have had radiation therapy within 14 days of 
enrollment.  
 
Expected number of 
patients 
Total: 125. 
Evaluable for all molecular studies: 100. 
Enrollment time table: 25 patients will be enrolled initially at Washington 
University. Once the molecular pipeline for routine exome analysis is 
established, at least 2 additional sites will be added.  
Study drug formulation 
and administration  
Decitabine (5-aza-2’-deoxycytidine) is a lyophilized white powder available 
for injection in 20-mL single-use vials containing 50 mg of drug. The 
product should be stored at room temperature. Prior to use, the test product is 
reconstituted in 10 mL of sterile water for injection. The reconstituted 
concentrate is further diluted with cold infusion solution to obtain a final 
concentration of decitabine of between 0.1 to 1.0 mg/mL. 
 
All patients will receive decitabine 20 mg/m2 given as a 1 hour continuous 
IV infusion on consecutive days 1-10 of each cycle; the dose should be 
calculated based on actual body weight at the beginning of each cycle.  
 
Once patients achieve morphologic complete remission with incomplete 
blood count recovery (CRi) (morphologic leukemia-free state, defined as less 
than 5% blasts in aspirate with marrow spicules and a count of  ≥ 200 
nucleated cells and no blasts with Auer rods, no persistence of 
extramedullary disease, and no persistent pre-treatment morphologic 
abnormalities, such as pseudo-Pelger-Hüet cells, ringed sideroblasts, or 
dysplastic megakaryocytes) administration may be decreased to 5 
consecutive days to limit myelosuppressive toxicity, per treating physician. 
After two cycles with bone marrow blast counts < 5% and no evidence of 
disease by cytogenetics or immunophenotype, patients may further decrease 
therapy to 3 consecutive days per cycle, per treating physician. 
Each cycle will be 28 ± 4 days. 
Duration of treatment 
Treatment may continue until one of the following criteria applies: 
 Death, 
 Disease progression (as defined in 3.7.1.3), 
 Intercurrent illness that prevents further administration of treatment, 
 Unacceptable adverse event(s), 
 Patient decides to withdraw from the study, or 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Version date: 08/01/12 
 
 General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the treating 
physician.  
Correlative studies  
Bone marrow samples for correlative studies will be collected prestudy, on 
Cycle 1 day 10 ± 1 and day 28 ± 4, and on day 28 ± 4 of subsequent even 
numbered cycles.  
Peripheral blood samples for correlative studies will be collected prestudy, 
and on Cycle 1 day 4 ± 1.   
 
1. Identify Patient-specific mutations 
We will perform exome sequencing of each patient’s day 0 bone 
marrow sample and skin sample. We will determine AML/MDS-
associated variants, defined as variants found in the bone marrow 
sample and not in the skin sample. We will correlate the presence of 
mutations with overall response. 
2. Velocity and depth of response. 
We will assess the rate of tumor clearance by measuring the allele 
frequency of patient-specific mutations in bone marrow samples 
during cycles 1 and 2. We will assess mutations in at least 24 
recurrently mutated genes (including, but not limited to: FLT3, 
DNMT3A, NPM1, TET2, WT1, IDH1, IDH2, RUNX1, NRAS, TP53, 
U2AF1, KRAS, PHF6, MSTP9, PTPN11, KIT, SMC3, PRUNE2, 
ETV6, CEBPA, SMC1A, ASXL1, STAG2, RAD21) as well as all 
patient-specific mutations discovered during exome sequencing. The 
leukemic allele frequency will be measured using the Illumnia 
MySeq or HiSeq platform using DNA capture by gene specific 
biotinylated-oligonucleotides, or by a comparable technique. Patient 
bone marrow samples will be assessed on day 0, day 10 ± 1, and day 
28 ± 4 of cycle 1, and on day 28 ± 4 of cycle 2 and compared with 
patient-specific skin samples (normal control). The velocity of 
response will be calculated as the natural log rate of patient-specific 
mutation allele frequency clearance. Depth of response will be 
defined as the lowest patient-specific mutation allele frequency in a 
bone marrow sample. The velocity and depth of tumor response will 
be correlated to: 
 
Overall response, 
 
Time to best response, 
 
Mutation status, 
 
Time to disease progression. 
 
3. Expression profile. 
Expression array profiling will be performed on bone marrow 
samples from day 0 and day 10 ± 1. We will correlate expression 
signatures, and changes in expression signatures, with: 
 
Overall response, 
 
Mutational status. 
 
4. Steady-state serum decitabine concentration. 
We will assess serum decitabine concentration on day 4 ± 1 and 
correlation with: 
 
Overall response. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Version date: 08/01/12 
 
5. Bone marrow DNA hypomethylation. 
We will assess the percent reduction in bone marrow methylcytosine 
content using LC-MS. This may serve as a better biomarker of 
adequate dosing than serum drug concentration. Patients will be 
evaluated on day 0 and day 10 ± 1 of cycle 1. The change in DNA 
methylcytosine content will be correlated to: 
 
Overall response, 
 
Change in expression signatures. 
 
  
Criteria for evaluation 
Efficacy-  
Bone marrow biopsies should be performed at baseline and at day 10 ± 1 and 
day 28 ± 4 of cycle 1 and at the end of subsequent even numbered 
cycles. Standard bone marrow procurement procedures will be 
followed for collection of the tissue. Jeffrey Klco, MD/PhD will act 
as a central reviewer for all bone marrow biopsies. 
 
Peripheral blood counts will be followed by weekly CBC with differential. 
After cycle 1, the frequency of routine CBC analysis may be 
decreased by the treating physician as clinically indicated.  
 
Patients will be assessed for response according to the IWG criteria:[1]  
Morphologic complete remission (CR)– Defined as morphologic 
leukemia-free state, defined as less than 5% blasts in aspirate with 
marrow spicules and a count of  ≥ 200 nucleated cells and no blasts with 
Auer rods or any persistence of extramedullary disease, and no evidence 
for dysplasia. When erythroid precursors constitute less than 50% of bone 
marrow nucleated cells, the percentage of blasts is based on all nucleated 
cells; when there are 50% or more erythroid cells, the percentage blasts 
should be based on the nonerythroid cells. The presence of mild 
megaloblastoid changes may be permitted if they are thought to be 
consistent with treatment effect. However, persistence of pretreatment 
abnormalities (eg, pseudo-Pelger-Hüet cells, ringed sideroblasts, 
dysplastic megakaryocytes) are not consistent with CR. In addition, ANC 
> 1000/μl and platelet count of ≥ 100,000/μl. Patient must be independent 
of transfusions. No duration requirement for this designation. 
Cytogenetic complete remission (CCR) – Defined as morphologic complete 
remission plus a reversion to a normal karyotype. For this study, reversion 
to a normal karyotype is defined as no clonal abnormalities detected in a 
minimum of 20 mitotic cells. Fluorescent in situ hybridization (FISH) 
may be used as a supplement to follow a specifically defined cytogenetic 
abnormality. 
Morphologic complete remission with incomplete blood count recovery (CRi) 
– Defined as morphologic complete recovery with the exception of 
neutropenia <1000/μl or thrombocytopenia <100,000/μl.  
Partial remission (PR) – Requires that the above criteria for complete 
remission be met, with the following exceptions: decrease of ≥50% in the 
percentage of blasts to 5-25% in the bone marrow aspirate. A value of 
5% blasts in bone marrow with Auer rods present may also be 
considered a partial remission.  
Treatment failure – Includes all patients who did not achieve a complete or 
partial remission.  


Genomic Predictors of Decitabine Response in AML/MDS 
 
Version date: 08/01/12 
 
Recurrence/morphologic relapse - Relapse following complete remission is 
defined as reappearance of blasts in the blood or the finding of ≥5% blasts 
in the bone marrow, not attributable to any other cause. New dysplastic 
changes are considered a relapse. If there are no blasts in the peripheral 
blood and 5-20% blasts in the bone marrow, bone marrow biopsy should 
be repeated in > 1 week to confirm relapse. 
Safety-- All patients receiving at least one dose of decitabine will be 
evaluated for safety. 
Statistical considerations 
This trial will be conducted as a phase II biomarker trial. 
 
This trial is designed to detect the most clinically meaningful mutational 
predictors of response. These are mutations that have sufficient positive 
predictive value to be used as a clinical assay. 
 
A power analysis was performed with Fisher’s exact test for mutation status 
and response to decitabine, assuming an expected 45% CR/CRi. With alpha = 
0.05, and a total anticipated enrollment of 100 evaluable patients, we 
anticipate 80% power to detect a 27% and 40% response difference between 
patients with and without a mutation, if the mutation occurs with a respective 
frequency of 30% and 15%. Such mutations would provide 80%  sensitivity 
to predict response to decitabine. These are values that would warrant 
diagnostic clinical testing prior to therapy.  
 
This trial is also designed to detect an improvement in clinical response 
compared to historical controls treated with 5 consecutive days of decitabine 
(20 mg/m2/day every 28 days)[2]. A power analysis was performed with 
Fisher’s exact test for response to decitabine compared to a historical rate of 
24% observed in 55 patients. With 100 evaluable patients enrolled, we 
anticipate 90% power with alpha = 0.1 to detect a 21% improvement in 
response (CR/ PR ≥ 45%). The reported CR/PR rate of the proposed 
treatment schedule (10 consecutive days of decitabine 20 mg/m2/day) is 
64%[3]; the expected CR/PR rate of 45% is a conservative estimation of 
expected outcomes.  
 
 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
Version date: 08/01/12 
 
Study Schema: 
 
 
Patients with progressive disease after two cycles (defined as a clear increase in bone marrow blast count, typically a 
> 50% increase in bone marrow blasts over baseline) should be removed from study. 
Patients with stable disease may continue on study and should undergo a diagnostic bone marrow biopsy at the end 
of even numbered cycles. 
After 2 cycles, if patients achieve morphologic CRi, administration will be decreased to 5 days to limit 
myelosuppressive toxicity (20 mg/m2 over 1 hour on Days 1-5 every 28 ± 4 days). After two cycles with 
morphologic CRi and no evidence of disease by cytogenetics or immunophenotype, patients may further 
decrease therapy to 3 days per cycle (20 mg/m2 over 1 hour on Days 1-3 every 28 ± 4 days). 
Sample collection for correlative studies: 
Bone marrow biopsies should be performed at baseline and at Day 10 ± 1 and Day 28 ± 4 of Cycle 1 and at 
the end of even numbered cycles. 
 
