BLockade of PD-1 Added to Standard Therapy to Target Measurable Residual Disease in Acute Myeloid Leukemia 1 (BLAST MRD AML-1): A Randomized Phase 2 Study of the Anti-PD-1 Antibody Pembrolizumab in Combination With Conventional Intensive Chemotherapy as Frontline Therapy in Patients With Acute Myeloid Leukemia
Phase: PHASE2
Conditions: Acute Myeloid Leukemia, Acute Myeloid Leukemia Arising From Previous Myelodysplastic Syndrome, Acute Myeloid Leukemia Post Cytotoxic Therapy, Secondary Acute Myeloid Leukemia
Sponsor: National Cancer Institute (NCI)

NCI Protocol #: 10300  
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i 
 
 
SUMMARY OF CHANGES – Protocol 
 
For Protocol Revision 20 to: BLockade of PD-1 Added to Standard Therapy to target 
Measurable Residual Disease in Acute Myeloid Leukemia 1 (BLAST MRD AML-1): A 
Randomized Phase 2 Study of the Anti-PD-1 Antibody Pembrolizumab in Combination with 
Conventional Intensive Chemotherapy as Frontline Therapy in Patients with Acute Myeloid 
Leukemia 
 
NCI Protocol #: 10300 
Local Protocol #: 2000028858 
 
NCI Version Date: 
04/10/2025 Protocol 
Date: 04/10/2025 
 
 
I. 
Update by Principal Investigator  
 
Section 
Comments 
All 
Updated Version Date in Header 
 
Title Page 
Revised Version# / Version Date  
5.8 
Updates to the CIMAC biomarker plan: 
 
Added the following to the Biomarker Plan table: 
• Corrected “end of treatment” to “relapse” for blood-based CIMAC assays 
• Added relapse timepoint to WES in bone marrow aspirate 
• Added TCRseq in peripheral blood 
• Updated TCRseq lab as Adaptive Biotechnologies working with MDACC 
CIMAC 
• Upated MDACC CIMAC Lab Co-PI’s. 
6.0 
Updated shipping addresses and lab contacts. 
Noted EDTA blood can also be used for TCRseq. 
 
 
 

 
 
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NCI Protocol #: 
10300 
 
Local Protocol #: 
2000028858 
 
ClinicalTrials.gov Identifier: NCT04214249 
 
TITLE: Blockade of PD-1 Added to Standard Therapy to target Measurable Residual Disease 
in Acute Myeloid Leukemia 1 (BLAST MRD AML-1): A Randomized Phase 2 Study of the Anti- 
PD-1 Antibody Pembrolizumab in Combination with Conventional Intensive Chemotherapy as 
Frontline Therapy in Patients with Acute Myeloid Leukemia 
 
Corresponding Organization: LAO-CT018 / Yale University Cancer Center LAO 
 
Principal Investigator: 
Amer Zeidan, M.B.B.S., M.H.S. 
Yale University 
333 Cedar Street, P.O. Box 208028 
New Haven, CT 06520-8028 
Telephone: 203-737-7078 
Fax (optional): 203-785-
7232 
amer.zeidan@yale.edu 
 
Co-Principal Investigator: 
Rory Shallis, M.D. 
Yale University 
333 Cedar Street, P.O. Box 208028 
New Haven, CT 06520-8028 
Telephone: 203-785-6074 
Fax: 203-785-7232 
rory.shallis@yale.edu 
 
                 Co-Principal Investigator:         Jan Bewersdorf, M.D. 
Yale University 
333 Cedar Street, P.O. Box 208028 
New Haven, CT  06520-8028 
Telephone: 203-785-4144 
Fax: 203-737-3401 
Jan.bewersdorf@yale.edu                                                        
 
Co-Laboratory Principal Investigator: 
Brent Wood, MD, PhD 
Children’s Hospital Los Angeles 
University of Southern California 
bwood@chla.usc.edu 
Telephone: 323-361-7462 
 
Co-Laboratory Principal Investigator: 
Jerald Radich, MD 

 
 
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Fred Hutchinson Cancer Research Center 
University of Washington 
jradich@fhcrc.org 
Telephone: 206-667-4118 
Fax: 206-667-6523 
 
Participating Organizations 
 
LAO-11030 / University Health Network Princess Margaret Cancer Center LAO 
 
LAO-CA043 / City of Hope Comprehensive Cancer Center LAO 
LAO-CT018 / Yale University Cancer Center LAO 
LAO-MA036 / Dana-Farber – Harvard Cancer Center LAO 
LAO-MD017 / JHU Sidney Kimmel Comprehensive Cancer Center LAO 
LAO-OH007 / Ohio State University Comprehensive Cancer Center LAO 
LAO-PA015 / University of Pittsburgh Cancer Institute LAO 
LAO-TX035 / University of Texas MD Anderson Cancer Center LAO 
LAO-NCI / National Cancer Institute LAO 
 
Statistician: 
Study Coordinator: 
Ondrej Blaha  
Anne Caldwell, RN, BSN, OCN 
300 George Street, Suite 511                                           Yale University 
 
New Haven, CT 06511                                                     37 College Street 
 New Haven, CT 06520-8028 
Telephone: 203-737-5906 
 Email: anne.caldwell@yale.edu 
Fax: 203-737-8880 
 
Email: Ondrej.blaha@yale.edu 
 
 
 
Responsible Research Nurse: 
Responsible Data Manager: 
Anne Caldwell, RN, BSN, OCN 
Anne Caldwell, RN, BSN, OCN  
333 Cedar Street, P.O.Box 208028 
333 Cedar Street, P.O. Box  New 
Haven CT, 06520-8028 
New Haven CT, 06520-8028 
anne.caldwell@yale.edu 
anne.caldwell@yale.edu 
 
NCI-Supplied Agent: Pembrolizumab (MK-3475) (NSC # 776864) 
Other Agents: Cytarabine (NSC # 63878) and either Idarubicin (NSC # 256439) or 
Daunorubicin (NSC # 82151) 
 
IND #: 
 
IND Sponsor: DCTD, NCI 
 
Protocol Type / Version # / Version Date: 
Original / April 16, 2019 
Revision 1 / June 20, 2019 
Revision 2 / July 23, 2019 

 
 
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Revision 3 / December 3, 2019 
Revision 4a / January 22, 2020 
Revision 5 / February 6, 2020 
Revision 6a / February 27, 2020 
Revision 6 / March 23, 2020 
Revision 7 / April 17, 2020 
Revision 8 / May 11, 2020 
Revision 9 / July 22, 2020 
Revision 10 / September 5, 2020 – Disapproved 
Revision 11 / October 24, 2020 
Revision 12 / October 11, 2021 
Revision 13 / October 18, 2022 
Revision 14 / February 8, 2023 
Revision 15 / April 11, 2023 
Revision 16 / September 18, 2023  
Revision 17 / May 28, 2024 
Revision 18 / October 23, 2024  
Revision 19 / November 26, 2024 
Revision 20 / April 10, 2025  
 

 
 
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TABLE OF CONTENTS 
 
1. 
OBJECTIVES ............................................................................................................................. 9 
1.1 
Primary Objectives .......................................................................................................... 9 
1.2 
Secondary Objectives ...................................................................................................... 9 
1.3 
Exploratory Objectives .................................................................................................... 9 
2. 
BACKGROUND ....................................................................................................................... 10 
2.1 
Study Disease ................................................................................................................ 10 
2.2 
CTEP IND Agent .......................................................................................................... 15 
2.3 
Other Agent(s) ............................................................................................................... 16 
2.4 
Rationale ....................................................................................................................... 18 
2.5 
Correlative Studies Background ................................................................................... 20 
3. 
PATIENT SELECTION ............................................................................................................ 27 
3.1 
Eligibility Criteria ......................................................................................................... 27 
3.2 
Exclusion Criteria .......................................................................................................... 29 
3.3 
Inclusion of Women and Minorities .............................................................................. 32 
4. 
REGISTRATION PROCEDURES ........................................................................................... 32 
4.1 
Investigator and Research Associate Registration with CTEP ...................................... 32 
4.2 
Site Registration ............................................................................................................ 33 
4.3 
Patient Registration ....................................................................................................... 36 
4.4 
General Guidelines ........................................................................................................ 38 
5 
BIOMARKER, CORRELATIVE, AND SPECIALSTUDIES ................................................ 38 
5.1 
Summary Table for Specimen Collection ....................................................................... 38 
5.2 
Specimen Procurement Kits and Scheduling ................................................................. 41 
5.3 
Specimen Tracking System Instructions ....................................................................... 42 
5.4 
Specimen Collection and Processing ............................................................................ 46 
5.5 
Shipping Specimens from Clinical Site to the EET Biobank ......................................... 50 
5.6 
Shipping of Specimens from Clinical Site to Brent Wood’s Lab, Department of 
Pathology and Laboratory, Children’s Hospital Los Angeles ...................................... 52 
5.8  Biomarker Plan ................................................................................................................... 55 
5.9 
Integral Laboratory Studies ........................................................................................... 62 
6.0 
Exploratory/Ancillary Correlative Studies .................................................................... 62 
7. TREATMENT PLAN ....................................................................................................................... 68 
7.1 
Agent Administration .................................................................................................... 68 
7.2 
General Concomitant Medication and Supportive Care Guidelines ............................... 74 
7.3 
Duration of Therapy ...................................................................................................... 78 
7.4 
Duration of Follow-Up .................................................................................................. 79 
7.5 
Criteria to Resume Treatment ....................................................................................... 80 
7.6 
Contraception and Pregnancy ........................................................................................ 80 
7.7 
Treatment Up to 2 Years ................................................................................................ 82 

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8 
DOSING DELAYS/DOSE MODIFICATIONS ....................................................................... 82 
8.1 
Pembrolizumab Dose Modification and Supportive Care Guidelines for Drug-Related 
Adverse Events (revised 1-10-2023) ............................................................................ 83 
Dose Modification and Toxicity Management Guidelines for Immune-related AEs and 
Infusion Reactions Associated with Pembrolizumab ................................................... 84 
8.2 
Commercial Agents ....................................................................................................... 94 
9 
PHARMACEUTICAL INFORMATION ................................................................................. 94 
9.1 
CTEP IND Agent .......................................................................................................... 95 
9.2 
Commercial Agents ....................................................................................................... 98 
10 
STATISTICAL CONSIDERATIONS .................................................................................... 102 
10.1 Study Design/Endpoints .................................................................................................. 102 
10.2 
Sample Size/Accrual Rate ............................................................................................ 104 
10.3  Stratification Factors ..................................................................................................... 105 
10.4 
Analysis of Secondary and Exploratory Endpoints ..................................................... 105 
Exploratory outcomes: ............................................................................................................ 111 
Part 1: Assess whether baseline biomarkers or the serial measurements of biomarkers are 
associated with response outcomes. ............................................................................ 112 
Part 2: Assess whether baseline biomarkers or the serial measurements of biomarkers are 
associated with survival outcomes. ............................................................................. 112 
Part 3: Compare the biomarkers effects between treatment arms, intensive chemo + pembro vs. 
intensive chemo only. .................................................................................................. 112 
Part 4: Evaluate the correlations between biomarkers. ........................................................... 112 
10.5 
For phase 2 protocols only: Reporting and Exclusions ............................................... 112 
10.6 Data Safety Monitoring Board ........................................................................................ 113 
11 
ADVERSE EVENTS: LIST AND REPORTINGREQUIREMENTS .................................... 114 
11.1 
Comprehensive Adverse Events and Potential Risks List (CAEPR) .......................... 114 
11.2 
Adverse Event Characteristics .................................................................................... 121 
11.3 
Expedited Adverse Event Reporting .......................................................................... 121 
11.4 
Routine Adverse Event Reporting .............................................................................. 124 
11.5 
Pregnancy .................................................................................................................... 125 
11.6 
Secondary Malignancy ................................................................................................ 125 
11.7 
Second Malignancy ..................................................................................................... 125 
12. STUDY CALENDAR ................................................................................................................... 126 
13            MEASUREMENT OF EFFECT ......................................................................................... 129 
13.1 
Definitions ................................................................................................................... 129 
13.2 
Hematologic Response ................................................................................................ 129 
13.3 
Other Response Parameters ......................................................................................... 131 
14 
STUDY OVERSIGHT AND DATA REPORTING / REGULATORY REQUIREMENTS . 131 
14.1 
Study Oversight ........................................................................................................... 131 
14.2 
Data Reporting ............................................................................................................ 131 
14.3 Data Quality Portal ......................................................................................................... 134 

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14.4 
Collaborative Agreements Language ......................................................................... 134 
14.5 
Genomic Data Sharing Plan ........................................................................................ 136 
14.6 
Incidental/Secondary Findings Disclosure Procedure ................................................. 136 
15 
REFERENCES ........................................................................................................................ 137 
APPENDIX A PERFORMANCE STATUS CRITERIA ................................................................. 145 
APPENDIX B FORMULA TO ESTIMATE RENAL FUNCTION USING SERUM CREATININE
 
146 
APPENDIX C PATIENT CLINICAL TRIAL WALLET CARD ................................................... 147 
APPENDIX D RESPONSE CRITERIA (PER ELN 2017 RECOMMENDATIONS) .................... 148 
Response criteria in AML Response ....................................................................................... 148 
APPENDIX E BONE MARROW CORE BIOPSY SAMPLES ....................................................... 150 
APPENDIX F SKIN PUNCH BIOPSY VERIFICATION FORM ............................................ 151 

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1. 
OBJECTIVES 
 
1.1 
Primary Objectives 
 
1.1.1 To assess the percentage of patients with minimal residual disease (MRD) negative 
complete remission (CR) (MRD-CR), or with minimal residual disease (MRD) 
negative complete remission with incomplete recovery (CRi) (MRD-CRi) as 
measured by flow cytometry at the end of first cycle of consolidation therapy with 
chemotherapy + Pembrolizumab (MK-3475) and compare between the two study 
arms. 
 
1.2 
Secondary Objectives 
 
1.2.1 Assess the rate of complete remission (CR) / complete remission with incomplete count 
recovery (CRi) as defined per European Leukemia Net 2017 response criteria at time of 
count recovery after induction therapy with chemotherapy + Pembrolizumab (MK-3475) 
(Dohner et al., 2017). 
 
1.2.2 Rates of complete remission with partial recovery count (CRh) and Hematologic 
improvement (HI) to red blood cells and platelets 
 
 
1.2.3 Assess the rates of MRD negativity at Day 14, MRD-negative CR at end of induction 
therapy and MRD negative CR after last consolidation cycle. 
 
 
1.2.4 Assess event free survival (EFS), measured from randomization to failure to achieve 
CR/CRi, relapse or death from any cause, and relapse free survival (RFS), calculated as 
the time from first documentation of CR/CRi to either disease relapse or death from any 
cause. 
 
 
1.2.5 Assess the duration of response (DOR, defined as the time from first CR/CRi to the date 
of the first documented relapse or death, whichever occurs first) and overall survival (OS), 
defined as time from randomization to death from any cause. 
 
 
1.2.6 Assess safety endpoints including proportion of patients who develop severe toxicity as 
defined in the protocol. 
 
 
1.3 
Exploratory Objectives 
 
1.3.1 MRD assessment by duplex sequencing (DS) and comparing DS and multiparameter 
flow cytometry for MRD detection as an exploratory biomarker. 
 
1.3.2 Assessment of immune-checkpoint expression and dynamic change of immune cell 
subsets in response to the combination of checkpoint-inhibition and backbone 
combination in AML. 

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1.3.3 High-throughput sequencing of the TCR Vb CDR3 regions on ﬂow cytometrically sorted t- 
cell subsets to assess the effect of immunotherapy on the diversity of the t-cell repertoire and 
assess for correlation to clinical outcomes. 
 
1.3.4 
Investigation of protein signatures and RNA signatures associated with response and 
efficacyusing O-link cytokine panel and RNA-seq, respectively 
 
1.3.5 Determination of mutational load by whole exome sequencing to assess for correlation with 
clinical outcomes, immune infiltrating profile, and T cell repertoire diversity and clonality. 
 
1.3.6 Correlate gut microbiome at baseline and changes in the microbiome with clinical response, 
both in standard chemotherapy and immunotherapy/chemotherapy therapy settings. 
 
 
1.3.7 MRD assessment using duplex sequencing strategy for circulating cell-free tumor DNA and 
correlation with long-term outcomes. 
 
 
2. 
BACKGROUND 
 
2.1 
Study Disease 
 
2.1.1 Acute myeloid leukemia (AML) and minimal residual disease (MRD) 
 
2.1.1.1 
AML remains a disease with a significant unmet clinic need 
 
Acute myeloid leukemia (AML) is a disease of myeloid precursors characterized by 
accumulation of immature myeloid progenitors, cells which lack the ability to differentiate, in 
the bone marrow interfering with normal hematopoiesis and leading to decreased production of 
normal blood cells (Dohner et al., 2015). AML is a disease of older adults with the median age 
at diagnosis is 67 years and one-third of patients are older than 75 years at time of diagnosis 
(Podoltsev et al., 2016). With the increasing population longevity, the number of newly 
diagnosed patients is increasing. In 2015, 20,830 patients were diagnosed with AML in the US, 
and more than 10,000 died from the disease. In patients who are younger than 60 years, the CR 
rates are 60% to 70% but the cure rates are only 35% to 40% (Dohner et al., 2010). However, 
older patients and those with adverse karyotypes have CR rates of 35% to 50% and cure rates of 
10% or less. AML, especially in older patients, is characterized by high rates of chemotherapy 
resistance and therefore novel therapeutic approaches are urgently needed. Despite an explosion 
in understanding of the biology of AML and recent approval of midostaurin and gemtuzumab, the 
general therapeutic strategy for non-acute promyelocytic leukemia (APL) fit patients with AML 
remains intensive induction and consolidation chemotherapy and has not substantially changed in 
the last 40 years (Yates et al., 1973; Dohner et al., 2010; Kadia et al., 2016). 
 
2.1.1.2 
Importance of measurable (also known as minimal) residual disease (MRD) in AML 
 
Adult AML patients who do not have morphologic evidence of less than 5% of blasts on marrow 

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examination after blood count recovery from induction chemotherapy are deemed to have 
attained a morphologic CR (Creutzig and Kaspers, 2004). However, without further therapy, the 
disease will recur in all patients and post-remission consolidation therapy remains a necessity 
demonstrating that residual leukemic cells remain in the bone marrow even after CR (Cassileth et 
al., 1988). The type of post-remission therapy is currently based on cytogenetic and molecular 
risk profiles (Dohner et al., 2010). Given that this risk assessment remains imperfect, there have 
been growing evidence regarding the value of achieving deeper responses and their assessment 
beyond morphologic assessment with the use of more sensitive laboratory measures 
(Paietta,2012). This state, referred to as MRD, can be detected by multicolor flow cytometry 
(MFC) as well as genetic testing (e.g. polymerase chain reaction [PCR] or next-generation 
sequencing [NGS]) in cases where acquired genetic mutations or translocations were present at 
diagnosis. 
Monitoring response with MRD has been proven to be very useful and is now considered a 
standard practice in acute lymphoblastic leukemia (ALL), APL and chronic myeloid leukemia 
(CML), although its optimal use for therapeutic decisions in AML remains unknown (Santamaria 
et al., 2007; Hughes et al., 2010; Borowitz et al., 2015). Nevertheless, monitoring of MRD in 
AML with MFC is gaining popularity as there is clear evidence it refines risk assessment, and 
allows for long term monitoring for detection of early relapse (Hourigan et al., 2017). In a study 
by the AML Cooperative Group, achievement of negative MRD by MFC during aplasia after 
induction was highly predictive for 5-year outcomes (Kohnke et al., 2015). In two large 
prospective studies, the AML16 trial in the United Kingdom, and the HOVON SAKK AML 42 
trial, undetectable MRD by MFC after 1 or 2 cycles of induction was significantly predictive for 
relapse risk and OS independent of other risk factors (Freeman et al., 2013; Terwijn et al., 2013). 
Although rates of MRD-negative CR achieved with induction therapy varies based on patient 
population studies and MRD detection assay used, in the two later clinical trials the percentage 
of AML patients with MRD-negative CR was only around 50%. Though some patients who 
achieve MRD-negative CR still relapse, and some patients who have detectable MRD after 
induction therapy can subsequently become MRD-negative and achieve long-term survival, 
MRD negativity after induction chemotherapy is associated with better outcomes. The optimal 
cutoff to define MRD, the technology used for detection, the optimal timing for assessment (after 
induction or consolidation), and the incorporation in clinical decision making all remain 
undefined and are being studied in prospective clinical trials. 
 
2.1.2 Immune Checkpoints and Immune Checkpoint Inhibition in AML 
 
2.1.2.1 
Role of the immune system activation in AML 
 
The lower relapse rate associated with allogeneic hematopoietic stem cell transplantation (HSCT) 
and the ability of donor lymphocyte infusion (DLI) to rescue some patients who have relapsed 
following HSCT both demonstrate the role the immune system plays as the most potent anti- 
leukemia therapy in AML (Bleakley and Riddell, 2004; Kolb, 2008). Allo-reactive T-cells confer a 
strong graft-versus-leukemia (GVL) effect, however, HSCT continues to be associated with poor 
long-term survival with 3-year OS of only 41% and DFS of 37% (Tauro et al., 2005). Furthermore, 
HSCT is associated with significant morbidity and mortality and therefore many AML patients are 
not eligible for the procedure. Additionally, early results from investigational vaccine approaches in 
AML targeting tumor-associated peptide antigens such as WT-1 and PR1 further support the 
potential therapeutic benefit of recruiting the endogenous immune system (Chaise et al., 2008; 
Rezvani et al., 2008). 

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2.1.2.2 
The role of the PD-1/PD-L1 pathway in AML 
 
Programmed death receptor-1 (PD-1) is a molecule expressed on activated T-cells and delivers 
downstream signaling that causes inhibition of T-cell proliferation, cytokine release, and 
cytotoxicity (Okazaki et al., 2013). Recent data showed that multiple types of tumor are capable 
of evading the immune system by expressing PD-1 ligand (PD-L1), which engages the PD-1 
receptor on the surface of cytotoxic T-cell leading to suppression of their activity (Pardoll, 2012). 
Immune checkpoint blockade with anti-PD-1 and anti-PD-L1 antibodies (and anti-cytotoxic T 
lymphocyte-associated protein 4 (CTLA-4) antibodies) has emerged as a novel promising 
approach to reverse this phenomenon (Postow et al., 2015). Immune checkpoint inhibition 
resulted in a significant and durable clinical activity in some patients with advanced solid 
malignancies including metastatic melanoma, non-small-cell lung cancer (NSCLC) and bladder 
cancer (Robert et al., 2014; Borghaei et al., 2015; Sharma et al., 2016) as well as in patients with 
Hodgkin’s lymphoma (Ansell et al., 2015). Several lines of evidence suggest that PD-1/PD-L1 
pathway is also of vital importance in AML. 
 
2.1.2.2.1 
Preclinical data: Immune checkpoint expression and inhibition in murine AML models 
 
The importance of the PD-1/PD-L1 pathway in immune evasion of AML has been demonstrated 
in several murine AML models whereby PD-L1 and PD-1 were upregulated in murine leukemia 
cells and PD-1 blockade suppresses in vivo leukemia cell proliferation and improved survival in 
AML bearing mice. (Zhang et al., 2009; Zhou et al., 2010). First, Zhang et al examined the 
expression of PD-L1 in the murine AML model C1498 (Zhang et al., 2009). The C1498 cell line 
was originally derived from a C57BL/6 mouse and most closely resembles AML. When murine 
C1498 AML cells were injected into syngenic C57BL/6 recipient mice intravenously (IV), 
leukemia cells grew progressively and evaded immune destruction. Low levels of PD-L1 
expression were found on C1498 cells grown in vitro. However, PD-L1 expression was up- 
regulated on C1498 cells after a 48-hour incubation with interferon-gamma (IFN-γ) or when 
grown in vivo suggesting that the cytokine milieu within the leukemia “microenvironment” was 
capable of stimulating PD-L1 up-regulation. PD-1 knock out mice (PD-1−/−) challenged with 
C1498 AML cells resulted in augmented antitumor T-cell responses measured by IFN-γ ELISpot 
assays performed from splenocytes isolated from wild-type and PD-1−/− mice 12 days after 
challenge with C1498 cells. This improved immune response resulted in a decreased AML 
burden in the blood and other organs of PD-1−/− mice and significantly longer survival compared 
to wild-type mice. Importantly, similar results were obtained with a PD-L1 blocking antibody, 
which decreased tumor burden, augmented an anti-leukemia immune response and prolonged 
survival of mice injected with C1498 AML cells. Second, Zhou et al used the same C1498 
murine AML model focusing on the role of PD-1/PD-L1 interaction in regulatory T-cell (Treg)- 
mediated immune suppression of adoptively transferred CTLs (Zhou et al., 2010). They showed 
that PD-1 is upregulated on endogenous CD8+ T-cells in the liver of AML-bearing mice and, 
consistent with Zhang’s study, that the percentage of IFN-γ–producing cells is significantly 
decreased in the PD-1-expressing CD8+ T-cell fraction compared with PD-1− fraction. AML 
progression caused significant accumulation of CD4+25+FoxP3+ Tregs in the liver of wild-type 
(WT) mice, whereas PD-1−/− mice had a constitutively elevated level of Tregs, and this was not 
altered by the presence of tumor. Tregs from PD-1−/− mice were unable to suppress CD8+ T-cell 
proliferation or IFN-γ secretion. Similarly, selective PD-1/PD-L1 blockade completely 
abrogated the ability of WT Tregs to suppress CD8+ T-cell proliferation IFN-γ production. These 

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data indicated that both enhanced CD8+ T-cell response as a result of diminished suppressive 
function of PD-1−/− Tregs coupled with augmented PD-1−/− CD8+ T-cells contribute to the 
resistance of tumor growth in PD-1−/− mice. Furthermore, they showed that anti–PD-L1 
monoclonal antibody (mAb) administration enhances the efficacy of adoptive CTL therapy: 
while CTL therapy alone was ineffective, and anti–PD-L1 mAb treatment alone modestly 
prolonged the survival of but did not rescue AML-bearing mice, combined CTL and anti–PD-L1 
mAb therapy in mice with advanced AML had an additive effect over either therapy alone and 
importantly resulted in 20% long-term survival. 
2.1.2.2.2 
Immune checkpoint expression in human AML samples 
 
The expression of immune checkpoint seems to be only modestly elevated on AML cells at baseline 
but increases significantly, once AML cells are exposed to IFN-γ or chemotherapy and epigenetic 
therapy, as well as at the time of relapse either after chemotherapy or after bone marrow transplant 
(Kronig et al., 2014; Sehgal et al., 2015). Kroenig et al demonstrated, in a cohort of 154 patients 
with AML, no significant increase in surface PD-L1 expression on leukemia cells at initial 
diagnosis compared to healthy controls (Kronig et al., 2014). However, stimulation with IFN-γ 
significantly increased PD-L1 expression on AML blasts but not in normal controls. Highest 
upregulation of PD‐L1 upon IFN‐γ as measured was observed in patients with CR and with 
relapse whereas only slight upregulation upon IFN‐γ was found in newly diagnosed patients. Yang 
et al showed that immune checkpoint expression was only modestly elevated on AML CD34+ cells 
at the time of diagnosis, however, was comparably higher when peripheral blood mononuclear cells 
(PBMCs) increased significantly once patients were treated with hypomethylating agents (Yang et 
al., 2014). Looking at the mRNA levels in CD34+ cells from 32 patients with AML, they found that 
15% had aberrant up-regulation (≥2- fold) in PD-L1, PD-1, and CTLA4, respectively. However, 
expression of PD-L1, PD-L2, PD-1, and CTLA4 in PBMCs from AML patients was significantly 
higher. Upregulation (≥2-fold) of PD-L1, PD-L2, PD-1, and CTLA4 was observed in 32%, 12.9%, 
39%, and 3% of AML samples, respectively. Furthermore, treatment of leukemia cells with 
decitabine resulted in a dose- dependent upregulation of the above genes and patients resistant to 
therapy had relatively higher increments in gene expression compared with patients who achieved 
response. Exposure to decitabine resulted in partial demethylation of PD-1 in leukemia cell lines and 
human samples. 
Another small study identified PD‐L1 to be preferentially expressed in AML‐M5 and at higher 
surface levels during relapse compared to that at first diagnosis in patients (Chen et al., 2008). 
Norde et al found that in patients who relapsed late after allogeneic transplant, despite the 
presence of circulating alloreactive T-cells to hematopoietic cell-restricted minor 
histocompatibility antigens, PD-L1 was highly expressed on the leukemic cells at baseline or 
upon stimulation with IFN-γ (Norde et al., 2011). Furthermore, stimulation of allogeneic CD3+ T- 
cells with the PD-L1-expressing AML cells led to significantly enhanced T-cell proliferation and 
cytokine production when performed in presence of anti-PD-1 antibody compared to isotype 
controls. 
 
Based on these studies, one can speculate that PD‐L1 expression is unlikely to be an oncogene‐ 
driven tumor immune escape mechanism but rather the result of an initially effective immune 
response against AML, which is later hampered by IFN‐induced upregulation of PD‐L1 
interacting with PD‐1‐expressing immune cells (Kronig et al., 2014). As a form of adaptive 
resistance to therapy, AML cells co‐opt the natural physiology of the PD‐1/PD‐L1 pathway for 
tissue protection in the face of inflammation, to protect itself from an anti-leukemia response. 
 

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2.1.2.2.3 
Early trials of checkpoint inhibition in AML 
 
A phase 1 trial using CT-011, a humanized antibody blocking with PD-1, in patients with 
advanced hematologic malignancies (AML, multiple myeloma (MM), Hodgkin lymphoma, and 
NHL) showed only limited activity (Berger et al., 2008). In a phase 2 trial, nivolumab has been 
combined with cytarabine and idarubicin for the treatment of 32 AML patients in the frontline 
setting (Ravandi et al., 2017). Treatment included 1 or 2 induction cycles of cytarabine 1.5 g/m2 
over 24 hours (Days 1-4) and idarubicin 12 mg/m2 (Days 1-3). Nivolumab 3 mg/kg was started 
on Day 24 ± 2 days and was continued every 2 weeks for up to a year. Of the 32 patients, 23 
patients (72%) achieved CR/CRi (19 CR, 4 CRi) and 9 patients went on to receive an allogeneic 
stem cell transplant (alloSCT). Among the patients proceeding to alloSCT the risk of graft 
versus host disease (GVHD) was not significantly increased. At baseline, bone marrow of non- 
responders had significantly higher percentage of CD4+ T-effector cells co-expressing the 
inhibitory markers PD-1 and TIM3 (p<0.05) and a trend towards a higher percentage of CD4+ T- 
effector cells co-expressing PD-1 and LAG3 compared to responders. 
 
In the relapsed-refractory setting, a phase 2 trial examined the combination of high dose 
cytarabine followed by Pembrolizumab (MK-3475) (Zeidner et al., 2017). Thirteen patients with 
relapsed-refractory AML were to receive age-adjusted high dose cytarabine (HiDAC) (<60 
years: 2 g/m2 IV, every 12 hours [Q12H], Days 1-5; ≥60 years: 1.5 g/m2 IV, Q12H, Days 1-5). 
This was to be followed by Pembrolizumab (MK-3475) 200 mg IV on Day 14 and maintenance 
phase Pembrolizumab (MK-3475) 200 mg IV every 3 weeks (Q3W) for up to 2 years until 
relapse/progression in case of a response. For the 10 evaluable patients at the time of abstract 
submission, overall response rate was 50% (CR/CRi: 4/10= 40%; partial response [PR]: 
1/10=10%) and 2 patients proceeded to alloSCT. Post-alloSCT, both patients developed steroid- 
responsive acute GVHD of skin, one patient developed a transient increase in hepatic enzymes 
that was responsive to steroids, and one patient developed moderate chronic GVHD. 
Furthermore, a combination of the PD-1 inhibitor with the hypomethylating agent azacytidine 
(AZA) was shown to lead to improved response rates in the relapsed-refractory setting (Daver et 
al., 2016). In this study, patients with AML who had failed prior therapy, received AZA 75 
mg/m2 on Days 1-7 with nivolumab 3 mg/kg on Days 1 and 14. Median OS was 9.3 months, 
which compared favorably to historical survival with AZA-based salvage protocols in a similar 
patient population. Patients, who achieved a response, had baseline higher levels of total CD3, 
CD8+ T-cells and a lower level of CD4+FoxP3+PD-1+Treg cell infiltrate in the bone marrow. The 
ratio of PD-1+CD8+ T-effector cells to PD-1+CD4+FoxP3+Treg cells was significantly higher in 
responders vs. non-responders. In summary, early results of phase 2 studies demonstrate the 
feasibility (with encouraging response rates) and safety of adding immune checkpoint blockade 
to chemotherapy or hypomethylating agents both in the frontline as well as the relapsed- 
refractory setting. 
 
2.1.3 The role of anthracycline-based chemotherapy in modulating the immune system 
 
Over the last years it has become apparent that chemotherapy does not only cause death of tumor 
cells by cytostatic effects alone but also by stimulating an immune response directed towards 
cancer cells by reinstating immune surveillance (Kroemer et al., 2013; Zitvogel et al., 2013). 
Chemotherapy has been demonstrated to augment the immune response against cancer through 
multiple mechanisms including improved antigen uptake and chemotactic response by 
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histocompatibility (MHC) complex class I and T-cell receptors, and increased susceptibility of 
tumor cells to immune-mediated cytotoxicity (Zitvogel et al., 2011; Galluzzi et al., 2012). The 
key in electing an immune response to cancer cells in response to chemotherapy is the induction 
of an immunogenic cell death (ICD) as opposed to a non-immunogenic cell death (non-ICD) like 
apoptosis (Kroemer et al., 2013). In order to induce ICD, chemotherapeutic agents need to 
induce pre-apoptotic exposure of calreticulin (CRT) at the cell surface by inducing endoplasmic 
reticulum (ER) stress (Panaretakis et al., 2009), the secretion of ATP during the blebbing phase 
of apoptosis (Michaud et al., 2011), and the cell death–associated release of the non-histone 
chromatin protein high-mobility group box 1 (HMGB1) (Apetoh et al., 2007). CRT release (ecto- 
CRT) from tumor cells is a potent “eat me” signal leading to an engulfment by macrophages and 
dendritic cells (Gardai et al., 2005). In AML patients, the spontaneous exposure of CRT by 
leukemic cells has been shown to predict antitumor T-cell responses and improved patient survival 
(Wemeau et al., 2010). Importantly, CRT only leads to an immunogenic cell death if it is not set 
off by the expression of “don’t eat me” signals like CD47 (Vonderheide, 2015). Importantly, the 
‘eat me’ signal ecto-CRT was shown to inversely correlate with the expression of the ‘don’t eat 
me’ signal CD47 in AML blasts (Wemeau et al., 2010). ATP released from tumor cells is a 
potent “find me” signal (chemotaxis) for dendritic cells and macrophages (Elliott et al., 2009). 
Interestingly, only a small selection of chemotherapeutic agents are able to induce ICD in cancer 
cells: when cancer cells were exposed to 24 different chemotherapeutic agents, only four agents 
(three anthracyclines and oxaliplatin) were able to induce ICD while all agents were able to induce 
apoptosis (Obeid et al., 2007). It seems that anthracyclines are particularly potent in inducing ICD, 
which is important for AML therapy since the backbone of AML 7+3 chemotherapy is 
anthracyclines (Fucikova et al., 2011). 
 