Peripheral blood samples should be collected on Day 4± 1 of Cycle 1. Collection should be obtained 45 ± 
15 minutes after decitabine infusion is initiated.
20 
mg/m
2/da
Cycle 1 
20 
mg/m
2/da
Cycle 2+ 
Day 1 
Day 28 
BM Bx 
PB 


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8 
Genomic Predictors of Decitabine Response in AML/MDS 
Table of Contents 
Section 
Page 
1.0 INTRODUCTION 
10 
1.1 
Rationale 
10 
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
12 
1.2.1 
Pharmacokinetics and Metabolism of Decitabine 
14 
1.2.2 
Experience with extended dosing schedules 
15 
1.3 
Correlative Studies 
16 
2.0 OBJECTIVES 
18 
2.1 
Primary Objective 
18 
2.2 
Secondary Objectives 
18 
3.0 PATIENT SELECTION 
19 
3.1 
Inclusion Criteria 
19 
3.2 
Exclusion Criteria 
19 
3.3 
Inclusion of Women and Minorities 
20 
4.0 REGISTRATION PROCEDURES 
21 
4.1 
Confirmation of Patient Eligibility 
21 
4.2 
Patient Registration in the Siteman Cancer Center Database 
21 
4.3 
Assignment of UPN 
21 
5.0 INVESTIGATIONAL PLAN 
22 
5.1 
Summary of Study Design 
22 
5.2 
Study Procedures 
22 
5.2.1 
Pre-study Procedures 
22 
5.2.2 
Baseline Evaluation 
23 
5.2.3 
Day 1 of Each Cycle 
23 
5.2.4 
Cycle 1 Days 1-10 
23 
5.2.5 
Cycle 1 Day 4 ± 1 
23 
5.2.6 
Cycle 1 Day 10 ± 1 
24 
5.2.7 
Weekly 
24 
5.2.8 
Every Other Week 
24 
5.2.9 
Cycle 1 and Even Cycles Day 28 ± 4 days 
24 
5.2.10 
End-of-Study Procedures 
24 
5.2.11 
Post-study Follow-up 
24 
5.3 
Duration of Therapy 
25 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
25 
5.4.1 
Chemotherapy 
25 
5.4.2 
Growth Factors 
25 
5.4.3 
Transfusions 
26 
5.4.4 
Prophylactic Antimicrobial Agents 
26 
5.4.5 
Radiotherapy 
26 
6.0 DOSE MODIFICATIONS 
27 
6.1 
Delay for Cytopenia or Infection 
27 


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6.2 
Delay for Organ Dysfunction 
27 
6.3 
Modification in Dose or Schedule 
27 
7.0 PHARMACEUTICAL INFORMATION 
28 
7.1 
Decitabine 
28 
7.1.1 
Study Drug Preparation 
28 
7.1.2 
Supplier 
28 
7.1.3 
Toxicities 
28 
8.0 CORRELATIVE STUDIES SAMPLE COLLECTIONS 
30 
9.0 EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
31 
9.1 
Response Criteria 
31 
9.2 
Guidelines for Evaluation of Disease 
32 
9.3 
Progressive Disease 
32 
9.4 
Other Secondary Efficacy Measures 
32 
9.4.1 
Overall Survival and Event-free Survival 
32 
9.4.2 
Duration of Remission and Relapse-free Survival for Patients Having Complete 
Remission 
32 
9.4.3 
Safety 
33 
10.0 ADVERSE EVENT REPORTING 
34 
10.1 Adverse Events (AEs) 
34 
10.2 Unanticipated Problems 
34 
10.3 Noncompliance 
34 
10.4 Serious Noncompliance 
34 
10.5 Protocol Exceptions 
35 
10.6 Reporting to the Human Research Protection Office (HRPO) and the Quality Assurance 
and Safety Monitoring Committee (QASMC) at Washington University: 
35 
10.7 Timeframe for Reporting Required Events: 
36 
11.0 DATA SAFETY MONITORING PLAN 
37 
12.0 STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
38 
12.1 Analysis Populations 
38 
12.2 Statistical Analysis 
38 
12.2.1 
Descriptive Analyses 
38 
12.2.2 
Primary Endpoint Analysis 
38 
12.2.3 
Secondary Endpoint Analyses 
39 
12.2.4 
Safety Analysis 
41 
12.2.5 
Description of Planned Subgroup Analyses 
41 
12.3 Sample Size 
41 
13.0 DATA SUBMISSION SCHEDULE 
42 
  


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1.0 
INTRODUCTION 
 
1.1 
Rationale   
 
Personalized therapy for cancer requires that 1) patient-specific factors (e.g. patient-
specific mutations, expression profiles, or pharmacogenomics) influence treatment 
algorithms, and 2) once therapy is initiated, in vivo responsiveness is used to guide further 
therapy. The modern era of genomics has provided significant insights into cancer 
pathogenesis by describing patterns of germline and somatic variants, as well as defining 
disease-specific patterns of gene expression. However, the application and integration of 
this data with therapeutics remains a difficult task. 
 
Decitabine is a commonly used single-agent therapy for patients with acute myeloid 
leukemia (AML) and/or myelodysplastic syndrome (MDS). Historically, it has yielded 
clinical complete responses in approximately 25% of patients across diverse clinical trials, 
it is well tolerated, and it can be given as in the outpatient setting. However, the majority 
of patients do not respond to decitabine, the molecular etiology of decitabine sensitivity is 
unknown, and there are no algorithms to predict patient responsiveness a priori or even 
within one month of starting therapy.  
 
AML and MDS are two closely related hematological disorders characterized by 
ineffective hematopoiesis and malignant expansion of clonal myeloid cells.  Both AML 
and MDS frequently occur in elderly patients and some cases are secondary and/or related 
to long-term bone marrow damage after radiation or chemotherapy. Current therapy for 
AML consists of cytotoxic chemotherapy followed by consolidation chemotherapy or stem 
cell transplantation. Using these approaches, current cure rates may be as high as 60% for 
patients younger than 60 with good risk cytogenetics (e.g. t(15;17), t(8;21), inv(16)).[4]  
Outcomes in patients ≥ 60 years old and with relapsed disease remain poor with 2 year 
survival of only ~25%.[5] Historically, the available treatment for MDS has been 
symptomatic and has not been shown to be effective in producing sustained improvement 
in hematopoiesis or in delaying leukemic evolution[6, 7].  
 
Current AML therapy is poorly tolerated in elderly and pre-treated patients due to 
hematologic and non-hematologic toxicities; following such therapy, patients typically 
require 3-4 weeks of hospitalization for treatment of neutropenic fevers and for regular 
transfusions. Even among the relatively healthy patients enrolled in clinical trials, induction 
mortality in the elderly can be as high as 20-40% following standard induction therapy,[8-
11] and moderate to severe non-hematologic toxicity occurs in at least an additional 30% 
of patients.[9, 12-14] Consequently, many older AML patients are not offered traditional 
intensive chemotherapy, but receive supportive care alone.[15]  More effective and less 
toxic therapies are needed to provide durable remissions in a significant proportion of 
elderly AML in populations. 
 
Decitabine has been studied in MDS patients and in elderly and/or relapsed AML patients. 
It has been found to be reasonably well tolerated, can be given in the outpatient setting, 
and has overall response rates of 20-45%.[2, 3, 16-20] Our own experience has been of 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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11 
CR/PR rates of 25%, [2] and this is typical of many other groups for both MDS and AML 
patients (reviewed in [21]  Decitabine is an S-phase depended drug.  [22-24] However, 
decitabine is usually administered in one hour daily infusions during 3 to 5 consecutive 
days (too short to expose decitabine to most AML blasts during at least 2 cycles of 
replication, which divide every 2 to 7 days).[25-27]  Recent experience with increasing the 
number of infusions per cycle from 5 consecutive days to 10 consecutive days has 
improved overall response rates to 67%.[3] Response to decitabine can be slow, sometimes 
requiring 3 to 5 cycles before maximum response is observed.[2] As a consequence, many 
patients are exposed to prolonged therapy, gaining little benefit, before determining 
resistance. There are currently no biomarkers that predict response to decitabine. 
 
Decitabine is a cytosine analogue that cannot be methylated. Incorporation of decitabine 
into DNA prevents DNA methylation at cytosine residues and derepresses epigenetically 
silenced gene expression. Therefore, the reduction in total genomic DNA methylcytosine 
content acts as a marker of effective decitabine dosing. Indeed, two groups have recently 
correlated effective methylcytosine reduction and gene expression derepression to 
response (Figure 1.1).[3, 28] 
 
 
 
   
Figure 1.1. From Shen et al JCO (2010) 28:611 and Blum et al JCO (2007) 25: 3884. 
 
We and others have recently used whole genome sequencing to identify recurrent, somatic 
mutations in AML and MDS patients in four genes that are involved in methylcytosine 
regulation: DNMT3A, IDH1, IDH2, and TET2.[29-37] Collectively, nearly half of all 
AML/MDS patients have a mutation in one of these four genes.  DNMT3A mutations are 
associated with an adverse outcome in AML patients treated with cytotoxic 
chemotherapy.[35, 38] However, it is not yet clear whether the presence of mutations in 
one or more of these genes is associated with response or resistance to decitabine. 
 
In this study, we will treat patients with the current state-of-the-art decitabine schedule.[3] 
We will perform exome sequencing on all patients and correlate response with mutation 
status, rate of mutation clearance, expression profile, serum decitabine levels, and DNA 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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12 
methylcytosine content reduction. This approach will comprehensively assess genomic and 
pharmacologic parameters that could clinically predict patient response to decitabine.  
 
In this study, we will combine analysis of patients with AML and MDS. Recent genomic 
analysis has shown that AML and MDS have shared mutations in many of the same genes, 
shared patterns of mutation acquisition, and shared patterns of mutation evolution.[39, 40] 
Furthermore, MDS, like AML, presents with clonal hematopoiesis, even when the 
morphologic blast count is zero.[36]Because this study will identify and track the rate of 
clearance of patient specific mutations, and because decitabine has been shown to have 
efficacy in both AML and MDS patients, [2, 3, 16-19, 41-45] combining analysis of AML 
and MDS patients is reasonable.  Asymptomatic MDS patients are excluded because these 
patients are typically followed with watchful-waiting rather than with chemotherapy. 
  