2.2 
CTEP IND Agent 
 
2.2.1 Pembrolizumab (MK-3475) 
 
Pembrolizumab (MK-3475) has high affinity and potent receptor-blocking activity for the 
programmed cell death 1 (PD-1) receptor, based on preclinical in vitro data (Investigator’s 
Brochure, 2018). Pembrolizumab (MK-3475) has an acceptable preclinical safety profile and is 
being advanced for clinical development as an IV immunotherapy for advanced malignancies. 
 
The importance of intact immune surveillance function in controlling outgrowth of neoplastic 
transformations has been known for decades (Disis, 2010). Accumulating evidence shows a 
correlation between tumor-infiltrating lymphocytes in cancer tissue and favorable prognosis in 
various malignancies. In particular, the presence of CD8+ T-cells and the ratio of CD8+ effector T- 
cells/FoxP3+Tregs correlates with improved prognosis and long-term survival in solid malignancies, 
such as ovarian, colorectal, and pancreatic cancer; hepatocellular carcinoma; malignant melanoma; 
and renal cell carcinoma. Tumor-infiltrating lymphocytes can be expanded ex vivo and re- 
infused, inducing durable objective tumor responses in cancers such as melanoma (Dudley et al., 
2005; Hunder et al., 2008). 
 
The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress immune 
control. The normal function of PD-1, expressed on the cell surface of activated T-cells under 
healthy conditions, is to down-modulate unwanted or excessive immune responses, including 
autoimmune reactions. PD-1 (encoded by the gene Pdcd1) is an Ig superfamily member related 
to cluster of differentiation 28 (CD28) and CTLA-4 that has been shown to negatively regulate 

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antigen receptor signaling upon engagement of its ligands (PD-L1 and/or PD-L2) (Greenwald et 
al., 2005; Okazaki et al., 2001). 
 
The structure of murine PD-1 has been resolved (Zhang et al., 2004). PD-1 and family members 
are type I transmembrane glycoproteins containing an Ig Variable–type (IgV type) domain 
responsible for ligand binding and a cytoplasmic tail responsible for the binding of signaling 
molecules. The cytoplasmic tail of PD-1 contains 2 tyrosine-based signaling motifs, an 
immunoreceptor tyrosine-based inhibition motif, and an immunoreceptor tyrosine-based switch 
motif. Following T-cell stimulation, PD-1 recruits the tyrosine phosphatases, SHP-1 and SHP-2, 
to the immunoreceptor tyrosine-based switch motif within its cytoplasmic tail, leading to the 
dephosphorylation of effector molecules such as CD3 zeta (CD3ζ), protein kinase C-theta 
(PKCθ), and zeta-chain-associated protein kinase (ZAP70), which are involved in the CD3 T-cell 
signaling cascade (Chemnitz et al., 2004; Sheppard et al., 2004; and Riley, 2009). The 
mechanism by which PD-1 down modulates T-cell responses is similar to, but distinct from, that 
of CTLA-4, because both molecules regulate an overlapping set of signaling proteins (Parry et 
al., 2005; Francisco, 2010). As a consequence, the PD-1/PD-L1 pathway is an attractive target 
for therapeutic intervention in AML. 
 
2.2.1.1 
Pembrolizumab (MK-3475) Background and Clinical Trials 
 
MK-3475 (pembrolizumab, Keytruda®), a humanized monoclonal antibody against the PD-1 
protein, has been developed by Merck & Co. for the treatment of cancer. Pembrolizumab (MK- 
3475) is approved for treatment of melanoma in several countries; in the United States (US) and 
European Union (EU) it is approved for the treatment of advanced (unresectable or metastatic) 
melanoma in adults. Pembrolizumab (MK-3475) has also been approved for treatment of NSCLC 
in several countries; in the US it is indicated for the treatment of patients with metastatic NSCLC 
whose tumors express PD-L1 as determined by a Food and Drug Administration (FDA)- 
approved test and who have disease progression on or after platinum-containing chemotherapy. 
Patients with NSCLC and epidermal growth factor receptor (EGFR) or anaplastic lymphoma 
kinase (ALK) genomic tumor 
aberrations should also have disease progression on FDA-approved therapy for these aberrations 
prior to receiving Pembrolizumab (MK-3475). P e m b r o l i z u m a b ( M K – 3 4 7 5 )  is 
approved in the US for the treatment of patients with recurrent or metastatic head and neck 
squamous cell carcinoma (HNSCC) with disease progression on or after platinum-containing 
chemotherapy. 
 
Pembrolizumab (MK-3475) has demonstrated initial clinical efficacy in single-arm monotherapy 
trials in patients with NSCLC, HNSCC, urothelial cancer, gastric cancer, triple negative breast 
cancer, and Hodgkin’s Lymphoma as determined by response rate. Ongoing clinical trials are 
being conducted in these tumor types as well as a number of other advanced solid tumor 
indications and hematologic malignancies. For study details please refer to the Investigator’s 
Brochure (2018). 
 
2.3 
Other Agent(s) 
 
2.3.1 “7+3” induction chemotherapy 
 
The most commonly used induction regimen for AML is the so-called “7+3” regimen, which has been 

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reviewed in detail before (Dohner et al., 2010). This regimen combines a seven-day continuous IV 
infusion of cytarabine (100 or 200 mg/m2 per day) with a short infusion or bolus of an anthracycline 
given on days 1 through 3. The most commonly used anthracycline in this regimen is daunorubicin, but 
idarubicin is sometimes used as well. The combination of “standard dose” cytarabine plus 
daunorubicin has been the historical standard for remission induction in AML. In this regimen, 
cytarabine is given by continuous IV infusion for 7 days plus daunorubicin by IV push or short 
infusion daily for the first three days. Standard dosing for patients less than 60 years old is: Cytarabine 
200 mg/m2 for 7 days plus daunorubicin 60-90 mg/m2 for 3 days. Idarubicin is administered at 12 
mg/m2/day for 3 days in combination with Cytarabine 200 mg/m2 for 7 days. 
 
2.3.1.1 
Pharmacokinetics 
 
Cytarabine is rapidly and widely distributed into tissues, including liver, plasma, and 
peripheral granulocytes. Rapid and extensive metabolism by cytidine deaminases occurs mainly 
in liver and kidneys. Cytarabine and its inactive metabolite are excreted in urine; 70-80% of 
cytarabine are eliminated in the urine within 24 hours (90% as inactive metabolite, 10% as intact 
drug). Initial cytarabine half-life elimination is 7 to 20 minutes; terminal cytarabine half-life 
elimination is 1 to 3 hours. 
 
Daunorubicin distributes widely into tissues, particularly the liver, kidneys, lung, spleen, and 
heart; does not distribute into the central nervous system (CNS). Daunorubicin is metabolized 
primarily hepatically to daunorubicinol (active), then to inactive aglycones, conjugated sulfates, 
and glucuronides. It is excreted in feces (40%) and urine (~25% as unchanged drug and 
metabolites). Initial daunorubicin half-life elimination is 45 minutes; terminal cytarabine half- 
life elimination is 18.5 hours. Idarubicin is metabolized hepatically to idarubicinol (active 
metabolite) and excreted primarily by biliary tract and in the urine (8 to 10%). The elimination half- 
life is 22 hours (range: 4 to 48 hours). 
 
2.3.1.2 
Efficacy 
 
Depending on age and patient selection, 70% to 80% of younger adults achieve a CR with these 
regimens. Most remissions happen after a single course. In the past, daunorubicin had been 
commonly employed at a dose of 45 mg/m2; however, randomized studies suggest that higher 
doses of daunorubicin (e.g., 60 to 90 mg/m2 per day) are more effective and no more toxic than 
45 mg/m2 and that 90 mg/m2 was no more effective, but more toxic than 60 mg/m2. 
 
2.3.1.3 
Safety data 
 
The combination of cytarabine plus daunorubicin or idarubicin results in severe pancytopenia in 
all patients and therefore requires transfusion support and antibiotics as needed. The median 
number of days with an absolute neutrophil count (ANC) less than 500 and a platelet count less 
than 50,000 are approximately 16 and 15, respectively. Common non-hematologic side effects 
seen in the majority of patients include stomatitis (mostly mild), alopecia, nausea, and vomiting 
(25% severe), and diarrhea (mostly mild to moderate). Cardiovascular side effects include 
cardiac failure (dose-related) and ECG abnormalities (transient; includes atrial premature 
contractions, S-T wave changes, supraventricular tachycardia, ventricular premature 
contractions; generally asymptomatic and self-limiting). 
 

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2.4 
Rationale 
 
Combining 7+3 induction chemotherapy with immune checkpoint inhibitor therapy could 
lead to synergy by combining the immunomodulatory abilities of checkpoint inhibition and 
traditional chemotherapy. Chemotherapy with anthracyclines as the backbone leads to 
immunogenic cell death of leukemic blasts resulting in antigen release and cross presentation of 
antigens by dendritic cells with a priming effect on CTL as discussed earlier (Kroemer et al., 
2013; Zitvogel et al., 2013). INF-γ leads to an activation of T-cells but also an increased 
expression of PD-L1 on leukemic blasts (Blank et al., 2004; Chen et al., 2012). Subsequently, 
CTLs are not able to kill leukemic blasts because they are inactivated by an increased 
expression of PD-L1 on leukemic blasts resulting in therapy resistant tumor cells or relapse after 
completion of induction chemotherapy. Adding the anti PD-1 antibody Pembrolizumab (MK- 
3475) to 7+3 induction chemotherapy may lead to more effective CTL-mediated destruction of 
leukemic blasts and thereby results in improved rates of CR without MRD and thereby lower 
relapse rates. 
 
Combination of checkpoint inhibition with chemotherapy and targeted therapy has been shown 
to be safe in solid tumors (Morrissey et al., 2016). For instance, in NSCLC nivolumab has been 
successfully combined with platinum doublet chemotherapy (Rizvi et al., 2016). Checkpoint 
inhibitors have been combined with chemotherapy (HiDAC followed by MK-3475 
[pembrolizumab]) (Zeidner et al., 2017) and epigenetic therapy (AZA and nivolumab) (Daver et 
al., 2017) in relapsed and refractory AML. Similarly, checkpoint inhibitors have been combined 
with chemotherapy (cytarabine, idarubicin, and nivolumab) (Ravandi et al., 2017) and epigenetic 
therapy (AZA and nivolumab) (Daver et al., 2017) for the treatment of AML patients in the 
frontline setting. The combination of checkpoint inhibitors with chemotherapy or epigenetic 
therapy was shown to be feasible and safe in the studies. Importantly, preliminary results from 
AML trials using anti-PD1 therapy in combination with intensive chemotherapy in the 
relapsed/refractory setting and high doses of cytarabine/idarubicin in frontline setting and in 
combination with hypomethylating agents among the patients who proceeded to HSCT after 
checkpoint inhibitor therapy found no signal for increased risk of GVHD or transplant-related 
toxicity (Ravandi et al., 2017; Zeidner et al., 2017). 
 
We hypothesize that standard anthracycline based induction chemotherapy in combination with 
the anti-PD-1 antibody Pembrolizumab (MK-3475) is associated with acceptable toxicity profile 
and leads to an improvement in the percentage of patients with MRD negative CR. We 
hypothesize that standard anthracycline induction in combination with the anti-PD-1 antibody, 
Pembrolizumab (MK-3475), would lead to an increase in the percentage of MRD negative CR 
from 50% to 75% after induction and one cycle of consolidation therapy. 

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Figure 1. Potential synergistic effect of 7+3 induction chemotherapy and checkpoint inhibition 
with anti PD-1 antibody Pembrolizumab (MK-3475) in inducing an immune response against 
AML blasts. Figure modified from Kroemer et al., (2013). 
 
2.4.1 Standard of care assessments: 
 
2.4.1.1 
Core Binding Factors 
 
The clinical and molecular heterogeneity of AML brings about varied response to induction 
chemotherapy, as well as drug resistance and disease relapse. The current risk-stratification 
systems (such as ELN-2017 and NCCN-2018) classify AML into favorable, intermediate, and 
adverse risk categories based on therapeutic sensitivity to standard chemotherapeutic approaches 
and likelihood of sustained remission after induction and consolidation. Core-binding factor 
(CBF) AML is a favorable risk AML category characterized by t(8;21)(q22;q22) and 
inv(16)(p13;q22)/t(16;16)(p13;q22) abnormalities, and despite mutational and cytogenetic 
heterogeneity among t(8;21) and inv(16), these entities share similarities in prognosis, are 
grouped together, and managed similarly (Appelbaum et al., 2006). In patients in favorable 
risk AML (Othus et al., 2014) treated with conventional chemo induction, clinical trial data have 
reported CR and long-term survival rates approaching 80-90% and 50-65%, respectively 
(Paschka et al., 2018; Schiller, 2005). Unlike the case with the favorable risk AML, intermediate 

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and adverse risk categories represent a particularly hard-to-treat disease population characterized 
by lower remission rates, high early mortality, and early relapse and death. Analysis and 
interpretation of outcomes between newer treatment approaches and traditional cytotoxic 
chemotherapy in AML must therefore be within the context of the underlying AML risk status. 
Therefore, we use CBF (by karyotype or FISH) as a stratification factor to account for 
heterogeneity in outcomes based on AML risk status. 
Some patients with AML require initiation of therapy quickly after diagnosis, and full 
metaphase karyotype results in some centers can take 2-3 weeks to result. To avoid this issue 
being an impediment to accrual to study or to cause unwanted delays in initiation of therapy in 
patients who need fast initiation of therapy, we will allow use of karyotype and/or FISH results 
(as well as FLT3 results) on samples from blood or marrow that were obtained up to 3 weeks 
before signing consent for purposes of eligibility and stratification. In any case, results from 
FISH or karyotype should show if CBF abnormalities are present by at time of randomization 
as the presence of CBF abnormalities is a required stratification factor. 
 
2.4.1.2 
2.4.1.2 FLT3 Mutations 
 
FLT3 represents the most common mutated gene in AML, with over 90% of AML expressing a 
FLT3. Mutations in the FLT3 can either be internal tandem duplications (FLT3-ITD) within the 
juxta-membrane domain or point mutations within the kinase domain (FLT3-TKD). FLT3-ITD 
mutations can be detected in 25-30% of AML cases and carry a worse prognosis (El Fakih et al., 
2018; Wang et al., 2005). Patients with newly diagnosed FLT3-ITD mutated AML who are 
treated with conventional chemotherapy have poorer survival outcomes, with 3-year survival 
estimates <20%, than do patients without these mutations due to their increased risk of relapse 
and inferior disease-free survival (Thiede et al., 2002). A number of other FLT3 inhibitors (such 
as quizartinib, gilteritinib, and midostaurin) are currently undergoing clinical development in 
phase 3 trials. In 2018, midostaurin received breakthrough therapy designation from the FDA 
for newly diagnosed FLT3-mutated AML, after demonstrating significantly improved OS in a 
randomized phase 2 study (RATIFY) of induction and consolidation chemotherapy combined 
with midostaurin or placebo (5-year OS: 51% vs. 43%; p=0·007) (Stone et al., 2017). A FLT3 
inhibitor (midostaurin) in combination with 7+3 is now the standard of care in the management 
of newly diagnosed FLT3-mutated AML. Since the current trial will not be incorporating a 
FLT3 inhibitor as a part of the treatment, we will exclude FLT3-mutated AML in this study. 
 
2.5 
Correlative Studies Background 
 
2.5.1 Assessment of MRD 
 
2.5.1.1 
MRD assessment by MFC as an integral biomarker 
 
The flow cytometric MRD assay is an established and validated assay and is used as the 
reference method for this comparison. While an integral biomarker in the study, Study-based 
therapeutic decisions will not be based on the results of the MRD flow assay. Patients with 
AML will have flow cytometry in the bone marrow (BM) at the time of study entry to determine 
leukemia- associated immunophenotypic abnormalities and then BM will be evaluated after one 
cycle of consolidation therapy (primary endpoint). 

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The central MRD studies will be performed solely in the flow cytometry laboratory located in the Department of 
Pathology and Laboratory Medicine Children’s Hospital Los Angeles directed by Dr. Brent Wood. This laboratory 
has more than 15 years of experience performing flow cytometric MRD assays on over 10,000 pediatric patients 
enrolled on past and ongoing Children’s Oncology Group (COG) B-ALL and T-ALL clinical trials. The laboratory 
also has a strong publication record for the flow cytometric detection of MRD in AML and performs such testing 
for Southwest Oncology Group (SWOG) trials in adults and for multiple biopharma clinical trials. 
 
The flow cytometry assay proposed for use in this trial is validated and multiple publications 
support its prognostic relationship to outcome in AML. The assay relies on the principle that 
immunophenotypic (IP) abnormalities exist in leukemia progenitor cells that distinguish them 
from normal progenitors (Wood, 2016). Cells are stained with antibodies that have been 
previously shown to be informative for this purpose, and that have been conjugated to different 
fluorochromes designed to maximize the resolution between normal and abnormal cells. 
Specifically, the antibody combinations used are outlined in the following table: 
 
Combination 
PB/ 
BV421 
FITC 
PE 
PE-TR 
PE-X 
PE-Cy7 
A594 
APC 
APC- 
A700 
APC- 
H7 
AMLM1 
HLA-DR CD15 
CD33 
CD19 
CD117 
CD13 
CD38 CD34 
CD71 
CD45 
AMLM2 
HLA-DR CD64 
CD123 
CD4 
CD14 
CD13 
CD38 CD34 
CD16 
CD45 
AMLM4 
HLA-DR CD56 
CD7 
CD5 
CD33 
CD38 CD34 
CD34 
 
Stained cells are analyzed on a LSRII flow cytometer (Becton Dickinson, San Jose, CA). The 
proportion of leukemic MRD cells is expressed as a percentage of white cells (CD45+) in each of 
the tubes. A total of 1,000,000 events is the acquisition target, with 100,000 white cell events 
being the minimum number of events required. With this number of events it is possible to 
detect leukemic cells with a routine sensitivity of 0.1%, better in a subset of cases. This assay 
was used on the SWOG (S0106) and other clinical trials. The flow cytometric assay for AML 
MRD has been used in over 2,000 children and adults with AML tested in our clinical reference 
laboratory since 2006 and is being used as the assay in multiple biopharma sponsored clinical 
trials. Precision studies performed in our laboratory on 3 replicates of 3 samples targeted to have 
1%; 0.1% and 0.01% MRD via serial dilution demonstrated CVs of 3.5%, 6.6% and 6.4%, 
respectively. The ~7% CV seen with samples with approximately 0.01% MRD supports the 
interpretation that the sensitivity of the assay is 0.01%. 
 
The current validation of the assay is for a threshold of 0.1% but there is a variable level of sensitivity 
of the assay in the range of roughly 0.01% - 0.1% depending on leukemic immunophenotype. We will 
use 0.1% as a threshold for MRD negative disease for purposes of the primary endpoint of the study 
as per ELN guidelines and using the current validation level threshold of the assay, but we will 
explore other thresholds such as 0.01% given the ability of the assay to detect this difference in some 
patients depending on the leukemia immunophenotype and also recognizing that the clinically 
relevant threshold is not well established yet. 
 
 
2.5.2 MRD assessment by duplex sequencing (DS) as an exploratory biomarker 
 
NGS provides a powerful new platform for MRD detection. Using NGS, millions of DNA 
fragments can be sequenced in parallel and pieced together to determine the sequence of large 

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regions of the genome. The sensitivity of conventional NGS, however, is limited by a relatively 
high error rate, which makes it extremely difficult to differentiate sequencing errors from true low- 
frequency mutations. If the accuracy of NGS could be further improved to meet or exceed that of 
conventional MRD detection methods, NGS would be an ideal tool for MRD detection because of 
its reproducible digital nature, generalizability for essentially all AML patients, and ease of 
implementation in any NGS equipped lab. Furthermore, rather than simply identifying the 
presence of MRD, NGS would provide genomic information about clonal composition and 
dynamics, which may improve the predictive value and clinical utility. 
 
Therefore, we want to apply DS to MRD detection in AML as an exploratory endpoint. The 
sensitivity of DS far surpasses single-stranded tagging methods and can detect mutations at a 
sensitivity greater than three orders of magnitude compared to NGS methods (Schmitt et al., 
2012). The error rate of <1 per 10 million base pairs sequenced (the probability of identical 
“jackpot” errors in the same position on both strands) is achieved by independently sequencing 
the two strands of each DNA molecule and building double-stranded consensus sequences. As 
the strands are complementary, true mutations will be found at the same position in both strands. 
 
The Radich Lab is working with TwinStrand (a University of Washington “spin off”) to use DS 
to sequence mutations in acute and chronic leukemia (Araki et al., 2016). This team has 
carefully selected a panel of 25 genes with the most common point mutations, insertions, 
deletions, fusions, and duplications associated with AML. They have developed probes targeting 
whole genes (e.g., RUNX1, TP53, etc.) or specific mutational hotspots (e.g., FLT3 point 
mutations and internal tandem duplications (ITD), NPM1 insertions, etc.). These genes are 
covered by a ~40 kilobase (kb) panel which comprises a median of 99% of all AML-associated 
mutations within the selected genes. A ~30 kb panel of genes will be used. Sequencing to a 
depth of 20,000x is expected to provide a 98% power to detect an allele frequency of 0.02% (1 in 
5000) and 86% power to detect alleles at 0.01% frequency (see Figure 2, below). 
 
Patients with AML will have DS performed on the bone marrow (BM) and PB at the time of study entry, 
at Day 14, at the end of induction therapy (EOI), after one cycle of consolidation therapy, and at the end 
of consolidation therapy (EOC), and at end of maintenance therapy/observation period for patients who 
do not progress. For patients who are willing to have BM done every 3 months during follow up, DS in 
BM and PB could be assessed during maintenance therapy/observation period. 
Figure 2. Sensitivity of Duplex Sequencing is a function of input DNA quantity and sequencing 
depth. Low-level mutations can be detected reliably at 20,000x based on statistical modeling 

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(Unpublished Data). 
 
2.5.2.1 
Comparing DS and MFC for MRD detection as an exploratory biomarker 
 
Unfortunately, standard MRD detection methods including MFC and RT-PCR have serious 
shortcomings in terms of sensitivity, applicability, and reproducibility. Thus, patients without 
detectable MRD still relapse. In the SWOG study, S0106 for newly diagnosed adult AML, 
MRD status by MFC was a strong predictor of survival, yet MRD predicted relapse-free survival 
with a poor c-statistic of 0.58 (0.5=no predictive value; 1.0=perfect prediction) (Petersdorf et al., 
2013). In addition, ~25% of patients without MRD prior to allogenic hematopoietic cell 
transplant (alloHSCT) relapse, while ~65% of those with MRD do not (Araki et al., 2016). 
Furthermore, optimal performance of MFC depends not only on immunophenotype, but also on 
flow cytometer, panel design, and operator experience. DS is not only more sensitive than MFC 
but also interpretation is fully digital and “automatic” and does not require manual review by a 
pathologist or technician and can be easily implemented an any NGS capable lab. Finally, DS 
will not simply reveal whether or not MRD is present, but the exact mutational nature of the 
clones comprising residual disease. 
 
In order to compare both approaches, DS will be performed on samples at a molecular depth of 
20,000x and the same samples will also be analyzed by 10-color MFC, although laboratory 
researchers conducting this assay will be blinded to MFC results. 
 
2.5.2.2 
Application of duplex sequencing strategy for circulating cell-free tumor DNA- 
based longitudinal minimal residual disease monitoring 
 
While measurable residual disease (MRD) is a well-recognized predictive biomarker for 
relapse in AML, the current standard of care MRD detection technique – namely multi- 
parameter flow cytometry (MFC) – does not accurately predict leukemic relapse, due to poor 
reproducibility and sensitivity, thereby limiting the utility of MFC-based MRD as a reliable 
surrogate end-point in AML clinical trials. With the growing, unmet need for more accurate 
MRD approaches in AML studies, the current application seeks to leverage the unprecedented 
accuracy and sensitivity of the sophisticated duplex sequencing technology to capture very 
low-level circulating tumor DNA (ctDNA), also known as circulating tumor free DNA 
(cfDNA). As a strategy to potentially more accurately predict early relapse. The proposed 
DS- based ctDNA MRD detection approach will not only serve as a proof-of-principle study 
supporting its utility as a powerful biomarker of relapse in AML but, if shown to be a more 
accurate predictor of relapse, will lay grounds for its implementation as a potential surrogate 
marker of long-term response in future AML clinical trials. 
 
For this assay, samples of 8mL will be collected at each time point in which MRD is collected 
from peripheral blood. This collection will be in Streck tubes that are provided to sites. Aside 
from the different type of tube. These sample will be collected, processed and shipped in the 
same manner and with the MRD tubes at the different time points. These samples will be sent 
to Dr. Brent Wood’s laboratory where they will be stored, and subsequently sent to Dr. 
Radich’s laboratory where the assays will be conducted. 

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2.5.3 Assessment of immune cell subsets and its correlation with the response to combine 
chemotherapy and anti-PD-1 directed therapy 
 
Currently, there is no single biomarker to predict the response to anti-PD-1 Ab therapy especially 
in myeloid leukemia. Two recent landmark studies suggest that the predicted response of anti- PD- 
1 is associated with PD-L1 expression in tumor-infiltrating immune cells (Herbst et al., 2014; 
Powles et al., 2014). These studies have led us to test the question about the expression of PD-L1 
on blasts from human AML BM and blood and its correlation with the response to combined anti- 
PD-1/chemotherapy in AML. 
 
The most reliable biomarker to predict anti-PD-1 Ab therapy includes tumor-infiltrating 
lymphocytes (TILs). However, further evaluation of TILs subpopulations and their interaction 
with other TME components will be necessary to identify other response-predictive factors. 
Compared to a murine model for linear T-cell differentiation, we have had to rely on cross- 
sectional assessment to define memory T-cell subsets. More than two markers (CD45RA, 
CCR7) enabled us to delineate memory T-cell compartments (Sallusto et al., 1999). For 
instance, we simply divide T-cells into four subsets: naïve (CD45RA+CCR7+), central memory 
(CD45RA-CCR7+), effector memory (CD45RA-CCR7-), and late effector (CD45RA+CCR7-). 
The ratio of TCM (CD45RA-CCR7+) to TEMRA (CD45RA+CCR7-) and of TEM (CD45RA- 
CCR7-) to TEMRA (CD45RA+CCR7-) will be assessed in BM and peripheral blood using flow 
cytometry. More recent preclinical data suggest that the frequency of CD8+, and CD8+/Tregs are 
two important predictive markers for anti-PD-1 therapy (Twyman-Saint Victor et al., 2015). 
Additionally, this study also demonstrated that exhausted CD8+ T-cells defined by the expression 
of PD-1 and Eomes are reinvigorated, which can be defined by increased Ki67 and GzmB, 
following anti-PD-1 and anti-CTLA-4 treatment in a murine model. CD47 is capable of 
interacting with its receptor signal-regulatory protein alpha (SIRPα) on macrophages to 
negatively regulate phagocytosis and blockade of CD47 expression results in phagocytosis of red 
blood cells (RBCs) (Oldenborg et al., 2000). CD47 functions as a ‘don’t eat me’ signal to ensure 
that autologous cells are not inappropriately phagocytosed. Intriguingly, two recent studies 
revealed that CD47 is upregulated on leukemia cells and represents an adverse prognosis factor 
in AML (Jaiswal et al., 2009; Majeti et al., 2009). The ‘eat me’ signal ecto-CRT was shown to 
correlated inversely with the expression of the ‘don’t eat me’ signal CD47 in AML blasts 
(Wemeau et al., 2010). We will determine whether other exploratory immune biomarkers 
predict the response to anti-PD-1 Ab therapy in combination with chemotherapy by assessing 
immune cell lineage markers as well as functional markers in fresh BM and blood from AML 
patients using high throughput mass spectrometry(CyTOF). 
 
2.5.4 Multiplex Immunoflorescence (mIF) 
 
Immunohistochemistry is one of the most acceptable tools to measure PD-1 and PD-L1 
expression in tumor microenvironment (TME). Human BM core biopsy will be decalcified for 
immunohistochemistry (IHC). We will compare IHC/IF findings and its quantitative analyses by 
using Multiplex Immunoflorescence (mIF) (will be describe below) with flow cytometric data in 
BM aspirate and blood. All these procedures will be performed with a collaboration with Dr. 
Ignacio I Wistuba at MDACC CIMAC. For multiplex IF image analysis platform, we will use 
the tyramide signal amplification (TSA) methodology through the Opal™ workflow and 
chemistry which allows simultaneous staining of multiple biomarkers within a single paraffin 
tissue section. The multispectral microscopy Vectra™ and Vectra-Polaris™ scanner systems 

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(Akoya Biosciences, Waltham, MA, USA) including the image analysis software (InForm, 
Akoya Biosciences) allows the analysis of the multispectral images generated by this staining 
methodology. Multiplex ImmunoFluorescence (IF) staining will be performed on FFPE tumor slides 
using the following panels and antibodies: 
1. Panel 1 (PD-L1/PD-1 and TIL Panel): PD-L1, CD68, PD-1, CD8, CD3, PanCytokeratin, DAPI. 
 
2. Panel 2 (T cell Activation/ Regulatory Panel): FOXP3, Granzyme B, CD45RO, CD8, CD3, 
PanCytokeratin, DAPI. 
 
 
2.5.5 Dynamic change of immune cell subsets: 
 
We will possibly have limited number of cells in the BM aspirate. Therefore, we will analyze 
the immune profile combining a high-throughput technique that will allow us to study more than 
20 markers in a single cell analysis using CyTOF. These techniques will provide information 
about the architecture of TME (i.e. BM) and the distribution of markers of interest detected with 
CyTOF. CyTOF-based single cell analysis is a new technology that combines flow cytometry 
with metal-conjugated antibodies detected by mass spectrometry permitting higher order 
multiplexing (up to 100 molecules) on a single cell analysis (Bendall et al., 2011). Additionally, 
after fixation and permeabilization, cells will be stained with anti-human Ki-67-151Eu (B56;BD 
Pharmingen Ab conjugated with lanthanide MaxPar Europium Chloride 151Eu using the MaxPar 
X8 Ab labeling kit) for 30 min at room temperature. Cells will be acquired on CyTOF 2 
instrument (DVS; Fluidigm Sciences). All data will be analyzed, and graphs generated using the 
DVS Cytobank software (Cytobank). 
 
2.5.6 Olink cytokine panel 
 
Inflammation is a key underlying factor for the pathophysiology of a wide range of diseases, 
including malignancies. The Olink inflammation biomarker panel provides a high-throughput, 
multiplex immunoassay enabling analysis of 92 inflammation-related protein biomarkers across 
96 samples simultaneously. The objective is to investigate protein signatures associated with 
response and efficacy. We will perform this procedure on the bone marrow aspirate in 
collaboration with Dr. Sacha Gnjatic at Mount Sinai CIMAC. 
 
2.5.7 Whole Exome Sequencing 
 
The determination of the mutation load, a total number of nonsynonymous point mutations, by 
whole exome sequencing (WES) was shown to be useful in predicting the treatment responses 
to cancer immunotherapy (Lyu, 2018). Tumor mutational burden (TMB), along with PD-L1 
expression, is a useful biomarker for immune checkpoint blockade selection across certain 
cancer types and TMB determination correlated with WES (Chan, 2019). We will use massively 
parallel sequencing technology to sequence the genomic DNA of tumor cells (leukemic bone 
marrow) and normal cells (germline) obtained from patients with AML at baseline. Mutational 
load by WES will be correlated with clinic-pathological parameters such as response to 
treatment, survival and immune infiltrating profile, and T cell repertoire diversity and clonality. 
 
2.5.8 RNA-seq 
The MD Anderson CIMAC uses Agilent RNA isolation products for RNAseq. RNA will be 

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prepared using suitable purification system depending on sample source (fresh or FFPE). RNA 
integrity of FFPE RNA will be assessed using either the Agilent 4200 TapeStation and High 
Sensitivity RNA ScreenTape or the Agilent 2100 Bioanalyzer and RNA 6000 Pico Chip. Either 
method will employ the region analysis method to determine the percentage of RNA in the 
sample that is >200 nt for each sample to be processed. It is necessary to have RNA molecules 
>200 nt for efficient library construction and this value for each sample will determine 
appropriate conditions at various steps in the workflow. 
 
2.5.9 TCR-seq 
 
One mechanism by which checkpoint inhibitors are thought to elicit their effects by broadening 
the TCR repertoire. We will perform high-throughput sequencing of the TCR Vb CDR3 regions 
on ﬂow cytometrically sorted T-cell subsets to assess the effect of immunotherapy on the 
diversity of the T-cell repertoire and assess for correlation to clinical outcomes. TCR sequencing 
analysis may be performed using DNA from tumor tissues as well as PBMC. Briefly, 500 ng 
tumor DNA or 3-6 µg PBMC DNA will be subjected to high throughput TCR Vβ CDR3 
sequencing on an Illumina HiSeq sequencer with at least 5-fold coverage by ImmunoSEQ™ 
sequencing (Adaptive Biotechnologies, Seattle, WA). TCR diversity and clonality (defined as 1- 
(entropy)/log2(#) of productive unique sequences, where the entropy term takes into account the 
varying clone frequency) will be calculated using a software by Adaptive Technologies. T-cell 
repertoire diversity and clonality will be correlated with clinic-pathological parameters such as 
response to treatment, survival, and immune infiltrating profile, as well as genomic profiles (total 
mutation burden, non-synonymous mutation burden, predicted neoantigen burden, clonal 
mutation burden and clonal predicted neoantigen burden). TCR profile generated from 
treatment-refractory tumors at the time of disease progression will be compared to data from pre- 
treatment tumor samples to explore the TCR repertoire evolution of these tumors under 
therapeutic pressure. The dynamic changes of TCR from PMBC, when longitudinal blood 
samples are available, will be correlated to response to immune checkpoint blockade or 
chemotherapy and survival. 
 
 
2.5.10 Gut microbiome characterization 
 
There is increasing evidence that dynamic changes within the microbiome can affect both immune and 
cancer cells. Imbalance in microbial communities promotes chronic inflammation and predisposes to 
cancer (Garett et al, 2015). Our collaborators have shown that microbiome is also involved in the 
metabolism of drugs and could explain person-to-person variability and responsiveness to therapy 
(Alexander et al, 2017, Zimmermann et al, 2019). Unlike in several solid tumors wherein, immune- 
check point blockade is used alone or in combination or with cytotoxic agents, the drug synergism to 
treat AML is unique. Majority of these combinations results in mucositis which helps microbes breach 
gastro-intestinal (GI) epithelial barrier. It is plausible that with each cycle of treatment, exposure of 
immune cells to time -prevalent oral and GI microbiome is subject to temporal variance. Extrapolating 
preclinical data, taxonomically unique microbes could further determine priming of antigen presenting 
cell interaction with immune-effector cells that ultimately determine clinical phenotype. Based on our 
current understanding on the mechanism of action of PD-1 inhibitors, our overall hypothesis is that 
gastro-intestinal microbiome holds salutary prognostic and therapeutic promise in patients with AML. 
Our preliminary hypothesis is that gut microbiome modulate clinical responses and toxicity in patients 
with AML receiving Check Point Inhibitors (CPI). To this end our proposal will test the longitudinal 

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relationships between the taxonomy and diversity of gut microbiome on clinical outcomes and toxicity. 
 