1.2 
Decitabine and Hypomethylating agents in AML/MDS 
 
Methylation of cytosine in the CpG dinucleotide by DNA methyltransferase leads to 
transcriptional silencing of genes during normal development and has emerged as a 
significant mechanism for the loss of tumor suppressor gene expression in human cancer, 
including AML and MDS.[46, 47] Like azacitidine, decitabine (5-aza-2’-deoxycitidine) is 
incorporated into DNA during S phase and forms covalent adducts with DNA 
methyltransferase. The irreversible inhibition of this enzyme results in loss of methylation 
and is associated with differentiation.[48]  Decitabine is an approximately 5-fold more 
potent inhibitor of DNA methyltransferase than azacitidine, which might explain why 
decitabine can induce differentiation of myeloid progenitors in vitro at lower doses than 
azacitidine. In addition to their differentiation inducing activity at low doses, both agents 
have a direct cytotoxic effect at higher doses (1-2 µM).[49]  
 
Decitabine has been investigated in hematologic malignancies using a range of doses and 
schedules.  It is currently FDA approved for the treatment of adults with MDS at a dose of 
15 mg/m2 every 8 hours for 3 days, based on a Phase III study that demonstrated improved 
outcomes in the treatment arm compared to patients treated with supportive care alone.[15]  
However, the overall response rate in this trial was modest (17%). Of note, 18% of the 
patients in this trial had 20-30% bone marrow blasts, meeting the current WHO criteria for 
AML.  Subsequent phase I and II studies in AML, using a variety of low-dose decitabine 
regimens, have reported overall response rates of 22-49%.[2, 3, 16-19, 41-45] Comparisons 
of multiple low dose schedules have noted improved efficacy in the MDS population using 
a dose of 20 mg/m2 on Days 1-5 of a 28-day cycle.[50, 51] Because of these results, we 
have recently explored this dosing schedule in the treatment of elderly patients with 
AML.[52] In this study, we observed an overall response rate of 24%, and an additional 
44% of patients achieved stable disease during a median of 5 monthly cycles.[48] In this 
study, we noted that decitabine does not act quickly and the median time to response was 
3 months. These results are similar to the recently published experience in Germany 
treating 227 patients with 15 mg/m2 over 3 hours TID for 3 days every 6 weeks, which 
resulted in a CR/PR rate of 26%.[16]  
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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13 
A recent study using a prolonged administration schedule (20 mg/m2 IV over 1 hour on 
consecutive days 1-10) showed improved response (64% CR/PR) and improved time to 
respond (50% of patients had bone marrow blasts < 5% after 1 cycle of therapy).[3]  These 
results are an improvement over previous experience by other groups using decitabine to 
treat elderly AML patients or patients with relapsed AML, and this experience is 
summarized below: 
Trials of decitabine in AML 
Status 
Other Therapy 
DAC Dose 
(mg x days) 
Evaluable 
CR 
de novo 
daunomycin1 
90 mg/m2 x 5 
6 
100% 
Salvage 
None4 
0.75-80 mg/kg 
16 
  
None5 
37-81 mg/kg 
6 
33% 
None7.  Ara-C 
Refractory 
Various 
4 
0 
AMSA7 
250-500 mg/m2 x 6 
10 
80% 
idarubicin or 
AMSA3 
250 mg/m2 x 6 
56 
39% 
Elderly 
None6 
270-360 mg/m2 x 3 
10 
30% 
None2 
45 mg/m2 x 3 
4 
50% 
None8 
20 mg/m2day 1-5,8-12 
50 
23% 
None9 
20 mg/m2 x 5 
55 
25% 
 
None10 
50 mg/m2 CIVI x 3 
29 
48% 
 
None11 
20 mg/m2 x 10 
53 
64% 
 
ATRA cycle 212 
15 mg/m2 TID x 3 
227 
26% 
1 Schwartsmann G.  Leukemia 1997; 11(Suppl 1):S28-31. 
2 Lubbert M. ASH 2002 Abstract #4601. 
3 Willemze R.  Leukemia.  1997; 11(Suppl 1):S24-27 
4 Rivard GE.  Leukemia Research 1981; 5:453-462. 
5 Momparler RL.  Pharmac Therapy 1985; 30:277-286.  
6 Petti MC.  Leukemia.  1993; 7(Suppl 1):36-41.  
7 Richel DJ.  Br. J. Cancer 1991; 64:144-148 
8 Issa, JP. Blood 2004. 103: 1635. 
9 Cashen AF. ASH Annual Meeting. 2008 abstract 560. 
10 Wijermans PW. Leukemia 1997. 11: Suppl 1, 19-23. 
11 Blum, W. PNAS 2010; 107:7473. 
12 Lubbert. Haematologica 2011. ePub 11/04/2011. 
 
In general, decitabine is well tolerated. Unlike conventional cytotoxic chemotherapy, 
decitabine can be administered in the outpatient setting. However, AML and MDS are 
aggressive hematologic malignancies. Cytopenias commonly result from AML/MDS 
inhibition of normal hematopoiesis, and often do not resolve until a complete response is 
achieved. Thus, neutropenic sepsis and thrombocytopenic bleeding remain significant 
problems regardless of therapy, especially during the first 4-8 weeks. In our experience 
treating elderly AML patients, neutropenia occurred in 24%, sepsis in 9%, and bacteremia 
in 7%.  Similar results have been observed in MDS patients.[20, 53, 54] As a result, death 
is not uncommon in the first 8 weeks of therapy, occurring in 15-17% of patients, and most 
commonly resulting from infection and cytopenias related to disease and therapy.[16, 52, 
55] While unsettling, these numbers are better than many historical patients treated with 
cytotoxic chemotherapy, and suggest a need for continued improvements in therapy.[9, 12]  


Genomic Predictors of Decitabine Response in AML/MDS 
 
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14 
 
1.2.1 Pharmacokinetics and Metabolism of Decitabine 
 
In human subjects, after IV administration, decitabine displayed a distribution phase 
with a half-life of 7 minutes and a terminal phase elimination half-life of 10-35 
minutes as measured by bioassay.[56, 57]   The short plasma half-life is due to rapid 
inactivation of decitabine by deamination by liver cytidine deaminase.[23] Total 
body clearance (Clp) in humans was high with a mean value of 126 mL/min/kg.[58]  
 
Among five adult patients with advanced solid tumors administered seven courses 
of 100 mg/m2 of decitabine by 1-hour infusions,[56] maximum plasma 
concentration ranged from 1 to 4 µM. Disappearance from plasma was biphasic 
with a rapid initial decline to less than 0.1 µM during the first hour post infusion 
and a slower decline over the next two hours to 6 nM, the level of detectability. 
Plasma half-life (meanSE) was estimated to be approximately 71 minutes in the 
initial phase (t½α) and 355 minutes in the second phase (t½β). The area under the 
plasma concentration-time curve was 40888 ng.hour/mL for these patients, with a 
peak plasma concentration of 2.010.44 µM (0.440.1 µg/mL), while mean volume 
of distribution (Vdss) and CLp was 4.591.42 L/kg and 12621 mL/min/kg, 
respectively. The time course of DAC concentrations was similar in 1 of 2 patients 
administered 75 mg/m2, while DAC was usually undetectable in the plasma of 
patients administered 25 to 60 mg/m2. 
 
Using a longer infusion time (up to 40 hours), Rivard et al.[57] reported a plasma 
concentration of 0.1 to 0.4 g/mL. With infusion times of 40-60 hours, at an infusion 
rate of 1 mg/kg/h, plasma concentrations of 0.43-0.76 µg/mL were achieved.[59]  The 
steady-state plasma concentration at an infusion rate of 1 mg/kg/h was estimated to 
be 0.2-0.5 µg/mL. The half-life after discontinuing the infusion was 12-20 min.[57]  
 
Urinary excretion of unchanged decitabine was low, ranging from less than 0.01% to 
0.9% of the total dose, and there was no relationship between excretion and dose or 
plasma drug levels. High clearance values and a total urinary excretion of <1% of the 
administered dose suggest that decitabine is eliminated rapidly and largely by 
metabolic processes, including extrahepatic metabolism.[60] 
 
The steady state plasma concentrations (Css) ranged from 0.31 to 0.39 µg/mL (1.27 
to 1.60 µM) during the infusion of 100 mg/m² decitabine over a 6-hour period.[61] 
The range of concentrations during a 600 mg/m2 infusion over an 8-hour period was 
0.41-16 g/mL(1.68 to 4.76 µM).[60] These plasma concentrations are in the same 
range as the in vitro concentrations that activated the expression of tumor 
suppressor gene p16 in three human non-small-cell lung cancer (NSCLC) cell 
lines.[61]  
 
Steady state decitabine levels of 0.1 to 0.2 µM were achieved in solid tumor patients 
dosed with 30 mg/m²/day and 40 mg/m²/day by continuous intravenous infusion 
over 72 hours.[62]  


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15 
 
Summary of published steady state serum levels are below and are in proportion to 
infusion rate (R2 = 0.98, p < 0.0001):[63] 
 
Rate 
Serum 
Concentration 
mg/m2/hr 
ng/ml 
nM 
5 
55 
241 
7.5 
80 
351 
15 
180 
789 
21 
182.4 
800 
22.5 
350 
1535 
42 
456 
2000 
84 
1140 
5000 
 
In our experience treating primary patient blasts in vitro, concentrations of 100 – 
500 nM are required maximize methylcytosine reduction without causing cellular 
toxicity. Because decitabine incorporation into DNA is S-phase dependent, 
exposure of cells during cell division is necessary for efficacy. 
  