 
 
 
3. 
PATIENT SELECTION 
 
3.1 
Eligibility Criteria 
 
3.1.1 Newly diagnosed and pathologically-confirmed AML, confirmed by a bone marrow 
aspirate and/or biopsy and/or peripheral blood with ≥20 % myeloid blasts. Bone marrow biopsy, or 
aspirate or peripheral blood that were obtained up to 3 weeks before signing consent are 
allowed for purposes of confirming AML diagnosis for eligibility purposes. Secondary AML 
that is arising from prior myelodysplastic syndrome [MDS] as well as t-AML) are also allowed. 
Clarifications: . AML arising from myeloproliferative neoplasms (MPN), MPN/MDS overlap 
(including chronic myelomonocytic leukemia [CMML]) or another myeloid malignancy are NOT 
allowed. Note 1: Patients must have evidence of bone marrow involvement on aspirate or biopsy. 
Patients with only extramedullary disease and no bone marrow involvement will be excluded. 
N o t e 2 : Every effort should be made to get an aspirate for central flow assessment at 
screening and all subsequent required time points, but in cases where an aspirate cannot be 
collected-including dry taps-the patient will not be excluded and assessments will be performed 
on PB which should be collected at every time that BM is collected. Note 3: Some patients with 
AML require initiation of therapy quickly after diagnosis, and full metaphase karyotype results 
in some centers can take 2-3 weeks to result. To avoid this issue being an impediment to 
accrual to study or to cause delays in initiation of therapy in patients who need fast initiation of 
therapy, we allow use of karyotype and/or FISH results (as well as FLT3 results) on samples 
from blood or marrow that were obtained up to 3 weeks before signing consent for purposes of 
eligibility and stratification. In any case, results from FISH or karyotype should show if CBF 
abnormalities are present by time of randomization as the presence of CBF abnormalities is a 
required stratification factor. 
 
3.1.2. Age ≥18 and ≤75 years. 
 
Because no dosing or AE data are currently available on the use of Pembrolizumab (MK- 
3475) in patients <18 years of age, children are excluded from this study, but will be eligible 
for future pediatric trials. 
 
3.1.3. ECOG performance status ≤ 2, see Appendix A.  
 
3.1.4. The patient has to be eligible to receive intensive “7+3” induction chemotherapy as 
judged by the treating physician. 
 
 
3.1.5. Prior use of hypomethylating agents (HMA), lenalidomide, erythropoiesis-stimulating 
agents (ESAs), and growth factors is allowed if used to treat prior MDS. AML must be 
previously untreated except as outlined below (hydroxyurea, or ATRA, or 
leukapheresis). Note: One dose of prophylactic intrathecal therapy is allowed during or 
before screening if a lumbar puncture is performed to rule out CNS involvement. 
 

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3.1.6. Hydroxyurea/leukapheresis allowed for control of hyperleukocytosis but hydroxyrea 
must be discontinued day prior to start of chemotherapy. 
 
3.1.7. Patients must have adequate organ and marrow function as defined below within 3 days prior 
to the first day of 7+3. 
 
 
System 
Laboratory Value 
Renal 
Creatinine OR 
measured or calculated creatinine 
clearance (CrCl) a,b (See Appendix B) 
≤1.5 × ULN OR 
≥60 mL/min for patient with 
creatinine levels >1.5 × institutional ULN 
Hepatic 
Total bilirubin 
≤1.5 × ULN OR 
Direct bilirubin ≤ULN for patients with total 
bilirubin levels >1.5 × ULN 
AST (SGOT) and ALT (SGPT) 
≤3 × ULN OR 
≤5 × ULN for patients with liver metastases 
Coagulation 
International normalized ratio (INR) or 
prothrombin time (PT) 
Activated partial thromboplastin time 
(aPTT) 
≤1.5 × ULN unless patient is receiving 
anticoagulant therapy as long as PT or PTT is 
within therapeutic range of intended use of 
anticoagulants 
≤1.5 × ULN unless patient is receiving 
anticoagulant therapy as long as PT or PTT is 
within therapeutic range of intended use of 
anticoagulants 
a Creatinine clearance (CrCl) should be calculated per institutional standard. 
b Glomerular filtration rate (GFR) can also be used in place of creatinine or CrCl. 
 
3.1.8. Patients with a known history of being Human Immunodeficiency Virus (HIV) positive 
may participate IF they meet the following eligibility requirements: 
1. They must be stable on their anti-retroviral regimen, and they must be healthy 
from an HIV perspective. 
2. Patients must have an undetectable HIV viral load. 
 
3.1.9. Patients with a known history of hepatitis C virus (HCV) infection must have been treated 
and cured. For patients with HCV infection who are currently on treatment, they are 
eligible if they have an undetectable HCV viral load. For patients with evidence of 
chronic hepatitis B virus (HBV) infection, the HBV viral load must be undetectable on 
suppressive therapy, if indicated. 
 
3.1.10. Patients who have undergone major surgery must have recovered adequately from the 
toxicity and/or complications from the intervention prior to starting therapy. 
 
3.1.11. Patients with known history or current symptoms of cardiac disease, or history of treatment 
with cardiotoxic agents, should have a clinical risk assessment of cardiac function using the 

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New York Heart Association Functional Classification. To be eligible for this trial, patients 
should be class 2B or better. 
3.1.12. Female patients of childbearing potential must have a negative urine or serum pregnancy test 
within 72 hours prior to receiving the first dose of 7+3 treatment. If the urine test is positive or 
cannot be confirmed as negative, a serum pregnancy test will be required. A female of 
childbearing potential is any woman, regardless of sexual orientation or whether they have 
undergone tubal ligation, who meets the following criteria: 1) has not undergone a 
hysterectomy or bilateral oophorectomy; or 2) has not been naturally postmenopausal for at 
least 12 consecutive months (i.e., has had menses at any time in the preceding 12 
consecutive months). 
 
Female patients of childbearing potential must be willing to use an adequate method of 
contraception as outlined in Section 6.6 (Contraception and Pregnancy) for the course of 
the study through 120 days after the last dose of study medication. 
 
Male patients with female partners of childbearing potential must agree to use an adequate 
method of contraception as outlined in Section 6.6 (Contraception and Pregnancy), starting 
with the first dose of study therapy through 120 days after the last dose of study therapy. 
 
NOTE: Abstinence is acceptable if this is the usual lifestyle and preferred contraception 
for the patient. 
 
3.1.13. Ability to understand and the willingness to sign a written informed consent document. 
Participants with impaired decision-making capacity (IDMC) who have a legally- 
authorized representative (LAR) and/or family member available will also be eligible. 
 
 
 
3.2 
Exclusion Criteria 
 
3.2.1. Patients with a known additional malignancy that is progressing or requires active 
treatment. Exceptions include basal cell carcinoma of the skin or squamous cell carcinoma 
of the skin that has undergone potentially curative therapy or in situ cervical cancer. 
 
Prior treatment with the following is not allowed: 
• Patients who have received anthracyclines for treatment of a prior, unrelated, 
curatively-treated malignancy which would limit their ability to receive 7 + 3 
chemotherapy treatment on study. 
• Anti-PD-1, anti-PD-L1, or anti-PD-L2, for a prior, unrelated, curatively-treated 
malignancy, within last 3 months of enrollment in the study. 
• Anti-cancer mAb within 4 weeks, for a prior, unrelated, curatively-treated 
malignancy, prior to study registration or have not recovered (recovery defined as 
baseline or ≤ grade 1) from AEs due to agents administered more than 4 weeks 
earlier. 
• Experimental treatment within 4 weeks prior to study registration. 

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3.2.2. Patients who have had chemotherapy (except hydroxyurea and all trans retinoic acid 
[ATRA] which are allowed but have to be stopped the day before induction therapy starts), 
targeted small molecule therapy (aside from imatinib, dasatinib, or nilotinib), or curative- 
intent radiotherapy within 4 weeks (6 weeks for nitrosoureas or mitomycin C), for a prior 
curatively treated malignancy, prior to entering the study. 
 
3.2.3. Patients who have received prior anthracyclines not to exceed 150 mg/m2 of daunorubicin 
or equivalent for treatment of a prior, unrelated, curatively-treated malignancy which 
would limit their ability to receive 7 + 3 chemotherapy treatment on study. 
 
3.2.4. Patients with a cardiac ejection fraction less than 50% as determined by Echocardiogram or 
MUGA scan. 
 
3.2.5. Other active primary malignancy (other than non-melanomatous skin cancer or carcinoma 
in situ of the cervix) requiring treatment or limiting expected survival to ≤2 years. 
 
NOTE: If there is a history of prior malignancy, they must not be receiving other 
specific treatment (other than hormonal therapy for their cancer). 
 
3.2.6. Patients who have FLT3-mutated AML. 
FLT3-ITD or TKD mutations are defined as a mutation with a ratio of mutant to wild-type 
allele ≥ 0.05 or variant allele fraction of ≥ 5% by PCR or next generation sequencing from 
either bone marrow or peripheral blood. 
Note 1: FLT3, karyotype, or FISH results from bone marrow or peripheral blood that were 
performed up to 3 weeks before initiation of trial therapy are acceptable for eligibility 
determination or therapy stratification as long as they are performed in a CLIA certified 
laboratory. Note 2: Patients are stratified based on age (younger than 65 vs. 65 and older),, 
presence of core-binding abnormalities by FISH or karyotype (yes/no), and by having t- AML 
or AML arising from prior/antecedent MDS (yes/no). 
 
3.2.7. Patients who have not recovered from AEs due to prior anti-cancer therapy (i.e., have not 
returned to baseline or have residual toxicities > grade 1) with the exception of ≤ grade 2 
neuropathy and alopecia. 
 
NOTE: Participants must have recovered from all radiation-related toxicities, not require 
corticosteroids, and not have had radiation pneumonitis. A 1-week washout is permitted 
for palliative radiation (≤2 weeks of radiotherapy) to non-CNS disease. 
 
3.2.8. Patients currently participating and receiving study therapy or have participated in a study of 
an investigational agent and received study therapy or used an investigational device within 4 
weeks of the first dose of treatment are ineligible. 
 
3.2.9. History of hypersensitivity to Pembrolizumab (MK-3475) or any of its excipients, or 
other agents used in this study. 
 
3.2.10. Current use of corticosteroids. 

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EXCEPTION: Low doses of steroids (<10 mg of prednisone or equivalent dose of other 
steroid) used for treatment of non-hematologic medical condition (e.g., chronic adrenal 
insufficiency) is permitted. 
 
3.2.11. Patients who underwent prior allogenic transplant 
 
3.2.12. Patient with a history or current evidence of any condition, therapy, or laboratory abnormality 
that might confound the results of the trial, interfere with the subject’s participation for the full 
duration of the trial, or is not in the best interest of the subject to participate, in the opinion of 
the treating investigator. 
 
3.2.13. Patient with known active CNS disease and/or carcinomatous meningitis before study 
enrollment. Assessment of the CSF is not required to enroll in the study unless there is 
clinical suspicion for CNS involvement. However, if CSF assessment is performed for any 
reason, there should be no evidence of active leukemia in the CSF as per investigator 
judgement. Up to one dose of prophylactic intrathecal chemotherapy is allowed prior to 
study enrollment Subjects with previously treated brain metastases may participate 
provided they are stable (without evidence of progression by imaging for at least four 
weeks prior to the first dose of protocol treatment and any neurologic symptoms have 
returned to baseline), have no evidence of new or enlarging brain metastases, and are not 
using steroids for at least 7 days prior to protocol treatment. This exception does not include 
carcinomatous meningitis which is excluded regardless of clinical stability. 
 
3.2.14. Patients with active autoimmune disease except for patients with hypothyroidism and vitiligo 
that has required systemic treatment in the past 2 years (i.e., with use of disease modifying 
agents, corticosteroids or immunosuppressive drugs). Replacement therapy (e.g., thyroxine, 
insulin, or physiologic corticosteroid replacement therapy for adrenal or pituitary 
insufficiency, etc.) is not considered a form of systemic treatment. 
 
3.2.15. Patients with a known history of non-infectious pneumonitis that required the use of 
steroids or current pneumonitis. 
 
3.2.16. Patients with active, uncontrolled infection as deemed by the treating investigator. . 
 
3.2.17. Patients with a known history of active TB (Bacillus Tuberculosis). 
 
3.2.18. Patients with uncontrolled intercurrent illness. 
 
3.2.19. Patients with psychiatric illness/social situations that would limit compliance with study 
requirements. 
 
3.2.20. Pregnant women are excluded from this study because Pembrolizumab (MK-3475) is 
humanized antibody with the potential for teratogenic or abortifacient effects. Because there 
is an unknown but potential risk for adverse events in nursing infants secondary to 
treatment of the mother with Pembrolizumab (MK-3475), breastfeeding should be 
discontinued if the mother is treated with Pembrolizumab (MK-3475). These potential risks 
may also apply to other agents used in this study. 

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3.2.21. Patient who have received a live vaccine within 30 days of planned start of study therapy. 
 
NOTE: Seasonal influenza vaccines for injection are generally inactivated flu vaccines 
and are allowed; however intranasal influenza vaccines (e.g., Flu-Mist®) are live 
attenuated vaccines, and are not allowed. 
 
3.2.22. Active hemolytic anemia requiring immunosuppressive therapy or other pharmacologic 
treatment. Patients who have a positive Coombs test but no evidence of hemolysis are NOT 
excluded from participation. 
 
3.2.23. Patients with clinically significant disseminated intravascular coagulation (DIC), which 
cannot be managed with supportive care including transfusions, as assessed by 
treating physician, will be excluded from study. 
 
3.2.24. Patients with no bone marrow involvement will be excluded (i.e., those with only 
extramedullary disease). 
 
3.2.25. Patients with acute promyelocytic leukemia will be excluded. 
 
3.3 
Inclusion of Women and Minorities 
 
NIH policy requires that women and members of minority groups and their subpopulations be 
included in all NIH-supported biomedical and behavioral research projects involving NIH- 
defined clinical research unless a clear and compelling rationale and justification establishes to 
the satisfaction of the funding Institute & Center (IC) Director that inclusion is inappropriate 
with respect to the health of the subjects or the purpose of the research. Exclusion under other 
circumstances must be designated by the Director, NIH, upon the recommendation of an IC 
Director based on a compelling rationale and justification. Cost is not an acceptable reason for 
exclusion except when the study would duplicate data from other sources. Women of 
childbearing potential should not be routinely excluded from participation in clinical research. 
Please see http://grants.nih.gov/grants/funding/phs398/phs398.pdf. 
 
 
4. 
REGISTRATION PROCEDURES 
 
4.1 Investigator and Research Associate Registration with CTEP 
 
Food and Drug Administration (FDA) regulations require sponsors to select qualified 
investigators. National Cancer Institute (NCI) policy requires all individuals contributing to 
NCI-sponsored trials to register with their qualifications and credentials and to renew their 
registration annually. To register, all individuals must obtain a Cancer Therapy Evaluation 
Program (CTEP) Identity and Access Management (IAM) account at 
https://ctepcore.nci.nih.gov/iam. In addition, persons with a registration type of Investigator 
(IVR), Non-Physician Investigator (NPIVR), or Associate Plus (AP) (i.e., clinical site staff 
requiring write access to Oncology Patient Enrollment Network (OPEN), Rave, or acting as a 
primary site contact) must complete their annual registration using CTEP’s web-based 
Registration and Credential Repository (RCR) at https://ctepcore.nci.nih.gov/rcr. 

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RCR utilizes five-person registration types. 
• IVR: MD, DO, or international equivalent, 
• NPIVR: advanced practice providers (e.g., NP or PA) or graduate level researchers (e.g., 
PhD), 
• AP: clinical site staff (e.g., RN or CRA) with data entry access to CTSU applications 
(e.g., Roster Update Management System [RUMS], OPEN, Rave,), 
• Associate (A): other clinical site staff involved in the conduct of NCI-sponsored trials, 
and 
• Associate Basic (AB): individuals (e.g., pharmaceutical company employees) with 
limited access to NCI-supported systems. 
 
RCR requires the following registration documents: 
 
Documentation Required 
IVR NPIVR AP 
A 
AB 
FDA Form 1572 
✔ 
✔ 
Financial Disclosure Form 
✔ 
✔ 
✔ 
NCI Biosketch (education, training, employment, license, 
and certification) 
✔ 
✔ 
✔ 
GCP training 
✔ 
✔ 
✔ 
Agent Shipment Form (if applicable) 
✔ 
CV (optional) 
✔ 
✔ 
✔ 
 
An active CTEP-IAM user account and appropriate RCR registration is required to access all 
CTEP and Cancer Trials Support Unit (CTSU) websites and applications. In addition, IVRs and 
NPIVRs must list all clinical practice sites and Institutional Review Boards (IRBs) covering their 
practice sites on the FDA Form 1572 in RCR to allow the following: 
 
• Addition to a site roster, 
• Assign the treating, credit, consenting, or drug shipment (IVR only) tasks in OPEN, 
• Act as the site-protocol Principal Investigator (PI) on the IRB approval, and 
• Assign the Clinical Investigator (CI) role on the Delegation of Tasks Log (DTL). 
 
In addition, all investigators act as the Site-Protocol PI, consenting/treating/drug shipment, or as 
the CI on the DTL must be rostered at the enrolling site with a participating organization (i.e., 
Alliance). 
 
Additional information is located on the CTEP website at 
https://ctep.cancer.gov/investigatorResources/default.htm. For questions, please contact the 
RCR Help Desk by email at RCRHelpDesk@nih.gov. 
 