 
 
 
1.2.2 Experience with extended dosing schedules 
 
Blum et al recently published their experience treating elderly AML patients with 
a 10-day course of decitabine 20 mg/m2/day as a 1-hour infusion.[3] This prolonged 
exposure course was designed to increase the number of AML blast divisions 
exposed to decitabine per cycle. Toxicities were very similar to a similar regiment 
given as a 3 and 5 day course,[2, 16] but the overall response (CR/PR) was much 
improved (64% vs 25%). In all three studies, the toxicities were primarily myeloid 
suppression and the resultant infectious and bleeding complications associated with 
leukemic and pharmacologic hematopoietic suppression. Furthermore, Blum et al 
noted that the percent methylcytosine reduction was improved (22% vs 11%) as 
 


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16 
was the time to blast clearance (50% of patients who ultimately achieved a CR had 
≤ 5% bone marrow blasts after 1 cycle vs 0% of patients after 2 cycles). Finally, 
like Shen et al, they observed a correlation between the extent of methylcytosine 
reduction and response.[28]  This study suggests that improvement in response 
rates and time to response can be achieved by prolonged administration of 
decitabine. Further, that DNA methylcytosine reduction can be achieved with 
prolonged exposure to decitabine and that this might be a patient-specific biological 
marker of adequate dosing. 
  
Scandura et al. recently published a study of infusional decitabine prior to 
cytarabine + daunorubicin induction therapy for AML.[64] They escalated the 
duration of infusional therapy and reached their planned goal of 20 mg/m2/day x 7 
days of continuous therapy given prior to cytarabine and daunorubicin. They noted 
similar toxicities (primarily hematopoietic and the associated neutropenic 
infections), but that these were not different from their experience giving cytarabine 
+ daunorubicin alone. 
 
1.3 
Correlative Studies 
 
The goals of the correlative studies include: 
 
1. To correlate patient-specific mutations with response to decitabine. 
We will perform exome sequencing of each patient’s Day 0 bone marrow sample and 
skin sample. We will determine AML/MDS-associated variants found in the bone 
marrow, but not skin sample. We will correlate the presence of mutations with response 
(especially mutations in DNMT3A, IDH1, IDH2, and TET2). This study is powered to 
identify mutations with >80% sensitivity to predict decitabine response if used as a 
clinical assay. 
 
2. To correlate the velocity and depth of response with response to decitabine. 
We will assess tumor burden by allele frequency of patient-specific mutations in bone 
marrow samples collected on day 0, 10±1, 28±4, and 56±4. We will assess mutations 
in at least 24 recurrently mutated genes (including, but not limited to: FLT3, DNMT3A, 
NPM1, TET2, WT1, IDH1, IDH2, RUNX1, NRAS, TP53, U2AF1, KRAS, PHF6, 
MSTP9, PTPN11, KIT, SMC3, PRUNE2, ETV6, CEBPA, SMC1A, ASXL1, STAG2, 
RAD21) as well as all patient-specific mutations discovered during exome sequencing. 
The leukemic allele frequency will be measured using the Illumnia MySeq or HiSeq 
platform using DNA capture by gene specific biotinylated-oligonucleotides. Patient 
bone marrow samples will be assessed on day 0, day 10 ± 1, and day 28 ± 4 of cycle 1, 
and day 28 ± 4 of cycle 2 and compared with patient specific skin samples (normal 
control). The velocity of response will be calculated as the natural log rate of patient-
specific mutation allele frequency clearance. Depth of response will be defined as the 
lowest patient-specific mutation allele frequency in a bone marrow sample. The 
velocity and depth of tumor response will be correlated to: 
 Achievement of clinical CR, 
 Time to best response, 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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17 
 Mutational status, 
 Time to disease progression. 
Because all patient-specific exome-discovered mutations will be assessed a second 
time in the primary bone marrow sample as well as the Day 10 ± 1, 28 ± 4, and 56 ± 4 
bone marrow samples, this study will also serve to validate all exome-discovered 
mutations. This is important, because exome sequencing is associated with a 20-50% 
false-discovery rate depending on type I error tolerated (mutation-calling algorithms 
are typically set for greater sensitivity than specificity).  Thus, many mutations 
discovered during exome sequencing are incorrect.  This second analysis of these 
mutations will server to exclude these false positive calls. 
 
3. To correlate the bone marrow expression profile on Day 0 and Day 10 ± 1 with response 
to decitabine. 
We will correlate expression signatures, and changes in expression signatures, with: 
 Achievement of clinical CR, 
 Mutation status. 
 
4. To correlate steady-state serum decitabine concentration on Day 4 ± 1 with: 
 
Achievement of CR. 
 
5. To correlate the extent of bone marrow DNA hypomethylation on Day 10 ± 1 with 
response to decitabine. 
We will assess the percent reduction in bone marrow methylcytosine content using LC-
MS. Patients will be evaluated on Day 0 and Day 10 ± 1 of Cycle 1. The change in 
DNA methylcytosine content will be correlated to: 
 Achievement of clinical CR, 
 Change in expression signature. 
This will determine whether inadequate blast exposure to decitabine correlates with 
drug resistance. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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18 
2.0 
OBJECTIVES 
 
2.1 
Primary Objective 
 
To determine whether patient-specific mutations correlate with clinical overall response to 
decitabine in patients with AML or MDS. 
 
2.2 
Secondary Objectives 
 
- 
To determine whether 10 consecutive days per cycle of decitabine improves outcomes 
compared with historical controls treated with 5 consecutive days per cycle of 
decitabine. 
 
- 
To determine whether other biomarkers correlate with clinical overall response to 
decitabine in patients with AML or MDS. These will include:  
 
1. The velocity and depth of patient-specific mutation clearance; 
2. The bone marrow expression profile and change in expression profile during 
decitabine treatment; 
3. The steady-state serum decitabine concentrations; 
4. The decrease in bone marrow methylcytosine content in response to decitabine.  
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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3.0 
PATIENT SELECTION 
 
3.1 
Inclusion Criteria 
 
One of the following: 
 Patient must have non-M3 AML and be > 60 years of age OR 
 non-M3 AML with relapsed disease OR 
 Symptomatic MDS with one of the following: 
□ Symptomatic anemia with either hemoglobin < 10.0 g/dL or requiring RBC 
transfusion 
□ Thrombocytopenia with a history of two or more platelet counts < 50,000/mcL 
or a significant hemorrhage requiring platelet transfusions 
□ Neutropenia with two or more ANC < 1,000/mcL 
 
All of the following: 
1. Patient must have an ECOG performance status ≤ 2. 
2. Patient must have >10% disease burden measured by cytomorphology, flow 
cytometry, or cytogenetics. 
3. Patient must have peripheral white blood cell count < 50,000/mcl. 
4. Patient must have adequate organ function, defined as: 
a. 
Total bilirubin < 1.5 x ULN 
b. 
AST/ALT < 2.5 x ULN 
c. 
Serum creatinine < 2.0 x ULN  
5. Patient must have undergone ≤ 2 cycles of prior hypomethylating agent (decitabine 
or azacitidine). 
6. Patient must be enrolled in HRPO# 201011766 (“Tissue Acquisition for Analysis 
of Genetic Progression Factors in Hematologic Malignancies”). 
7. Patient must be > 18 years of age. 
8. Patient must be able to understand and willing to sign an IRB-approved written 
informed consent document. 
 
3.2 
Exclusion Criteria 
 
1. Patient must not be pregnant or nursing. 
2. Patient must not have known CNS leukemia.  
3. Patient must not have a history of positive HIV serology.  
4. Patient must not have a history of positive Hepatitis C serology. 
5. Patient must not have any uncontrolled intercurrent illness including, but not 
limited to, ongoing or active infection, ongoing or active GVHD, congestive heart 
failure of NYHA class 3 or 4, unstable angina pectoris, cardiac arrhythmia, or 
psychiatric illness/social situation that would limit compliance with study 
requirements. 
6. Patient must not have had radiation therapy within 14 days of enrollment.  
 
 


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3.3 
Inclusion of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible for this trial. 
 


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4.0 
REGISTRATION PROCEDURES 
 
Patients must not start any protocol intervention prior to registration through the Siteman 
Cancer Center. 
 
The following steps must be taken before registering patients to this study: 
 
1. Confirmation of patient eligibility 
2. Registration of patient in the Siteman Cancer Center database 
3. Assignment of unique patient number (UPN) 
 
4.1 
Confirmation of Patient Eligibility 
 
Confirm patient eligibility collecting the information listed below  
1. The registering MD’s name 
2. Patient’s race, sex, and DOB 
3. Three letters (or two letters and a dash) for the patient’s initials 
4. Copy of signed consent form 
5. Completed eligibility checklist, signed and dated by a member of the study team 
6. Copy of appropriate source documentation confirming patient eligibility 
 
4.2 
Patient Registration in the Siteman Cancer Center Database 
 
All patients must be registered through the Siteman Cancer Center database. 
 
4.3 
Assignment of UPN 
 
Each patient will be identified with a unique patient number (UPN) for this study.  Patients 
will also be identified by first, middle, and last initials.  If the patient has no middle initial, 
a dash will be used on the case report forms (CRFs).  All data will be recorded with this 
identification number on the appropriate CRFs. 
 


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5.0 
INVESTIGATIONAL PLAN 
 
5.1 
Summary of Study Design 
 
This is an open-label, single-arm, Phase II, biomarker study of patients with AML or MDS 
who are treated with 10 consecutive days of decitabine. FAB-M3 AML is excluded from 
this trial, because other well-tolerated options exist for the treatment of FAB-M3 acute 
promyelocytic leukemia. AML patients who are < 60 years old and have not received 
cytotoxic induction chemotherapy are also excluded, because cytotoxic therapy offers a 
curative possibility for these patients.  MDS patients without cytopenias are also excluded 
because these patients can be followed with watchful-waiting. 
 
All patients will undergo bone marrow biopsies prior to therapy, on Day 10 ± 1, and on 
Day 28 ± 4 of Cycles 1 and 2 for comprehensive genomic correlative studies. Patients will 
also provide peripheral blood samples on Day 4 ± 1 for pharmacokinetic studies. 
 
Patients will receive decitabine 20 mg/m2/day as a 1-hour infusion on consecutive Days 1-
10 in 28-day cycles.  The dose will be calculated based on actual patient weight at the 
beginning of each cycle. 
 
After 2 cycles, if patients achieve CRi, the administration of decitabine will be decreased 
to 5 days. After two cycles with CRi and no evidence of disease by cytogenetics or 
immunophenotype, patients will further decrease therapy 3 consecutive days.  The dose of 
decitabine will remain 20 mg/m2/day over 1 hour regardless of decreases in the number of 
days per cycle of administration. 
 
Patients who achieve stable disease, partial response, or complete response will continue 
on decitabine therapy (receiving cycles of 3-10 days every 28 days (i.e., Days 1-10, Days 
1-5, or Days 1-3), depending on response) until time of progression, unacceptable toxicity, 
or patient decision to discontinue study drugs. 
 
It will be a logistical goal of this trial consistently to return preliminary exome results to 
the treating physician by the end of Cycle 1 and validated results by the end of Cycle 2. 
However, this logistical goal is not a primary or secondary aim of this study. Given the 
limited data regarding mutation presence and drug response, we do not anticipate that the 
results will directly influence therapeutic decisions made by the treating physician. 
However, the development of comprehensive genomic pipelines and integration of this 
data into a clinical trial setting will be required before personalized medicine can be 
realized. 
 
5.2 
Study Procedures 
 
5.2.1 Pre-study Procedures 
 
Pre-study procedures must take place no more than 14 days prior to the first dose 
of decitabine. 


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 Bone marrow morphologic assessmenta 
 Bone marrow or peripheral blood for flow cytometry 
 Bone marrow for cytogenetics.  
a Standard bone marrow procurement procedures will be followed for collection of the tissue.  
 
5.2.2 Baseline Evaluation 
 
Baseline evaluations must take place no more than 7 days prior to the first dose of 
decitabine and include: 
 
1. Medical history 
2. Physical exam including height, weight, vital signs, and ECOG performance 
score. 
3. Concomitant medications 
4. Record transfusion requirements for 8 weeks before first dose of study drug 
5. CBC with differential and platelets 
6. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
7. Urine βHCG pregnancy test if appropriate 
 
5.2.3 Day 1 of Each Cycle 
 
1. Record adverse events  
2. Physical exam, including weight and ECOG performance score 
3. Vital signs, including blood pressure, pulse and temperature 
4. CBC with differential and platelets 
5. Serum chemistries, including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, LDH 
 
5.2.4 Cycle 1 Days 1-10 
 
Decitabine administration, 20 mg/m2 per day given as a 1-hour continuous 
intravenous infusion.  The dose will be calculated based on actual patient weight at 
the beginning of each cycle.  Patients may be premedicated with lorazepam, 
prochlorperazine, palonosetron, or ondansetron at the treating physician’s 
discretion. 
 
5.2.5 Cycle 1 Day 4 ± 1 
 
Obtain 3 mL peripheral blood for correlative studies. Sample must be obtained 45 
minutes ± 15 minutes after beginning the infusion, before the decitabine infusion is 
complete, and should be obtained from the opposite arm as the infusion.  The 
sample should be collected in a 3 mL K3EDTA tube pre-loaded with 8 mcL of 
tetrahydrouridine (THU 500 mcg/mL), an inhibitor of cytidine deaminase.  The 


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sample should be processed by the Tissue Procurement Core Facility at the Siteman 
Cancer Center.  Serum should be separated and stored per protocols. 
  
5.2.6 Cycle 1 Day 10 ± 1 
 
Obtain bone marrow biopsy for response and correlative studies. a 
a Standard bone marrow procurement procedures will be followed for collection of the tissue.  
 
5.2.7 Weekly 
 
1. CBC with differential and platelets. After the first cycle, this may be changed 
to every other week at the discretion of the treating physician. 
2. Record adverse events. 
 
5.2.8 Every Other Week 
 
Serum chemistries, including bicarbonate, calcium, glucose, total protein, albumin, 
BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, ALT, alkaline 
phosphatase, LDH. After the first cycle, this may be changed to every month at the 
discretion of the treating physician. 
 
5.2.9 Cycle 1 and Even Cycles Day 28 ± 4 days  
 
1. Bone marrow biopsy and aspirate collection.a    
2. Clinical samples for response determination should be obtained at the end of 
Cycles 1, 2 and 4. Patients who receive > 4 cycles should continue to undergo 
bone marrow biopsies on even cycles for clinical evaluation (or as clinically 
indicated). 
a Standard bone marrow procurement procedures will be followed for collection of the tissue.  
 
5.2.10 End-of-Study Procedures  
 
1. CBC with differential and platelets.  
2. Record adverse events.  
3. Physical examination including weight, vital signs, and ECOG performance 
score.  
4. Serum chemistries including bicarbonate, calcium, glucose, total protein, 
albumin, BUN, potassium, chloride, sodium, creatinine, total bilirubin, AST, 
ALT, alkaline phosphatase, and LDH.  
5. Bone marrow collection, with samples sent for routine pathology.a 
a Standard bone marrow procurement procedures will be followed for collection of the tissue.  
 
5.2.11 Post-study Follow-up 
 
Patients are to be followed according to standard patient care procedures for 6 
weeks following the date of the last decitabine dose to monitor for toxicity, survival, 


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and response. Thereafter, the investigator or designee will record information on 
subsequent therapy, disease progression, and/or death every 2 months for 2 years 
after first dose of study drug or until the patient is lost to follow-up or dies. 
 
5.3 
Duration of Therapy 
 
If at any time the constraints of this protocol are considered to be detrimental to the 
patient’s health and/or the patient no longer wishes to continue protocol therapy, the 
protocol therapy should be discontinued and the reason(s) for discontinuation documented 
in the case report forms. 
 
In the absence of treatment delays due to adverse events, treatment may continue 
indefinitely or until one of the following criteria applies: 
 
 Documented and confirmed disease progression 
 Death 
 Adverse event(s) that, in the judgment of the investigator, may cause severe or 
permanent harm or which rule out continuation of study drug 
 General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment in the judgment of the investigator 
 Suspected pregnancy 
 Serious non-compliance with the study protocol 
 Lost to follow-up 
 Patient withdraws consent 
 Investigator removes the patient from study 
 The Siteman Cancer Center decides to close the study 
 
Patients who prematurely discontinue treatment for any reason will be followed as 
indicated in the study calendar. 
 
5.4 
Concomitant Therapy and Supportive Care Guidelines 
 
5.4.1 Chemotherapy 
 
Patients cannot receive any chemotherapy within 21 days of study enrollment and 
must have recovered from any treatment-related toxicities. Patients may have 
received hydrea prior to enrollment for a WBC > 50,000/l but must stop hydrea at 
least one day prior to initiating decitabine. Other than decitabine, patients cannot 
receive chemotherapy during this trial.  
 
5.4.2 Growth Factors 
 
Erythroid growth factors should not be routinely administered to patients during the 
study. 
 


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Thrombopoietin receptor agonists should not be routinely administered to patients 
during the study. 
 
Filgrastim (G-CSF) or sargramostim (GM-CSF) may be administered to patients 
with septic shock or febrile neutropenia at the discretion of the treating physician. 
 
5.4.3 Transfusions 
 
 Packed red blood cells transfusion:   
Two units of packed RBCs are to be administered when the hemoglobin drops 
under 8 g/dL. Transfusion administration when the patient’s hemoglobin is 8-
10 g/dL may be done according to the treating physician’s clinical judgment.  
 
 Platelet transfusion:   
One unit of single-donor platelets will be administered when platelets are < 
20,000/L. 
 
5.4.4 Prophylactic Antimicrobial Agents  
 
Antimicrobial agents will be administered at the discretion of the treating physician. 
 
5.4.5 Radiotherapy 
 
Patients cannot receive radiotherapy within 14 days prior to study enrollment or 
while on study. 
 


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6.0 
DOSE MODIFICATIONS 
 
6.1 
Delay for Cytopenia or Infection 
 
As cytopenias are a defining feature of AML and MDS, their presence alone does not 
generally warrant dose modifications or delays. Patients will receive four cycles of therapy 
without adjustment for any cytopenia. 
 
Patients with febrile neutropenia (temperature ≥ 38.5° with ANC < 1,000/l) or systemic 
infections should have treatment held until clinical resolution of the infection. A patient 
will meet criterion for discontinuation of the study if the patient is observed to have a delay 
of > 8 weeks between any two cycles.  
 
6.2 
Delay for Organ Dysfunction 
 
If renal dysfunction (creatinine ≥ 3 X institutional upper limit of normal) or hepatic 
dysfunction (bilirubin ≥ 2.5 mg/dl or ALT/AST ≥ 3 X institutional upper limit of normal) 
occurs, therapy may not resume until the abnormalities have resolved to below the limits 
stated above. If renal or hepatic dysfunction occurs during a treatment course, the next dose 
of decitabine should be held, and serum chemistries should be checked twice per week.  
Decitabine can be resumed when creatinine, bilirubin, ALT and AST are below the limits 
listed above.  
 
For grade 3-4 non-hematologic toxicities, decitabine treatment should be delayed until 
recovery to baseline or to grade ≤ 1. For toxicities felt to be related to decitabine, decitabine 
may be reintroduced with a 50% dose reduction, or at the discretion of the investigator. 
 
6.3 
Modification in Dose or Schedule 
 
Once patients achieve morphologic complete remission with incomplete blood count 
recovery (CRi) (morphologic leukemia-free state, defined as less than 5% blasts in aspirate 
with marrow spicules and a count of  ≥ 200 nucleated cells and no blasts with Auer rods, 
any persistence of extramedullary disease, or persistent pre-treatment morphologic 
abnormalities, such as pseudo-Pelger-Hüet cells, ringed sideroblasts, or dysplastic 
megakaryocytes), administration may be decreased to 5 consecutive days to limit 
myelosuppressive toxicity, per treating physician. After two cycles with bone marrow blast 
counts < 5% and no evidence of disease by cytogenetics or immunophenotype, patients 
may further decrease therapy to 3 consecutive days per cycle, per treating physician. 
 