4.2 Site Registration 

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IRB Approval 
 
Sites participating with the NCI Central Institutional Review Board (NCI CIRB) must submit the 
Study Specific Worksheet for Local Context (SSW) to the CIRB using IRBManager to indicate 
their intent to open the study locally. The NCI CIRB’s approval of the SSW is automatically 
communicated to the CTSU Regulatory Office, but sites are required to contact the CTSU 
Regulatory Office at CTSURegPref@ctsu.coccg.org to establish site preferences for applying 
NCI CIRB approvals across their Signatory Network. Site preferences can be set at the network 
or protocol level. Questions about establishing site preferences can be addressed to the CTSU 
Regulatory Office by emailing the email address above or calling 1-888-651-CTSU (2878). 
 
In addition, the Site-Protocol PI (i.e., the investigator on the IRB/REB approval) must meet the 
following five criteria to complete processing of the IRB/REB approval record: 
• Holds an Active CTEP status, 
• Rostered at the site on the IRB/REB approval (applies to US and Canadian sites only) and 
on at least one participating roster, 
• If using NCI CIRB, rostered on the NCI CIRB Signatory record, 
• Includes the IRB number of the IRB providing approval in the Form FDA 1572 in the 
RCR profile, and 
• Holds the appropriate CTEP registration type for the protocol. 
 
Additional Requirements 
 
Additional requirements to obtain an approved site registration status include: 
• An active Federal wide Assurance (FWA) number, 
• An active roster affiliation with the Lead Protocol Organization (LPO) or a Participating 
Organization, and 
• Compliance with all protocol-specific requirements (PSRs). 
 
4.2.1. Downloading Regulatory Documents 
 
Download the site registration forms from the protocol-specific page located on the CTSU 
members’ website. Permission to view and download this protocol and its supporting documents 
is restricted based on person and site roster assignment. To participate, the institution and its 
associated investigators and staff must be associated with the LPO or a Participating 
Organization on the protocol. 
 
• Log on to the CTSU members’ website (https://www.ctsu.org) using your CTEP-IAM 
username and password, 
• Click on Protocols in the upper left of your screen. 
o Enter the protocol number in the search field at the top of the protocol tree, or 
o Click on the By Lead Organization folder to expand, then select LAO-CT018, and 
protocol number 10300, 

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• Click on Documents, select Site Registration, and download and complete the forms 
provided. (Note: For sites under the CIRB initiative, IRB data will load automatically to 
the CTSU as described above.) 
 
4.2.2. Protocol Specific Requirements For 10300 Site Registration 
 
Upon site registration approval in RSS, the enrolling site may access OPEN to complete 
enrollments. The enrolling site will select their credentialed provider treating the subject in the 
OPEN credentialing screen, and may need to answer additional questions related to treatment in 
the eligibility checklist. 
 
• Specimen Tracking System Training Requirement: 
o All data entry users (Clinical Research Associate role) at each participating site will 
need to complete the Theradex-led training. 
o Theradex will provide a certificate of completion, which will need to be submitted to 
the CTSU through the Regulatory Submission Portal. 
o The training is a one-time only requirement per individual. If an individual has 
previously completed the training for another ETCTN study, the training does not 
need to be completed again nor does the certificate of completion need to be 
resubmitted to the CTSU. However, new versions of the Specimen Tracking System 
may require new training. 
o This training will need to be completed before the first patient enrollment at a given 
site. 
o Please contact STS Support at Theradex for the training 
(STS.Support@theradex.com,, Theradex phone: 609-799-7580). 
 
4.2.3. Submitting Regulatory Documents 
 
Submit required forms and documents to the CTSU Regulatory Office via the Regulatory 
Submission Portal on the CTSU website. 
 
To access the Regulatory Submission Portal, log on to the CTSU members’ website  
Regulatory  Regulatory Submission. 
 
Institutions with patients waiting that are unable to use the Regulatory Submission Portal should 
alert the CTSU Regulatory Office immediately at 1-866-651-2878 in order to receive further 
instruction and support. 
 
Delegation of Tasks Log (DTL) 
 
Each site must complete a protocol-specific DTL using the DTL application in the Delegation 
Log section on the CTSU members’ website. The Clinical Investigator (CI) is required to review 
and electronically sign the DTL prior to the site receiving an Approved site registration status 
and enrolling patients to the study. To maintain an approved site registration status the CI must re- 
sign the DTL at least annually and when a new version of the DTL is released; and activate new 
task assignments requiring CI sign-off. Any individual at the enrolling site on a 

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participating roster may initiate the site DTL. Once the DTL is submitted for CI approval, only 
the designated DTL Administrators or the CI may update the DTL. Instructions on completing 
the DTL are available in the Help Topics button in the DTL application and include a Master 
Task List, which describes DTL task assignments, CI signature, and CTEP registration 
requirements. 
 
 
4.2.4. Checking Site Registration Status 
 
You can verify your site’s registration status on the members’ side of the CTSU website. 
 
• Log on to the CTSU members’ website 
• Click on Regulatory at the top of your screen 
• Click on Site Registration 
• Enter your 5-character CTEP Institution Code and click on Go 
 
Note: The status shown only reflects institutional compliance with site registration 
requirements as outlined above. It does not reflect compliance with protocol 
requirements for individuals participating on the protocol or the enrolling investigator’s 
status with the NCI or their affiliated networks. 
 
4.3 Patient Registration 
 
4.3.1 OPEN/IWRS 
 
The Oncology Patient Enrollment Network (OPEN) is a web-based registration system available 
on a 24/7 basis. OPEN is integrated with CTSU regulatory and roster data and with the Lead 
Protocol Organization (LPOs) registration/randomization systems or Theradex Interactive Web 
Response System (IWRS) for retrieval of patient registration/randomization assignment. OPEN 
will populate the patient enrollment data in NCI’s clinical data management system, Medidata 
Rave. 
 
Requirements for OPEN access: 
 
• A valid CTEP-IAM account. 
• To perform enrollments or request slot reservations: Be on an LPO roster, ETCTN 
Corresponding roster, or Participating Organization roster with the role of Registrar. 
Registrars must hold a minimum of an AP registration type. 
• If a DTL is required for the study, the registrar(s) must hold the OPEN Registrar task on 
the DTL for the site. 
• Have an approved site registration for a protocol prior to patient enrollment. 
 
To assign an Investigator (IVR) or Non-Physician Investigator (NPIVR) as the treating, 
crediting, consenting, drug shipment (IVR only), or receiving investigator for a patient transfer in 
OPEN, the IVR or NPIVR must list the IRB number used on the site’s IRB approval on their 
Form FDA 1572 in RCR. If a DTL is required for the study, the IVR or NPIVR must be 
assigned the appropriate OPEN-related tasks on the DTL. 

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Prior to accessing OPEN, site staff should verify the following: 
 
• Patient has met all eligibility criteria within the protocol stated timeframes, and 
• All patients have signed an appropriate consent form and HIPAA authorization form (if 
applicable). 
 
Note: The OPEN system will provide the site with a printable confirmation of registration and 
treatment information. Please print this confirmation for your records. 
 
Access OPEN at https://open.ctsu.org or from the OPEN link on the CTSU members’ website. 
Further instructional information is in the OPEN section of the CTSU website at 
https://www.ctsu.org or https://open.ctsu.org. For any additional questions, contact the CTSU 
Help Desk at 1-888-823-5923 or ctsucontact@westat.com. 
 
 
 
4.3.2 Special Instructions for Patient Enrollment 
 
This Study will use the ETCTN Specimen Tracking System (STS). 
 
• All biospecimens collected for this trial must be submitted using the ETCTN Specimen 
Tracking System (STS) unless otherwise noted. 
• The system is accessed through special Rave user roles: “CRA Specimen Tracking” for 
data entry at the treating institutions and “Biorepository” for users receiving the 
specimens for processing and storage at reference labs and the Biorepository. 
• Please refer to the Medidata Account Activation and Study Invitation Acceptance link on 
the CTSU website under the Rave/DQP tab. 
Important: Failure to complete required fields in STS may result in a delay in 
sample processing. Any case reimbursements associated with sample submissions will 
not be credited if samples requiring STS submission are not logged into STS. 
 
Detailed instructions can be found in Section 5.3. 
 
4.3.3 OPEN/IWRS Questions? 
 
Further instructional information on OPEN is provided on the OPEN link of the CTSU website 
at https://www.ctsu.org or at https://open.ctsu.org. For any additional questions contact the 
CTSU Help Desk at 1-888-823-5923 or ctsucontact@westat.com. 
 
Theradex has developed a Slot Reservations and Cohort Management User Guide, which is 
available on the Theradex website: http://www.theradex.com/clinicalTechnologies/?National- 
Cancer-Institute-NCI-11. This link to the Theradex website is also on the CTSU website OPEN 
tab. For questions about the use of IWRS for slot reservations, contact the Theradex Helpdesk at 
609-619-7862 or Theradex main number 609-799-7580; CTMSSupport@theradex.com. 
 

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4.4 General Guidelines 
 
Following registration, patients should begin protocol treatment within 7 days. Issues that would 
cause treatment delays should be discussed with the Principal Investigator. If a patient does not 
receive protocol therapy following registration, the patient’s registration on the study may be 
canceled. The Study Coordinator should be notified of cancellations as soon as possible. 
 
 
5 
BIOMARKER, CORRELATIVE, AND SPECIALSTUDIES 
 
5.1 Summary Table for Specimen Collection 
 
Time 
Point 
Specimen2 
Send Specimens To: 
Baseline 1 
• 5 mL bone marrow aspirate (mandatory) 
Local CLIA Labs 
• 10 mL bone marrow aspirate (mandatory) 
• 10 mL blood in EDTA lavender top vacutainer tube 
(mandatory) 
Brent Wood’s lab, 
Children’s Hospital Los 
Angeles 
• 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred 
Hutchinson Cancer 
Research Center  
• 1 bone marrow biopsy core (processed to FFPE 
blocks) (mandatory) 
• 16 mL bone marrow aspirate sent unprocessed in 
EDTA tubes (mandatory) 
• 30 mL blood in sodium heparin Green top 
vacutainer tubes (mandatory) 
• Snap Frozen Skin Punch Biopsy 
• Stool (OMNIgene-GUT kit) 
NCI Early-Phase and 
Experimental Clinical 
Trials Biospecimen Bank 
(EET Biobank) 
Induction therapy phase – Between Days 14 – 21 
• 10 mL bone marrow aspirate (mandatory) 
• 10 mL blood in EDTA lavender top vacutainer tube 
(mandatory) 
 
Brent Wood’s lab, 
Children’s Hospital Los 
Angeles 
• 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred 
Hutchinson Cancer 
Research Center  
End of induction phase 

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39 
 
 
• 10 mL bone marrow aspirate (mandatory) 
• 10 mL blood in EDTA lavender top vacutainer tube 
(mandatory) 
 
Brent Wood’s lab, 
Children’s Hospital Los 
Angeles 
• 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred 
Hutchinson Cancer 
Research Center  
Consolidation therapy phase – Post Cycle 1 
 
• 10 mL bone marrow aspirate (mandatory) 
• 10 mL blood in EDTA lavender top vacutainer tube 
(mandatory) 
 
Brent Wood’s lab, 
Children’s Hospital Los 
Angeles 
• 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred 
Hutchinson Cancer 
Research Center  
• Stool (OMNIgene-GUT kit) 
EET Biobank 
End of consolidation phase 1 
Time 
Point 
Specimen2 
Send Specimens To: 
• 10 mL bone marrow aspirate (mandatory) 
• 10 mL blood in EDTA lavender top vacutainer tube 
(mandatory) 
 
Brent Wood’s lab, 
Children’s Hospital Los 
Angeles 
• 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred 
Hutchinson Cancer 
Research Center  
• 1 bone marrow biopsy core (processed to FFPE 
blocks) (mandatory) 
• 16 mL bone marrow aspirate sent unprocessed in 
EDTA tubes (mandatory) 
• 30 mL blood in sodium heparin Green top 
vacutainer tubes (mandatory) 
EET Biobank 
Maintenance therapy/observation phase – Every 3 months (±7 days) 
• 10 mL bone marrow aspirate (optional – only if 
patients have a standard-of-care biopsy that 
coincides with this collection time point.) 
• 10 mL blood in EDTA lavender top vacutainer tube 
(mandatory) 
 
Brent Wood’s lab, 
Children’s Hospital Los 
Angeles 

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40 
 
 
• 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred 
Hutchinson Cancer 
Research Center  
After 6 months of Treatment 
• 1 bone marrow biopsy core (processed to FFPE 
blocks) (optional) 
• 16 mL bone marrow aspirate sent unprocessed in 
EDTA tubes (optional) 
• 30 mL blood in sodium heparin Green top 
vacutainer tubes (mandatory) 
EET Biobank 
After 1 year of Treatment 
• 1 bone marrow biopsy core (processed to FFPE 
blocks) (optional) 
• 16 mL bone marrow aspirate sent unprocessed in 
EDTA tubes (optional) 
• 30 mL blood in sodium heparin Green top 
vacutainer tubes (mandatory) 
EET Biobank 
 
Relapse 
• 1 bone marrow biopsy core (processed to FFPE 
blocks) (optional) 
• 16 mL bone marrow aspirate sent unprocessed in 
EDTA tubes (mandatory) 
• 30 mL blood in sodium heparin Green top 
vacutainer tubes (mandatory) 
EET Biobank 
End of maintenance/observation phase* 
• 10 mL bone marrow aspirate (mandatory) 
• 10 mL blood in EDTA lavender top vacutainer tube 
(mandatory) 
 
Brent Wood’s lab, 
Children’s Hospital Los 
Angeles 
• 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred 
Hutchinson Cancer 
Research Center  
 
1 Sample priority in cases of limited aspirate will be for MRD testing by flow cytometry and DS 
(one tube of 10 cc or whatever amount can be extracted). Every effort should be made to get an 
aspirate for MRD assessment at all the above required time points, but in cases were an aspirate 
cannot be collected-including dry taps-the patient will not be excluded and assessments will be 
performed on PB which should be collected at every time that BM is collected as described 
above. 
2 All bone marrows performed (aspirate and/or biopsy) should be evaluated for disease status 
locally at the institution. The bone marrow report must be uploaded to the STS and provided to 
the EET Biobank, when available. 

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3 For skin punch biopsies, the Skin Punch Biopsy Verification Form (Appendix F) must be  
completed and sent with the tissue to the EET Biobank.   
 
*For patients who do not progress. 
 
5.2 Specimen Procurement Kits and Scheduling 
 
5.2.1 Specimen Shipping Kits 
 
Kits for the submission of frozen skin punch tissue, and for collection and shipment of stool to the EET 
Biobank can be ordered online via the Kit Management system: (https://kits.bpc-apps.nchri.org).  
 
Users at the clinical sites will need to set up an account in the Kit Management system and select a 
specific clinical trial protocol to request a kit. Please note that protocol may include more than one 
type of kit. Each user may order two kits per kit type per day (daily max = 6 kits). Kits are shipped 
ground, so please allow 5-7 days for receipt. A complete list of kit contents for each kit type is 
located on the Kit Management system website. 
 
It is preferred that the sites maintain 2 stool kits on site and 5 Streck tubes on site to prevent delays 
of screening for patients who might need to start study therapy quickly. 
 
− Institutional supplies must be used for all other specimen collection, processing, and 
shipment, with the exception of Streck tubes which will be supplied to the sites on 
request. 
− Please request a supply upon site activation. Email lbeppu@fredhutch.org, and 
jradich@fredhutch.org to request shipment of Streck tubes. 
− Additional questions regarding Streck tube supply may be directed to Lan Beppu in Dr. 
Radich lab at Fred Hutch at 206-667-6966. 
− Aside from the different type of tube, these DS ctDNA-based longitudinal MRD monitoring 
samples will be collected, processed, and shipped in the same manner as Dr. Wood’s MRD 
tubes at the different time points listed in Section 5.1 Summary Table for Specimen 
Collection. These Streck tube samples will be sent directly to Dr. Radich’s laboratory where 
the assays will be conducted. 
 
5.2.2 Scheduling of Specimen Collections to the EET Biobank 
 
Please adhere to the following guidelines when scheduling procedures to collect tissue: 
• 
Bone marrow core biopsies and skin punch biopsies may be collected and processed to 
FFPE block any day. FFPE blocks may be shipped Monday through Thursday. 
• 
Fresh blood and bone marrow specimens may be collected and shipped Monday 
through Friday. 
• 
Specimens submitted frozen such as skin punch biopsy tissue can be 
collected on any day but must be stored frozen until shipped to the EET 
Biobank on Monday through Thursday. In the event that frozen specimens 
cannot be shipped immediately, they must be maintained in a -70°C to -80°C 
freezer. 
• 
Stool samples may be collected any day and may be shipped on Monday through 
Thursday. 

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5.2.3. Scheduling of Specimen Collections for Brent Wood’s lab, Children’s Hospital Los Angeles. 
 
Do not draw specimens on Fridays for delivery on Saturday. One EDTA "BM" tube (10 mL of 
BM aspirate) and one EDTA "PB" tube (10 mL of peripheral blood) should be shipped overnight 
with a cold pack (do not allow the samples to freeze). 
 
5.2.4. Scheduling of Specimen Collection for Radich laboratory, Fred Hutchinson Cancer 
Research Center 
Please adhere to the following guidelines when scheduling procedures to collect blood in Streck 
tubes: 
1. We can receive samples Monday-Friday, do not ship samples on Fridays for Saturday receipt. 
Exceptions can be made on case-by-case basis for Friday shipping but should be discussed in 
advance of performing screening bone studies with the study chair and approved. 
2. Samples should be shipped within 5 days of collection, ideally no later than 48 hrs after 
collection. 
3. Samples do not need to be refrigerated; it can be kept at ambient temperature. 
4. Our shipping address is: 
Radich Laboratory 
Fred Hutchinson Cancer Research Center 
1100 Fairview Avenue North 
Room D4-385 
Seattle, WA 98109 
Email: lbeppu@fredhutch.org 
 
 
5.3 Specimen Tracking System Instructions 
5.3.1. Specimen Tracking System Overview and Enrollment Instructions 
For the ETCTN STS, the following information will be requested: 
• Protocol Number 
• Investigator Identification 
o Institution and affiliate name 
o Investigator’s name 
• Eligibility Verification: Patients must meet all the eligibility requirements listed in 
Section 3. 
• Additional Requirements: 
o Patients must provide a signed and dated, written informed consent form. 
 