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7.0 
PHARMACEUTICAL INFORMATION 
 
7.1 
Decitabine 
 
Decitabine is an antimetabolite that can replace cytosine in DNA but, unlike cytosine, it 
cannot be methylated. The exact mechanism of decitabine activity has not been determined 
and may involve multiple pathways. Decitabine is thought primarily to act by correcting 
DNA methylation, a major mechanism for gene expression. In some cancer cells, 
hypermethylation blocks the activity of tumor suppressor genes, which regulate cell 
division and differentiation to prevent malignant transformation. When suppressor gene 
activity is blocked, cell division becomes unregulated, leading to the formation of 
neoplastic cells. Decitabine regulates DNA methylation (specifically targets the 
methyltransferase, DNMT1), effectively demethylating and reactivating different tumor 
suppressor genes. Because hypermethylation occurs early in the malignant transformation 
of cells, decitabine may have activity in chemoprevention. Other possible mechanisms for 
decitabine activity include cytotoxicity due to incorporation into DNA similar to other 
antimetabolites, methylation-independent induction of gene expression, and degradation of 
DNA methyltransferase 1. 
 
7.1.1 Study Drug Preparation  
 
OSHA Guidelines for handling cytotoxic drugs outlined in the American Journal 
of Hospital Pharmacy must be followed. 
 
Decitabine (5-aza-2’-deoxycytidine) is supplied as a lyophilized powder for 
injection, 50 mg in 20-ml vials. When reconstituted with 10 ml of sterile water for 
injection, each ml will contain 5 mg of decitabine and 6.8 mg of KH2PO4. The 
reconstituted solution should be diluted to 0.1-1.0 mg/mL with pre-chilled 0.9% 
sodium chloride intravenous infusion, which has been cooled to 2-8oC. 
Reconstitution of the powder results in a rapidly decomposing solution. Unless used 
within 15 minutes of reconstitution, the diluted solution must be prepared using 
cold (2—8 degrees C) infusion fluids and stored at 2—8 degrees C (36—46 degrees 
F) for a maximum of 7 hours. 
Decitabine will be administered per dosing schedule every 28 ± 4 days.  
 
Decitabine may be administered either as an inpatient or an outpatient.  
 
7.1.2 Supplier 
 
Decitabine is available commercially. 
 
7.1.3 Toxicities 
 
Likely (>15%) 
Cytopenias: 
neutropenia, 
thrombocytopenia, 
anemia, 
leukopenia, 
febrile 
neutropenia.  


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Bleeding: petechiae, ecchymosis. 
Infections: pneumonia. 
Constitutional: fatigue, pallor, fever, rigors, arthralgia, cough. 
GI: nausea/vomiting, constipation, diarrhea, decreased appetite. 
Electrolyte abnormalities: hyperglycemia, hypoalbuminemia, hypomagnesemia, 
hypokalemia, hyponatremia.  
Cardiac: cardiac murmur. 
Neurologic: headache, insomnia, dizziness. 
Dermatologic: rash. 
 
Less likely (1-15%)  
Hematologic: thrombocythemia, hematoma. 
Other infections: cellulitis, candidal infection, urinary tract infection, sinusitis. 
GI: abdominal 
pain, hyperbilirubinemia, 
stomatitis, 
dyspepsia, ascites, 
hemorrhoids, oral ulceration, dysphagia, abdominal distention, gastro-esophageal 
reflux disease/pyrosis (heartburn), and glossodynia. 
Electrolyte abnormalities: hyperkalemia, and dehydration. 
Urologic: dysuria, increased urinary frequency.  
Dermatologic: pruritis, alopecia, urticaria, edema. 
 
Rare (<1%) 
Cardiac: arrest/respiratory arrest, atrial fibrillation, and tachycardia. 
 
  
 
  
 


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8.0 
CORRELATIVE STUDIES SAMPLE COLLECTIONS 
 
Bone marrow samples will be collected at Baseline, on Cycle 1 Day 10 ± 1, and on Day 28 ± 4 of 
Cycle 1 and of even number cycles for correlative studies. Bone marrow aspirate (6-10 ml) will be 
collected and divided between two EDTA (lavender top) tubes. The bone marrow samples and 
peripheral blood samples should be delivered by hand to Sandra McDonald, M.D. PhD, director 
of the Tissue Procurement Core Facility at the Siteman Cancer Center.  At the Tissue Procurement 
Core Facility, patient identification will be removed from the samples, and the cells will be stored 
as frozen pellets in DMSO at 107 per tube. Subsequently, the samples will be thawed, and cells 
will be assayed. 
 
Peripheral blood sample (3 mL) will be collected on Cycle 1 Day 4 ± 1.  Sample must be obtained 
45 minutes ± 15 minutes after beginning the infusion, before the decitabine infusion is complete, 
and should be obtained from the opposite arm as the infusion.  The sample should be collected in 
a 3 mL K3EDTA tube pre-loaded with 8 mcL of tetrahydrouridine (THU 500 mcg/mL), an 
inhibitor of cytidine deaminase.  The sample should be processed by the Tissue Procurement Core 
Facility at the Siteman Cancer Center.  Serum should be separated and stored per protocols. 
 
 


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9.0 
EFFICACY, SAFETY AND CORRELATIVE STUDY MEASURES 
 
A secondary endpoint of this study is to estimate morphologic complete remission rate. 
  
9.1 
Response Criteria 
 
Patients will be assessed for response according to the IWG criteria:1  
 
Morphologic complete remission (CR) – Defined as morphologic leukemia-free state, 
defined as less than 5% blasts in aspirate with marrow spicules and a count of ≥200 
nucleated cells and no blasts with Auer rods or any persistence of extramedullary disease. 
When erythroid precursors constitute less than 50% of bone marrow nucleated cells, the 
percentage of blasts is based on all nucleated cells; when there are 50% or more erythroid 
cells, the percentage blasts should be based on the nonerythroid cells. The presence of mild 
megaloblastoid changes may be permitted if they are thought to be consistent with 
treatment effect. However, persistence of pretreatment abnormalities (eg, pseudo-Pelger-
Hüet cells, ringed sideroblasts, dysplastic megakaryocytes) are not consistent with CR.  In 
addition, ANC >1000/μl and platelet count of ≥100,000/μl. Patient must be independent of 
transfusions. No duration requirement for this designation. 
 
Cytogenetic complete remission (CCR) – Defined as morphologic complete remission plus 
a reversion to a normal karyotype. For this study, reversion of a normal karyotype is 
defined as no clonal abnormalities detected in a minimum of 20 mitotic cells. 
 
Morphologic complete remission with incomplete blood count recovery (CRi) – Defined as 
morphologic complete recovery with the exception of neutropenia <1000/μl or 
thrombocytopenia <100,000/μl.  
 
Partial remission (PR) – Requires that the above criteria for complete remission be met, 
with the following exceptions: decrease of ≥50% in the percentage of blast to 5-25% in the 
bone marrow aspirate. A value of 5% blasts in bone marrow with Auer rods present may 
also be considered a partial remission.  
 
Treatment failure – Includes all patients who did not achieve a complete or partial 
remission following 4 cycles of therapy.  
 
Recurrence/morphologic relapse - Relapse following complete remission is defined as 
reappearance of blasts in the blood or the finding of ≥5% blasts in the bone marrow, not 
attributable to any other cause. New dysplastic changes are considered a relapse. If there 
are no blasts in the peripheral blood and 5-20% blasts in the bone marrow, bone marrow 
biopsy should be repeated in > 1 week to confirm relapse. 
 
 


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9.2 
Guidelines for Evaluation of Disease 
 
Bone marrow biopsy should be performed at baseline for both the correlative study and 
clinical evaluation. Bone marrow biopsy should be performed on Day 28 ± 4 of cycle 1 
and on Day 28 ± 4 of each even cycle for clinical disease assessment. 
 
Peripheral blood counts will be followed by weekly CBC with differential and platelets.  
After the first cycle, this may be changed to every other week or monthly at the discretion 
of the treating physician. 
 
9.3 
Progressive Disease 
 
Progressive disease is defined as a clear increase in bone marrow blast count, typically a 
greater than 50% increase compared with baseline. Patients with progressive disease will 
be removed from the study. 
 
Patients may also be removed from the study for progressive disease based on evidence of 
new extramedullary disease or based on the clinical judgment of the treating physician. 
 
Patients with a > 50% increase in peripheral blast count with an increase of the total 
peripheral white blood cell count to > 10,000/l should undergo evaluation by bone 
marrow biopsy regardless of the cycle number. 
 
9.4 
Other Secondary Efficacy Measures 
 
9.4.1 Overall Survival and Event-free Survival 
 
Overall survival is defined as the date of first dose of study drug to the date of death 
from any cause.  Patients still alive at the time of the final analysis will be right-
censored. 
 
Event-free survival is defined as the interval from the date of first dose of study 
drug to date of treatment failure, recurrence, death due to any cause, or loss to 
follow up. 
 
9.4.2 Duration of Remission and Relapse-free Survival for Patients Having 
Complete Remission 
 
Duration of complete remission is defined as the interval from the date that 
complete remission is documented to the date of recurrence, death due to any cause, 
or loss to follow-up.  
 
Relapse-free survival is defined only for those patients achieving a complete 
remission. It is defined as the interval from the date of first documentation of a 


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leukemia-free state to the date of recurrence, death due to any cause, or loss to 
follow up.   
  
9.4.3 Safety 
 
Safety evaluation will include assessments of adverse experiences, medical history, 
physical examinations, vital signs, and laboratory assessments at baseline and 
throughout the study period. Local laboratories may be used for this study.  
 


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10.0 
ADVERSE EVENT REPORTING  
 
10.1 
Adverse Events (AEs) 
 
Definition:  any unfavorable medical occurrence in a human subject including any 
abnormal sign, symptom, or disease. 
 
Grading:  the descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for all 
toxicity reporting.  A copy of the CTCAE version 4.0 can be downloaded from the CTEP 
web site:  http://ctep.cancer.gov/reporting/ctc.html. 
 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the terms 
listed that should be used are those provided by the Department of Health and Human 
Services’ Office for Human Research Protections (OHRP).  A copy of this guidance can 
be found on OHRP’s website: (http://www.hhs.gov/ohrp/policy/AdvEvntGuid.htm). 
 
10.2 
Unanticipated Problems 
 
Definition: 
 
 unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document; and (b) the 
characteristics of the subject population being studied; 
 related or possibly related to participation in the research (in this guidance 
document, possibly related means there is a reasonable possibility that the incident, 
experience, or outcome may have been caused by the procedures involved in the 
research); and 
 suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
 
10.3 
Noncompliance 
 
Definition: failure to follow any applicable regulation or institutional policies that govern 
human 
subjects 
research 
or 
failure 
to 
follow 
the 
determinations 
of 
the 
IRB.  Noncompliance may occur due to lack of knowledge or due to deliberate choice to 
ignore regulations, institutional policies, or determinations of the IRB. 
 