Upon enrolling a patient, IWRS will communicate with OPEN, assigning two separate and 
unique identification numbers to the patient, a Universal patient ID (UPID) and a Treatment 
patient ID. The UPID is associated with the patient and used each and every time the patient 
engages with the portion of this or any other protocol that uses the ETCTN Specimen Tracking 
System. The UPID contains no information or link to the treatment protocol. IWRS will maintain 
an association between the UPID for ETCTN biobanking and molecular characterization and any 
treatment protocols the patient participates in, thereby allowing analysis of the molecular 

NCI Protocol #: 10300  
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43 
 
 
characterization results with the clinical data. 
 
Immediately following enrollment, the institutional report for the diagnosis (e.g., bone marrow 
pathology report) under which the patient is being enrolled must be uploaded into Rave when 
available.  The report must include the collection date, and the IWRS-assigned UPID and 
patient study ID for this trial. 
Important: Remove any personally identifying 
information, including, but not limited to, the patient’s name, date of birth, initials, 
medical record number, and patient contact information from the institutional 
pathology report prior to submission. 
 
Additionally, please note that the STS software creates pop-up windows when reports are 
generated, so you will need to enable pop-ups within your web browser while using the software. 
 
For questions regarding the Specimen Tracking System, please contact the STS Support at 
STS.Support@theradex.com. 
 
The Shipping List report must be included with all sample submissions. 
5.3.2 Specimen Labeling 
 
5.3.2.1 Blood or Bone Marrow Specimen Labels 
 
Include the following on blood specimens (including whole blood or bone marrow, and frozen, 
processed blood products – like serum and plasma): 
• 
Patient Study ID 
• 
Universal Patient ID(UPID) 
• 
Specimen ID (automatically generated by Rave) 
• 
Time point 
• 
Specimen type (e.g., blood, serum) 
• 
Collection date. 
• 
BM Laterality (e.g., Right (R) or Left (L) Side if both submitted) – to be added by 
hand 
 
5.3.2.2 Tissue or Skin Punch Biopsy Specimen Labels 
 
Include the following on all tissue specimens or containers (e.g., formalin jar): 
• 
Patient Study ID 
• 
Universal Patient ID(UPID) 
• 
Specimen ID (automatically generated by Rave) 
• 
Time point 
• 
Specimen type (e.g., Bone Marrow Aspirate etc.) 
• 
BM Core Biopsy Laterality (e.g., Right (R) or Left (L) Side if both submitted) – to be 
added by hand 
• 
Surgical pathology ID (SPID) number 
• 
Collection date 
 
5.3.2.3 
Stool Specimen Labels 
Include the following on all stool  specimens or containers (e.g., vials): 

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44 
 
 
• Patient Study ID 
• Universal Patient ID (UPID) 
• Specimen ID (automatically generated by Rave) 
• Time point 
• Specimen type (e.g., stool etc.) 
• 
Collection date 
 
5.3.2.4 
Example of Specimen Label Generated by STS 
 
STS includes a label printing facility, accessed via the Print Label CRF in the All Specimens folder. A 
generated PDF is emailed to the user as a result of saving that form. 
 
The following image is an example of a tissue specimen label printed on a label that is 0.5” high and 
1.28” wide. 
 
 
 
 
The QR code in the above example is for the Specimen ID shown on the second line. 
 
Labels may be printed on a special purpose label printer, one label at a time, or on a standard laser 
printer, multiple labels per page. Theradex recommends the use of these low temperature waterproof 
labels for standard laser printers: https://www.labtag.com/shop/product/cryo-laser-labels-1-28-x-0-5- 
cl-23-colors-available/ 
 
The last line item on the label includes the following data points joined together: 
1. Tissue only: Primary (P), Metastatic (M), Normal (N) tissue indicated at the beginning of 
the specimen ID; this field is blank if not relevant (e.g., for blood) 
2. Block ID or blank if not relevant. 
3. SPID (Surgical Pathology ID) or blank if none 
4. An optional alpha-numeric code that is protocol specific and is only included if the 
protocol requires an additional special code classification. 
 
Space is provided at the bottom of the label for the handwritten date and optional time. The last 
line on the example label is for the handwritten date and optional time. 
 
 
5.3.3. Overview of Process at Treating Site 
 
5.3.3.1 OPEN Registration 

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All registrations will be performed using the Oncology Patient Enrollment Network (OPEN) 
system. OPEN communicates automatically with the Interactive Web Response System (IWRS) 
which handles identifier assignments, any study randomization and any prescribed slot 
assignments. If specimen analysis is required to determine eligibility, the protocol will be setup 
with multi-step registration. 
 
Registration with eligibility specimen analysis: 
1. Site enters first step data into OPEN. 
2. IWRS receives data from OPEN, generates the Patient Study ID and the Universal 
Patient ID, both of which are sent back to OPEN. 
3. IWRS sends first step registration data, including the IDs and a TAC of “NOT 
REG” directly to Rave. 
4. The specimen tracking system in Rave is utilized for the specimen that contributes 
to eligibility determination. 
5. Site enters second and any subsequent step data into OPEN including results of 
specimen analysis. 
6. IWRS receives all data from OPEN, then sends it onto Rave with either the 
treatment TAC or a TAC of “SCRN FAIL”. 
7. In addition to the specimen tracking forms completed to determine eligibility, data 
entry for screen failure patients should include Histology and Disease, all forms in 
the Baseline folder, any lab forms connected to eligibility determination, and Off 
Treatment/Off Study. 
 
Any data entry errors made during enrollment should be corrected in Rave. 
 
5.3.3.2. Rave Specimen Tracking Process Steps 
 
Step 0: Log into Rave via your CTEP-IAM account, then navigate to the appropriate 
participant. 
Step 1: Complete the Histology and Disease form (but do not upload reports until a 
specimen label can be applied to them) and the Baseline forms regarding Prior 
Therapies. Enter the initial clinical specimen data: 
• Specimen Tracking Enrollment CRF: Enter Time Point, Specimen Category, 
Specimen Type, Block number, Tissue type, Surgical Path ID, number of labels 
needed (include extra labels to apply to reports to be uploaded). CRF generates 
unique Specimen ID. 
 
Step 2: Print labels using the Print Labels CRF located in the All Specimens folder, then 
collect specimen. 
• Label specimen containers and write collection date and time on each label. For bone 
marrow samples, also write R for right or L for left on the label. 
• After collection, store labeled specimens as described in Section 5.3.2. 
• Apply an extra specimen label to each report before scanning. Return to the 
Histology and Disease form to upload any initial Pathology, Radiology, Bone 
Marrow, Molecular Reports (up to 4), Surgical (or Operative) reports and Tissue 
Biopsy Verification form (when applicable). Return to Specimen Tracking 

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Enrollment CRF to upload any molecular report (one per specimen) and/or 
specimen specific pathology or related report (one per specimen), and/or the Skin 
Punch Biopsy Verification Form (when applicable). Uploaded reports should 
have protected health information (PHI) data, like name, date of birth, mailing 
address, medical record number or social security number (SSN), redacted. Do not 
redact SPID, block number, diagnosis, or relevant dates (such as collection date) 
and include the UPID and patient study ID on each document (either by adding a 
label or hand-writing).  
 
Step 3: Complete specimen data entry. 
• Specimen Transmittal Form: Enter Collection date and time and other required 
specimen details. 
 
Step 4: When ready to ship, enter shipment information. 
• Shipping Status CRF: Enter tracking number, your contact information, 
recipient, number of sample containers and ship date once for the first 
specimen in a shipment. 
• Copy Shipping CRF: In the specimen folders for additional specimens (if any) that 
will be shipped with the initial specimen, please use the Copy Shipping form to 
derive common data into additional Shipping Status forms. A few unique fields will 
still need to be entered in Shipping Status. 
 
Step 5: Print shipping list report and prepare to ship. 
• Shipping List report is available at the site level. 
• Print two copies of the shipping list, one to provide in the box, the other for your 
own records. 
• Print pathology or other required reports to include in the box. Be sure the printed 
copy includes the specimen label. 
 
Step 6: Send email notification. 
• For only one of the specimens in the shipment, click “Send Email Alert” 
checkbox on the Shipping Status CRF to email recipient. 
 
Step 7: Ship the specimen(s). 
Step 8: Monitor the Receiving Status form located in each specimen folder for 
acknowledgment of receipt and adequacy. 
 
5.4 Specimen Collection and Processing 
 
5.4.1. 
Pre-Analytic Information 
Collection site must record all preanalytical information and enter the following into a specimen 
tracking system (STS) used by each trial network or record and provide with shipping manifest: 
1. Time/date blood or tissue sample collection was made as Time/Date Specimen 
Collected. 
2. Ischemia start time (time when sample was devascularized OR estimated time of 
surgery)—Tissue Collection Time/Date. 

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3. Ischemic end time for each tissue core and surgical segment (time when sample was 
moved to preservative such as formalin or dry ice)—Tissue Processing (Formalin 
Start) Time/Date. 
4. Completion of formalin fixation should be recorded as Formalin End Time/Date in 
the STS (or under “comments” if field is not available). 
5. Start of 70% Ethanol dehydration should be recorded as Ethanol Start Time/Date in 
STS (or under “comments” if field is not available) 
6. Time when fixed tissue, held in Ethanol, was placed into an automated processor 
should be recorded as Ethanol End Time/Date in the STS (or under “comments” if 
field is not available). 
7. Core # for each core needle biopsy obtained. Each core should be recorded in the 
STS as a separate specimen with a unique Specimen ID that captures the 
chronological order in which the biopsy cores were obtained. 
 
5.4.2 Bone Marrow Aspirate Collection 
 
5.4.2.1 
Collection of Bone Marrow Aspirate 
 
Bone Marrow Aspiration Procedure: 
 
 
1. Label EDTA tubes according to the instructions in section 5.3.2. 
 
2. For time points with submissions to both Brent Wood’s lab, Children’s Hospital Los Angeles, 
and the EET Biobank: Obtain 26 mL of bone marrow aspirate into a single tube and aliquot 
10 mL in the first EDTA tube (mandatory, to ship to Dr. Wood’s Lab) and aliquot 16 mL 
into two EDTA tubes (~8 mL in each, to ship to EET Biobank) (mandatory). Mix the 
EDTA tubes, by inversion, 8-10 times. Ship the first tube on the day of collection 
(whenever possible) according to instructions in section 5.5. This tube will be sent to 
Brent Wood’s lab for MRD studies. The second and third tubes will be sent to the 
EET Biobank for immune correlative studies. 
 
3. For time points with only shipping to Dr. Wood’s Lab: obtain 10 mL of bone marrow 
aspirate and aliquot into an EDTA tube (optional during the maintenance phase). Mix the 
EDTA tubes, by inversion, 8-10 times. Ship this tube on the day of collection (whenever 
possible) according to instructions in section 5.5. This tube will be sent to Brent Wood’s 
lab for MRD studies. 
 
4. 
For time points submissions sent only to the EET Biobank: obtain 16 mL of bone 
marrow aspirate and aliquot ~8 mL into each EDTA tube (optional). Mix the EDTA 
tubes, by inversion, 8-10 times. This tube will be sent to the EET Biobank for 
immune correlative studies. 
 
5. Ship on the day of collection. If liquid specimens cannot be shipped on the day of 
collection, then store tubes at 4ºC until shipment. 
 

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**For baseline bone marrow, obtain an additional 5 mL of bone marrow aspirate and aliquot in 
EDTA tube (to be sent to local CLIA labs for cytogenetics, next generation sequencing including 
CBF genetic alterations and FLT3 mutations). 
 
Send all tubes after confirming their specimen labels to the respective laboratories. 
 
 
5.4.3 Bone Marrow Core Biopsy Collection and Processing 
 
5.4.3.1 
Collection of Bone Marrow Core Biopsy 
 
Bone marrow core biopsies should be collected as per institutional guidelines and standard 
operating procedures. 
 
5.4.3.2 
Processing of Bone Marrow Core Biopsy 
 
Each bone marrow core biopsies should be processed into separate FFPE blocks (1 core per block) 
for shipment per institutional guidelines and standard operating procedures. Bone marrow core 
biopsies should be decalcified in 10% Formic acid, after fixing with neutral buffered-formalin, for 
4-6 hours prior to embedding. However, the institutions may follow their respective institutional 
protocol guidelines if this reagent is not available. This sample is sent to the EET Biobank. 
 
1. Neutral-buffered formalin must be used as fixative (no acid-based products). 
2. Samples must be fixed in formalin for 12-24 hours and embedded directly at the 
collection site. Embedding must be completed within 72 hours of adding 70% 
ethanol to tissue. 
3. Sites must use automated tissue processors and not use microwave tissue processors 
(Refer to Appendix E). 
4. Sites should follow embedding protocols where the total processing time from 70% 
ethanol to block embedding exceeds 4 hours. 
 
5.4.4 Flash Freezing of Skin Punch Biopsies 
 
1. Tissue should be frozen as soon as possible.  Optimally, freeze within 30 minutes from 
biopsy collection. 
2. Prior to tissue collection: 
a. Label cryovial(s) according to instructions in Section 5.3.2.2.  
b. Place cryovial(s) on dry ice to freeze. The vials should appear frosty when ready.  
3. Obtain one ≥5mm skin punch biopsy. 
4. Immediately place tissue in foil and allow to completely freeze (using either direct contact 
with dry ice, or liquid nitrogen vapor).  
5. Gently remove the frozen tissue from the foil. If the tissue is sticking to the foil, then gently 
run a finger over the back of the foil to loosen the tissue.  
6. Using clean forceps place each tissue core in a separate pre-chilled cryovial. Tissue should 
move freely in the vial. 
7. Place the tissue in a -70 to -80°C freezer.  Keep frozen until shipment to the EET Biobank. 
 

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5.4.5 Blood Collection` 
 
5.4.5.1 Collection of Blood in EDTA Tubes for Shipment to Brent Wood’s lab, Children’s 
Hospital Los Angeles 
 
1. Label EDTA tubes according to the instructions in section 5.3.1. 
2. Collect 10 mL of blood in 1 EDTA tubes and gently invert tube 8-10 times to mix. 
3. Identify the peripheral blood tube by writing the letters “PB” on the tube. 
4. This blood sample is sent to Brent Wood’s lab, Children’s Hospital Los 
Angeles and can be shipped at ambient temperature. 
5. Ship on day of collection (whenever possible) according to instructions in section 5.5. 
6. If blood cannot be shipped on the day of collection (e.g., a late scheduled collection), 
then refrigerate until shipment. 
 
5.4.5.2 Collection of Blood in Streck Tubes for Shipment to Radich laboratory, Fred 
Hutchinson Cancer Research Center 
1. Label Streck tubes according to the instructions in section 5.3.1 
2. Collect 8-10 mL of blood in 1 Streck tube and gently invert tube 8-10 times to mix 
3. Identify the peripheral blood tube by writing the letters “PB” on the tube. 
4. This blood sample is sent to Radich laboratory, Fred Hutchinson Cancer 
Research Center, and can be shipped at ambient temperature. 
5. Ship on day of collection (whenever possible) according to instructions in section 5.5. 
 
5.4.5.3. 
Collection of Blood in Green Top Sodium Heparin Tubes 
 
1. Label Green Top tubes according to the instructions in section 5.3.1 
2. Collect 30 mL of blood in 3 Heparin tubes and gently invert tube 8-10 times to mix. 
3. Identify the peripheral blood tube by writing the letters “PB” on the tube. 
4. Blood samples being sent to the EET Biobank can be shipped at ambient 
temperature. 
5. Ship on day of collection (whenever possible) according to instructions in section 5.5. 
6. If blood cannot be shipped on the day of collection (e.g., a late scheduled collection), 
then refrigerate until shipment. 
7. An external sample label should be fixed to the shipping container to alert the 
Biorepository of blood sample collection time and date (this helps to identify and 
prioritize received samples that have processing time requirements). 
8. Blood should be shipped ambient FedEx Priority Overnight to the biorepository 
where it is processed the day of receipt within 24 hours of collection (not to exceed 
48 hours). 
 
5.4.6 Collection of Stool in OMNIgene-GUT Tubes for Shipment to the EET Biobank 
 
Baseline and “Consolidation therapy phase – Post Cycle 1” timepoints will use OMNIgene GUT kits 
(OMR-200.100—shipped ambient) which include a DNA stabilizing solution. Collection kits will 
include directions for specimen collection and shipment as well as collection and shipping supplies. 
 

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1. Clinical site staff will explain to patients how to use the kits at the clinic or in the privacy 
of their home. The collection tube must be labeled by clinical site staff according to 
instructions in 5.3.2 prior to being provided to the patient. 
2. The patient will collect the stool sample and return the sample to the site staff. It is 
recommended that patients return the specimens within 24 hours, when collected at home. 
The stool specimen should be kept ambient until shipment. 
3. The stool and a shipping manifest should be shipped on day of collection (whenever 
possible) according to instructions provided in the kit and section 5.5.1.2. 
4. Collection site staff should not ship any samples that do not utilize the collection kit 
properly. 
 