10.4 
Serious Noncompliance 
 
Definition: noncompliance that materially increases risks, that results in substantial harm 
to subjects or others, or that materially compromises the rights or welfare of participants. 
 


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10.5 
Protocol Exceptions 
 
Definition: A planned deviation from the approved protocol that are under the research 
team’s control. Exceptions apply only to a single participant or a singular situation. 
 
Pre-approval of all protocol exceptions must be obtained prior to the event. 
 
10.6 
Reporting to the Human Research Protection Office (HRPO) and the Quality 
Assurance and Safety Monitoring Committee (QASMC) at Washington 
University: 
 
The PI is required to promptly notify the IRB of the following events: 
 
 Any unanticipated problems involving risks to participants or others which occur 
at WU, any BJH or SLCH institution, or that impacts participants or the conduct of 
the study. 
 Noncompliance with federal regulations or the requirements or determinations of 
the IRB. 
 Receipt of new information that may impact the willingness of participants to 
participate or continue participation in the research study. 
 
These events must be reported to the IRB within 10 working days of the occurrence of the 
event or notification to the PI of the event.  The death of a research participant that qualifies 
as a reportable event should be reported within 1 working day of the occurrence of the 
event or notification to the PI of the event. 
 
10.7 
Reporting to the FDA 
 
The conduct of the study will comply with all FDA safety reporting requirements.  
PLEASE NOTE THAT REPORTING REQUIREMENTS FOR THE FDA DIFFER 
FROM REPORTING REQUIREMENTS FOR HRPO/QASMC.  It is the 
responsibility of the investigator to report any unanticipated problem to the FDA as 
follows: 
 
 Report any unexpected fatal or life-threatening adverse experiences associated with 
use of the drug (i.e., there is a reasonable possibility that the experience may have 
been caused by the drug) by telephone or fax no later than 7 calendar days after 
initial receipt of the information.  A life-threatening adverse experience is defined 
as any adverse drug experience that places the subject (in the view of the 
investigator) at immediate risk of death from the reaction as it occurred, i.e., it does 
not include a reaction that, had it occurred in a more severe form, might have caused 
death. 
 Report any serious, unexpected adverse experiences, as well as results from animal 
studies that suggest significant clinical risk within 15 calendar days after initial 
receipt of this information.  A serious adverse drug experience is defined as any 


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adverse drug experience occurring at any dose that results in any of the following 
outcomes: 
o Death 
o A life-threatening adverse drug experience 
o Inpatient hospitalization or prolongation of existing hospitalization 
o A persistent or significant disability/incapacity (i.e., a substantial disruption 
of a person’s ability to conduct normal life functions) 
o A congenital anomaly/birth defect 
o Any other experience which, based upon appropriate medical judgment, 
may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed above 
An unexpected adverse drug experience is defined as any adverse drug experience, 
the specificity or severity of which is not consistent with the current investigator 
brochure (or risk information, if an IB is not required or available). 
 
All MedWatch forms will be sent by the investigator or investigator’s team to the FDA at 
the following address or by fax: 
 
Food and Drug Administration 
Center for Drug Evaluation and Research 
Division of Oncology Drug Products 
5901-B Ammendale Rd. 
Beltsville, MD 20705-1266 
FAX: 1-800-FDA-0178 
 
10.8 
Timeframe for Reporting Required Events:  
 
Reportable adverse events will be tracked for 30 days following the last day of study 
treatment. 
 
Deaths 
Any reportable death while on study or within 
30 days of study 
Immediately, within 24 hours, to PI 
and the IRB 
Any reportable death while off study  
Immediately, within 24 hours, to PI 
and the IRB  
Adverse Events/Unanticipated Problems 
Any reportable adverse events as described in 
Sections 10.1 and 10.2 (other than death) and 
10.7 
Immediately, within 24 hours to PI 
and within 10 working days to the 
IRB, and within 7 or 15 days to the 
FDA  
All adverse events regardless of grade and 
attribution should be submitted cumulatively  
Include in DSM report 
Noncompliance and Serious Noncompliance 
All noncompliance and serious noncompliance 
as described in Sections 10.3 and 10.4 
Immediately, within 24 hours, to PI 
and within 10 working days to the IRB 


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11.0 
DATA SAFETY MONITORING PLAN 
 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, the 
Principal Investigator will provide a Data and Safety Monitoring (DSM) report to the Washington 
University Quality Assurance and Safety Monitoring Committee (QASMC) semi-annually 
beginning six months after accrual has opened (if at least five patients have been enrolled) or one 
year after accrual has opened (if fewer than five patients have been enrolled at the six-month mark). 
 
The Principal Investigator will review all patient data at least every six months, and provide a 
semi-annual report to the QASMC. This report will include: 
 HRPO protocol number, protocol title, Principal Investigator name, data coordinator 
name, regulatory coordinator name, and statistician 
 Date of initial HRPO approval, date of most recent consent HRPO approval/revision, 
date of HRPO expiration, date of most recent QA audit, study status, and phase of study 
 History of study including summary of substantive amendments; summary of accrual 
suspensions including start/stop dates and reason; and summary of protocol exceptions, 
error, or breach of confidentiality including start/stop dates and reason 
 Study-wide target accrual and study-wide actual accrual 
 Protocol activation date 
 Average rate of accrual observed in year 1, year 2, and subsequent years 
 Expected accrual end date 
 Objectives of protocol with supporting data and list the number of participants who 
have met each objective 
 Measures of efficacy 
 Early stopping rules with supporting data and list the number of participants who have 
met the early stopping rules 
 Summary of toxicities 
 Abstract submissions/publications 
 Summary of any recent literature that may affect the safety or ethics of the study  
 
The study principal investigator and Research Patient Coordinator will monitor for serious 
toxicities on an ongoing basis. Once the principal investigator or Research Patient Coordinator 
becomes aware of an adverse event, the AE will be reported to the HRPO and QASMC according 
to institutional guidelines. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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38 
12.0 
STATISTICAL ANALYSIS METHODS AND SAMPLE SIZE 
 
12.1 
Analysis Populations 
 
Intent-to-Treat - The intent-to-treat (ITT) population will be defined as all patients who 
receive at least one dose of decitabine.  
 
Efficacy-Evaluable - The efficacy-evaluable (EE) population will be defined as all patients 
who receive at least one cycle of treatment.   
 
Safety - The safety population will be defined as all patients with at least one dose of 
decitabine. 
 
12.2 
Statistical Analysis 
 
12.2.1 Descriptive Analyses 
 
Patient Disposition: The number of patients discontinued, the reasons for 
discontinuation, and the number of cycles administered will be summarized. 
 
Demographics and Baseline Characteristics: Descriptive summary statistics will 
be provided for demographic and important baseline characteristics.  For 
continuous variables, the number of patients, mean, standard deviation, median, 
minimum and maximum will be provided.  For categorical variables, the number 
and percent of patients in each demographic/characteristic category will be 
summarized. 
 
12.2.2 Primary Endpoint Analysis 
 
The primary end-point of this phase II study is the correlation biomarkers with 
overall response in AML/MDS patients to decitabine. 
 
We will correlate the presence of patient specific mutations (especially mutations 
in DNMT3A, IDH1, IDH2, and TET2) with overall response rates. Mutation rates 
in these genes and in other patient-specific genes will be correlated with overall 
response and their respective 95% confidence intervals will be provided. 
 
Statistical considerations. The relationship between AML/MDS-associated 
mutations and the responsiveness to decitabine will be described by contingency 
tables and assessed using Chi-square test or Fisher’s exact test as appropriate. The 
differentially expressed genes between responders and non-responders will be 
determined using the significance analysis of microarray (SAM). Multivariate 
logistic regression will also be fit to adjust the potential confounding effects of other 
demographic and clinical characteristics. The resultant critical P values will be 
corrected for multiple testing.  Since the Bonferroni correction is known too 
conservative, a permutation analysis (e.g., 10,000 random assignments for 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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39 
responsive status) will be used to derive an empirical critical P value.  The new 
critical p-value is the number of permutation tests which yield a lower p-value than 
the original test divided by the total number of permutation tests.[61]   
 
A power analysis was performed using Chi-square test for the association between 
mutation and response to decitabine. The frequency of DNMT3A, IDH1/2, and 
TET2 mutations in AML and MDS patients are each between 15% and 30% in 
multiple cohorts.[29-37] Table 3 shows the minimal detectable differences of 
response rates between mutant and wild-type cases, assuming an expected overall 
45% CR/CRi rate and 80% power. For an average of 20% frequency of a mutated 
gene, for example, 100 patients will provide 80% power to detect 30% difference 
at a 2-sided 0.1 alpha error. The purpose of this study is to determine whether any 
of these mutations result in a 
clinically 
significant 
association 
that 
would 
warrant pre-treatment testing. 
We believe that a reasonable 
cut off would be if at least 
30% of clinical response 
could be attributed to the 
presence 
of 
a 
specific 
mutation. 
However, 
we 
anticipate that more power 
could be achieved as we 
assess patient groups with 
combined 
characteristics 
(e.g. DNMT3A mutation or 
loss of DNMT3A expression). 
In addition, we expect that 
the majority of the enrolled 
125 patients can be used in the analysis (since all of our molecular information will 
be acquired within 30 days of enrollment, we anticipate a drop-out rate of 20% or 
less). 
 
12.2.3 Secondary Endpoint Analyses 
 
The overall response rate (CR/CRi/PR) and complete response rate (CR/CRi) will 
be compared with historical controls[2], and the 95% confidence intervals will be 
provided. 
 
The rate of patient specific mutation clearance after 10, 30, and 60 days of therapy 
will be determined using an Illumina based platform. We will compare the rate of 
mutation clearance between the patients who achieve a CR/CRi after 4 cycles and 
those who do not. We anticipate that we will identify between 6 and 25 exonic 
SNVs per case (the range obtained with >200 AML samples sequenced to date with 
whole genome or exome sequencing), and that the expected 95% confidence 
Table 3. Detectable mutation associated response rates (80% power, α = 0.05) 
 
Total 
N 
% of patients 
with a given 
mutation  
Detectable 
response 
difference 
Sensitivity of mutation to 
predict response at minimum 
detectable response difference* 
100 
15%  
20%  
30% 
40% 
50% 
0.40 
0.35 
0.30 
0.27 
0.26 
80% 
75% 
67% 
61% 
58% 
* Assuming the overall response  rate (CR/Cri) of 45%   


Genomic Predictors of Decitabine Response in AML/MDS 
 
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40 
interval of the mean variant allele frequency will be ±2.56% and ±1.25%, 
respectively, if we assume an anticipated standard deviation of 3.2% within the 
same clone. The change of tumor burden over time and its association with clinical 
response will  be assessed using 2-way ANOVA for repeated measurement data, 
followed by the ad-hoc multiple comparisons for between-group differences (e.g., 
responders vs. non-responders) at given time points as well as the between-time 
differences within each group. The resultant critical P values will also be corrected 
using permutation analysis for multiple testing. 
 
The steady-state serum decitabine concentration on day 4 ± 1 will be measured and 
correlated with clinical overall response. 
 
The percent decrease in peripheral blood methylcytosine will be calculated on day 
10±1. We will compare this reduction with previously published data and between 
the patients who achieve CR/CRi and those who do not. The correlation of peak 
serum levels with response will be assessed using 2-sample t-test or Mann-Whitney 
non-parametric rank-sum test as appropriate. The change of total bone marrow 
DNA methylcytosine from baseline and its association with both steady-state serum 
drug levels and response will be assessed using 2-way ANOVA for repeated 
measurement data, followed by the ad-hoc multiple comparisons for between-group 
differences (e.g., responders vs. non-responders) and between-time differences. 
Similar analyses will be performed for the correlation of expression profiles and 
SNP analysis with pharmacokinetic data.   
 
Statistical power justification.  A power analysis was performed using two-sample 
t-test for between-group difference and paired t-test for the change over time. For 
tumor clearance, this will provide > 99% power to detect a 10% difference in the 
means at two separate time points for clones with 6 or more variants, and > 98% 
power to detect a 15% difference if there are 3 or more variants per clone with α = 
0.05 (power will decrease to 91% and 82%, respectively, in cases where the 
standard deviation is 4.7%, the maximum we have observed thus far). For 
pharmacologic correlations, we anticipate 85% power to determine a 25 ng/ml 
difference between the average Cmax of 45 evaluable responders and 55 evaluable 
non-responders with an anticipated average Cmax of 70 ng/ml and a standard 
deviation of 40 ng/ml within the entire cohort, and with α = 0.05.[22] We believe a 
less significant difference would not be sufficient to warrant application in clinical 
decision making (i.e. whether to continue therapy for more than 1 cycle). We 
anticipate 99% power to determine a difference of 20% in the reduction of bone 
marrow methylcytosine between 45 evaluable responders and 55 evaluable non-
responders with an expected standard deviation of 15% and α = 0.05.[28, 66-68] 
The difference in methylcytosine reduction previously observed was 30% between 
patients achieving CR/PR and those with only hematologic improvement.[28]  
 
Time to relapse and time to death will also be listed for each patient. 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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12.2.4 Safety Analysis 
 
Extent of Exposure – Duration (number of cycles/infusions) of decitabine doses 
(cumulative and intensity) will be summarized. 
 
Adverse Events - All adverse events will be coded using NCI CTCAE v. 4.0.  The 
incidence of treatment-emergent adverse events (number and percent of patients 
reporting the adverse event at least once during the study) will be summarized.  In 
addition, adverse events will be summarized by investigator attribution of 
relationship to study medication and by grade.  Similar summaries will be provided 
by treatment cycle. 
 
Clinical Laboratory Evaluation – Applicable laboratory parameters will be graded 
according to NCI CTCAE version 4.0. The incidence of maximum grade (number 
and percent of patients experiencing the maximum grade during the study) will be 
summarized for each laboratory parameter.  Similar summaries will be provided by 
treatment cycle.  Graphical displays of select hematological parameters will also be 
provided. 
 
Vital Signs, Physical Findings, and Other Safety Observations – Vital signs, 
findings from physical examinations, ECOG scores and weights will be presented 
in listing format. 
 
12.2.5 Description of Planned Subgroup Analyses 
 
The following subgroups (if sufficient numbers of patients) will be explored with 
respect to the primary and overall survival endpoints. 
 Age (60-69 years old vs ≥ 70 years old) 
 Cytogenetics 
 Patients with AML-associated mutations in DNMT3A, TET2, IDH1, or 
IDH2 vs those who do not have somatic variants in these genes. 
 
12.3 
Sample Size 
 
We anticipate initially enrolling 25 patients at Washington University. Once we establish 
a routine exome analysis with validation and calculation of the rate of patient-specific 
mutation clearance, we will enroll an additional 100 patients from at least 2 additional 
centers.  
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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13.0 
DATA SUBMISSION SCHEDULE 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to registration 
Patient Registration Form 
Disease Diagnosis Form 
Inclusion Criteria Worksheet 
Exclusion Criteria Worksheet 
 
Prior to starting treatment 
Treatment Record Form 
To be maintained throughout a patient’s treatment 
cycle.  The complete record is due at completion of 
treatment. 
Bone Marrow Sample Collection 
Form 
To be maintained throughout the course of the 
study at designated bone marrow sample 
collections time points (See Appendix 2). 
Pathology: Bone Marrow Biopsy 
To be completed upon the return of pathology 
report for each bone marrow biopsy. 
Study Calendar Form 
To be completed with the date of each time point 
upon occurrence. 
Labs Form 
To be completed upon the return of laboratory 
reports for each study time point (See Appendix 2) 
Dose Modifications 
To be maintained throughout the course of the 
study if dose modifications are performed. 
Therapy Response Form 
To be filled out upon remission (prior to, and/or on 
therapy) 
End of Study Form 
 
Due at the completion of the EOS visit, at the time 
of death, and/or at the time that the patient is lost to 
follow-up 
Follow Up Form 
 6 weeks post date of last decitabine dose. 
 Every 2 months for 2 years after the first dose of 
study drug. 
Record of Adverse Events 
At the time of any AE 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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43 
Appendix 1 
ECOG Performance Scale 
Activity Status 
Description 
0 
Asymptomatic, fully active, and able to carry on all predisease 
performance without restrictions. 
 
1 
Symptomatic, fully ambulatory but restricted in physically strenuous 
activity and able to carry out performance of a light or sedentary 
nature, eg, light housework, office work. 
 
2 
Symptomatic, ambulatory and capable of all self-care but unable to 
carry out any work activities.  Up and about more than 50% of 
waking hours: in bed less than 50% of day. 
 
3 
Symptomatic, capable of only limited self-care, confined to bed or 
chair more than 50% of waking hours, but not bedridden. 
 
4 
Completely disabled.  Cannot carry on any self-care.   Totally 
bedridden. 
 
5 
Dead 
 
 


Genomic Predictors of Decitabine Response in AML/MDS 
 
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Appendix 2 
Study Flow Chart 
Study Flow Chart 
Test 
Pre-
study 
Base-
line 
All 
Cycles 
Day 1 
All Cycles 
Cohort A 
Days 1-10a, b 
Cycle 1 day 
4 ± 1 
 
Cycle 1 
Day 10 ± 1 
and 28 ± 4 
Every 
week 
Every 
2 
weeks 
Even 
cycles 
Day 28±4 
End of 
Study 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Medical History 
 
X 
X 
 
 
 
 
 
 
 
Physical Exam 
 
X 
X 
 
 
 
 
 
 
X 
Concomitant 
Medications 
 
X 
 
 
 
 
 
 
 
 
Transfusion 
Requirementsc 
 
Xc 
 
 
Bone Marrow 
Aspirate and Biopsy 
Xd,e 
 
 
 
 
Xd 
 
 
Xd 
Xd 
Peripheral blood for 
correlative studies 
 
 
 
 
X 
 
 
 
 
 
Pregnancy test 
 
X 
 
 
 
 
 
 
 
 
CBC with differential 
and platelets 
 
X 
X 
 
 
 
Xf 
 
 
X 
Serum Chemistriesg 
 
X 
X 
 
 
 
 
Xf 
 
X 
Decitabine 
administrationa,b,h 
 
 
 
X 
 
 
 
 
 
 
Adverse Events  
 
 
 
Ongoing 
Post-study follow-up 
 
 
 
 
 
 
 
 
 
Xi 
a Once bone marrow blast count is ≤ 5% (measured by cytomorphology, immunophenotype, and cytogenetics), the 
number of days per cycle of decitabine may be decreased to 5 per treating physician discretion. 
b Once bone marrow blast count is ≤ 5% for two or more cycles and the peripheral blood counts have normalized, the 
number of days per cycle of decitabine may be decreased to 3 per treating physician discretion. 
c Care should be given to record the frequency of transfusions from 8 weeks prior to first dose of study drug. 
d Standard bone marrow procurement procedures will be followed for collection of the tissue. Prestudy samples should 
be collected within 14 days of first dose of study drug.  
e The bone marrow biopsy for correlative studies may be collected with the prestudy evaluation. If not, it should be 
done as part of the base-line evaluation 
f Once stable, these labs may be obtained every other week or once a month at the discretion of the treating physician. 
g Serum chemistries include bicarbonate, calcium, glucose, total protein, albumin, BUN, potassium, chloride, sodium, 
creatinine, total bilirubin, AST, ALT, alkaline phosphatase, and LDH. 
h Decitabine treatment will be 20 mg/m2 over a 1-hour IV infusion on consecutive Days 1-10 every 28-day cycle.   The 
dose will be calculated based on actual patient weight at the beginning of each cycle. 
i Patients will be followed for 6 weeks following the date of the last decitabine dose to monitor for toxicity, survival 
and response. Thereafter, the investigator or designee will record information on subsequent therapy, disease 
progression, and death, every 2 months for 2 years after first dose of study drug or until the patient is lost to follow-up.


Genomic Predictors of Decitabine Response in AML/MDS 
 
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45 
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