 
5.5 Shipping Specimens from Clinical Site to the EET Biobank 
5.5.1 General Shipping Information 
 
When kits are provided, the shipping container sent with kit contents should be used to ship specimens 
to the EET Biobank.  In winter months, please include extra insulation, such as bubble wrap, inside the 
shipping container. 
 
5.5.1.1 Required Forms for Specimen Submissions: 
 
Each document submitted with the specimen must be labeled with a label printed from the STS, 
or the Universal ID and Patient Study ID.  
 
Specimen 
Required Forms 
 
 
Bone Marrow Aspirate or Biopsy 
1.Shipping List 
2.Corresponding Bone Marrow Report 
 
Other (blood, blood product, stool) 
1. Shipping List 
Skin Punch Biopsy 
1.Shipping List 
2.Skin Punch Biopsy Verification Form 
(Appendix F) 
 
 
5.5.2. Specimen Shipping Instructions 
 
FFPE tissue and frozen specimens may be shipped on Monday through Thursday. 
 
Fresh blood or bone marrow may be shipped on Monday through Friday. Please select 
“Saturday Delivery” when shipping fresh blood on a Friday. 
Frozen specimens such as frozen skin punch tissue should be shipped on dry ice in an insulated 
shipping container provided by the EET Biobank. Keep frozen in a -70ºC to -80ºC  freezer until 

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shipment to the EET Biobank. 
 
 
5.5.2.1. Shipping Ambient Tissue, Blood and Bone Marrow 
 
1. Before packaging specimens, verify that each specimen is labeled according to the 
instructions above and that the lids of all primary receptacles containing liquid are tightly 
sealed 
2. Place the specimens in zip-lock bags. Use a separate bag for each specimen type. 
3. Place specimens into a biohazard envelope with absorbent material. Expel as much air as 
possible and seal the envelope securely. 
4. Place the biohazard envelope into a Tyvek envelope. Expel as much air as possible and 
seal securely. 
5. Place the specimen(s) and a copy of the shipping manifest and corresponding reports 
such as the bone marrow report into the shipping container. In winter months, please 
include extra insulation, such as bubble wrap, inside the shipping container, to prevent 
specimens from freezing. 
6. Place the lid and tape shut. 
7. Attach a shipping label to the top of the shipping container. 
8. Attach an Exempt Human Specimen sticker to the side of the container. Add a label 
indicating “CIMAC specimen” with collection date and time on the outside of the 
shipping container. 
9. Ship specimens via overnight courier to the address below. FedEx Priority Overnight is 
strongly recommended to prevent delays in package receipt. 
 
5.5.2.2  Shipping Frozen Specimens in a Single-Chamber Kit 
1. Before packaging specimens, verify that each specimen is labeled according to the 
instructions in 5.3 and that lids of all primary receptacles are tightly sealed.  
2. Place the specimens in zip-lock bags. Use a separate zip-lock bag for each specimen 
type and time point. 
3. Place the zip-lock bags in the biohazard envelope containing absorbent material. Expel 
as much air as possible and seal securely. 
4. Put the secondary envelope into a Tyvek envelope. Expel as much air as possible and 
seal securely. 
5. Place frozen specimens in the kit compartment with dry ice. Layer the bottom of the 
compartment with dry ice until it is approximately one-third full. Place the frozen 
specimens on top of the dry ice. Cover the specimens with additional dry ice until the 
compartment is almost completely full. When packaging specimens, ensure that you 
leave enough room to include at least 5 pounds of dry ice in the shipment. 
6. Insert a copy of the required forms into a plastic bag and place in the kit chamber. 
7. Place the Styrofoam lid on top to secure specimens during shipment. Do not tape 
the inner chamber shut. 
8. Close the outer lid of the Specimen Procurement Kit and tape it shut with durable sealing 
tape. Do not completely seal the container. 
9. Complete a FedEx air bill and attach to top of shipping container. 
10. Complete a dry ice label. 
11. Attach the dry ice label and an Exempt Human Specimen sticker to the side of the 

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shipping container. 
12. Ship specimens via overnight courier to the address below. FedEx Priority Overnight is 
strongly recommended to prevent delays in package receipt. 
 
5.5.2.3. Shipping Stool in an Ambient Shipper 
 
The clinical site will ship each specimen to the EET Biobank. The OMNIgene GUT collected 
samples can remain at ambient temperatures for a maximum of 60 days but should be shipped as 
soon as possible after collection. 
 
Detailed instructions for packaging and shipping the stool specimen are provided in the kit. 
 
 
5.5.3. Shipping Address 
 
Ship to the address below. Ship formalin-fixed and fresh blood specimens the same day of 
specimen collection. Do not ship specimens the day before a holiday. 
 
EET Biobank   
2200 International Street 
Columbus, OH 43228 
PH: (614) 722-3270 
FAX: (614) 722-2856 
Email: BPCMGLab@nationwidechildrens.org 
 
FedEx Priority Overnight service is very strongly preferred. 
 
NOTE: The EET Biobank FedEx Account will not be provided to submitting institutions. There is no central 
Courier account for this study. Sites are responsible for all costs for shipments to the EET Biobank. 
 
5.5.4. Contact Information for Assistance 
For all queries, please use the contact information below: 
EET Biobank  
Phone: (614) 722-3270 
E-mail: BPCMGLab@nationwidechildrens.org 
 
 
5.6 
Shipping of Specimens from Clinical Site to Brent Wood’s Lab, Department of 
Pathology and Laboratory, Children’s Hospital Los Angeles 
5.6.1.1. Specimen Shipping Instructions 
 
1 Specimen should be received by Department of Pathology and Laboratory Medicine within 48 
hours of collection. 
1. Specimen must be placed in a leak proof vacutainer. 
2. Multiple fragile vacutainers must be individually wrapped or separated to prevent 
contact. 

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3. The vacutainer must be placed into a leak proof Ziploc biohazard bag in such a way that 
under normal conditions of transport, they cannot break or leak. 
4. The Ziploc bag must be labeled with the universal biohazard symbol. 
5. Absorbent material such as absorbent pads or pillows must be placed in the Ziploc bag 
with sufficient capacity to absorb the entire contents of the vacutainer. 
6. The Ziploc bag must be placed into a rigid outer package with suitable cushioning 
materials. 
7. The outer packaging must be clearly and durably marked with the words “Diagnostic 
Specimen” and 2-inch diamond with “UN3373” inside of the diamond. 
8. The outer packaging must be marked with the name, address, and phone number of both 
sender and recipient. 
9. Specimen should be shipped with a cold pack (do not allow to freeze). 
 
The Department of Pathology and Laboratory Medicine is open Monday-Friday 8:00-4:30. Do not 
draw specimens on Fridays for delivery on Saturday. The laboratory will not be open to accept and 
test specimens. 
 
5.6.1.2. Shipping Address 
Send FedEx tracking number to: 
plmclinicalimmunologylaboratory@chla.usc.edu 
 
Send the specimens FedEx Priority Overnight to: 
 
Brent Wood, MD, PhD 
Department of Pathology and Laboratory Medicine Children’s Hospital Los Angeles 
4650 Sunset Blvd. 
Duque Bldg., 2nd Floor, Room 2-290 
Los Angeles, CA 90027 
Phone: (877) 543-9522 
 
An STS Shipping Manifest Form must be generated and shipped with all specimen submissions. 
 
5.6.1.3. Contact Information for Assistance 
 
Contact the Department of Pathology and Laboratory Medicine (877) 543-9522 or 
plmclinicalimmunologylaboratory@chla.usc.edu for assistance. 
 
5.7 
Shipping of Specimens from Clinical Site to Radich laboratory, Fred Hutchinson Cancer Research 
Center  
5.7.1.1    Specimen Shipping Instructions  
 
 
1 Specimen should be received by the Radich Laboratory, Fred Hutchinson Cancer Research 
Center within 5 days of collection, ideally no later than 48 hours after collection. 
2 Specimen must be placed in a leak proof vacutainer. 
3  Multiple fragile vacutainers must be individually wrapped or separated to prevent 
contact. 

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4 The vacutainer must be placed into a leak proof Ziploc biohazard bag in such a way that 
under normal conditions of transport, they cannot break or leak. 
5 The Ziploc bag must be labeled with the universal biohazard symbol. 
6 Absorbent material such as absorbent pads or pillows must be placed in the Ziploc bag 
with sufficient capacity to absorb the entire contents of the vacutainer. 
7 The Ziploc bag must be placed into a rigid outer package with suitable cushioning 
materials. 
8 The outer packaging must be clearly and durably marked with the words “Diagnostic 
Specimen” and 2-inch diamond with “UN3373” inside of the diamond. 
9 The outer packaging must be marked with the name, address, and phone number of both 
sender and recipient. 
10 Specimen should be shipped with a cold pack (do not allow to freeze). 
 
The Radich Laboratory, Fred Hutchinson Cancer Research Center is open Monday-Friday. Do 
not draw specimens on Fridays for delivery on Saturday. The laboratory will not be open to accept 
and test specimens. Exception can be made on a case-by-case basis for Friday shipping but 
should be discussed in advance of performing screening samples with the study chair and 
approved.  
 
5.7.1.2 Shipping Address 
Send FedEx tracking number to: 
lbeppu@fredhutch.org and 
jradich@fredhutch.org 
 
Send the specimens FedEx Priority Overnight to: 
 
Radich Laboratory 
Fred Hutchinson Cancer Research Center 
1100 Fairview Avenue North 
Room D4-385 
Seattle, WA 98109 
 
An STS Shipping Manifest Form must be generated and shipped with all specimen submissions. 
 
5.7.1.3  Contact Information for Assistance 
 
Contact the Radich Laboratory at 206-667-6966 or lbeppu@fredhutch.org.  
 

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5.8  Biomarker Plan 
 
List of Biomarker Assays in Order of Priority 
Note for participating sites: Please see Section 5.1 for details on specimens to collect.  The specimens tested are not always the same 
specimens that are submitted by the site, as processing of blood and tissue will occur at the Biobank prior to testing. 
 
Priority Biomarker 
Name 
Assay 
(CLIA: 
Y/N) 
Use in the Trial 
and Purpose 
Specimens 
Tested 
Collection Time Points Mandatory 
or Optional 
Assay Laboratory and Lab PI 
Core Biopsy Biomarkers 
1 
Multiplex 
Immunof 
lorescenc 
e 
MultiplexIF 
CLIA: N 
Exploratory 
Correlate with 
clinical response 
FFPE Bone 
Marrow Core 
Biopsy, 
decalcified 
Baseline, end of 
consolidation, after 6 
months of treatment, 
after 1 year of treatment, 
and at relapse 
M/O 
(optional for 
the after 6 
months of 
treatment 
and after1 
yearof 
treatment 
time points) 
CIMAC 
Ignacio Wistuba 
iiwistuba@mdanderson.org 
Dr. Gheath Al-Atrash 
galatras@mdanderson.org   
Dr. Cara Haymaker 
CHaymaker@mdanderson.org  
 
Bone Marrow Aspiration Biomarkers 
1 
CBF gene 
alterations 
Cytogenetics 
or 
Fluorescence 
In situ 
Hybridization 
(FISH) 
testing 
Integral 
Stratification 
factor 
Bone marrow 
aspirate 
Baseline and as 
clinically indicated 
M 
Local CLIA labs 
CLIA: Y 
2 
FLT3 
Mutations 
Molecular 
FLT3 assay 
(either FLT3- 
PCR testing 
or as a part of 
Next 
generation 
sequencing 
panel) 
Integral 
Exclusion factor 
N/A 
(collected as 
part of the 
CBF gene 
alterations 
assay) 
Baseline and as 
clinically indicated 
M 
Local CLIA labs 
CLIA: Y 

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Priority Biomarker 
Name 
Assay 
(CLIA: 
Y/N) 
Use in the Trial 
and Purpose 
Specimens 
Tested 
Collection Time Points Mandatory 
or Optional 
Assay Laboratory and Lab PI 
3 
MRD 
Status 
Multicolor 
flow 
cytometry 
(MFC) 
Integral 
Primary Endpoint 
Bone marrow 
aspirate 
Baseline 
M 
Department of Pathology and 
Laboratory Medicine Children’s 
Hospital Los Angeles 
Brent Wood 
bwood@chla.usc.edu 
CLIA: Y 
Consolidation therapy 
phase: 
Post Cycle 1. 
4 
MRD 
Status 
Duplex 
Sequencing 
(DS) 
CLIA: N 
Exploratory 
Exploratory 
Endpoint 
Bone marrow 
aspirate 
Baseline 
Induction therapy phase: 
Days 14 and end of 
induction. 
Consolidation therapy 
phase: 
Post Cycle 1 and end of 
consolidation. 
M/O 
(optional 
during the 
maintenance 
phase) 
To be shipped from Brent 
Wood’s lab after completion of 
flow cytometry studies 
Hematopathology Laboratory at 
Fred Hutchinson CancerResearch 
Center 
Jerald Radich 
jradich@fhcrc.org 
Maintenance therapy 
phase: 
Every 3 months (BM is 
optional but if done a 
BM sample should be 
sent) 
End of 
maintenance/observation 
phase for patients who 
do not progress. 

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Priority Biomarker 
Name 
Assay 
(CLIA: 
Y/N) 
Use in the Trial 
and Purpose 
Specimens 
Tested 
Collection Time Points Mandatory 
or Optional 
Assay Laboratory and Lab PI 
5 
Immune 
cell subset 
Analysis 
(CyTOF) 
CyTOF 
CLIA: N 
Exploratory 
Correlate with 
clinical responses 
Mononuclear 
cells from 
Bone marrow 
aspirate in 
EDTA 
Baseline, end of 
consolidation, after 6 
months of treatment, 
after 1 year of treatment, 
and at relapse 
M/O 
(optional for 
the after 6 
months of 
treatment 
and after1 
yearof 
treatment 
time points) 
MDACC CIMAC 
Dr. Ignacio I Wistuba 
iiwistuba@mdanderson.org 
Dr. Gheath Al-Atrash 
galatras@mdanderson.org   
Dr. Cara Haymaker 
CHaymaker@mdanderson.org 
6 
Olink 
Olink 
CLIA: N 
Exploratory 
Correlate with 
clinical responses 
Plasma from 
Bone marrow 
aspirate in 
EDTA 
Baseline, end of 
consolidation, after 6 
months of treatment, 
after 1 year of treatment, 
and at relapse 
M/O 
(optional for 
the after 6 
months of 
treatment 
and after 1 
year of 
treatment 
time points) 
Mt. Sinai CIMAC 
Sasha Gnjatic 
Sacha.gnjatic@mssm.edu 
7 
Whole 
Exome 
Sequencing 
WES (tumor) Exploratory 
Correlate with 
clinical response 
DNA 
from 
Mononuclear 
cells from 
bone marrow 
aspirate 
Baseline and at relapse 
M 
MDACC CIMAC 
Dr. Ignacio I Wistuba 
iiwistuba@mdanderson.org 
Dr. Gheath Al-Atrash 
galatras@mdanderson.org   
Dr. Cara Haymaker 
CHaymaker@mdanderson.org 
CLIA: N 
Whole 
Exome 
Sequencing 
WES 
(Germline) 
CLIA:N 
Exploratory 
Correlate with 
clinical response 
DNA 
from 
Skin punch 
biopsies for 
germline 
control 
Baseline 
M 
MDACC CIMAC 
Dr. Ignacio I Wistuba 
iiwistuba@mdanderson.org 
Dr. Gheath Al-Atrash 
galatras@mdanderson.org   
Dr. Cara Haymaker 
CHaymaker@mdanderson.org 

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8 
RNA-Seq 
RNA-Seq 
CLIA: N 
Exploratory 
Correlate with 
clinical response 
RNA from 
mononuclear 
cells from 
bone marrow 
aspirate in 
EDTA 
Baseline and at relapse 
M 
MDACC CIMAC 
Dr. Ignacio I Wistuba 
iiwistuba@mdanderson.org 
Dr. Gheath Al-Atrash 
galatras@mdanderson.org   
Dr. Cara Haymaker 
CHaymaker@mdanderson.org 

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Priority Biomarker 
Name 
Assay 
(CLIA: 
Y/N) 
Use in the Trial 
and Purpose 
Specimens 
Tested 
Collection Time Points Mandatory 
or Optional 
Assay Laboratory and Lab PI 
9 
TCR-Seq 
TCR-Seq 
CLIA: N 
Exploratory 
Correlate with 
clinical response 
DNA from 
mononuclear 
cells from 
bone marrow 
aspirate in 
EDTA 
Baseline, end of 
consolidation, after 6 
months of treatment, 
after 1 year of treatment, 
and at relapse 
M/O 
(optional for 
the after 6 
months of 
treatment 
and after1 
yearof 
treatment 
time points) 
Adaptive Biotechnologies 
working with MDACC CIMAC 
Dr. Ignacio I Wistuba 
iiwistuba@mdanderson.org 
Dr. Gheath Al-Atrash 
galatras@mdanderson.org   
Dr. Cara Haymaker 
CHaymaker@mdanderson.org 
 
 
Priority Biomarker 
Name 
Assay 
(CLIA: 
Y/N) 
Use in the Trial 
and Purpose 
Specimens 
Tested 
Collection Time Points Mandatory 
or 
Optional 
Assay Laboratory and Lab PI 
Blood-based Biomarkers 
1 
MRD Status 
Duplex 
Sequencing 
(DS) 
CLIA: N 
Exploratory 
Exploratory 
Endpoint 
Peripheral 
blood (EDTA) 
Baseline 
Induction therapy phase: 
Days 14 and end of 
induction. 
Consolidation therapy 
phase: 
Post Cycle 1 and end of 
consolidation. 
M 
To be shipped from Brent Wood’s 
lab after completion of flow 
cytometry studies 
Molecular Oncology Laboratory at 
Fred Hutchinson CancerResearch 
Center 
Jerald Radich 
jradich@fhcrc.org 
Maintenance therapy 
phase: 
Every 3 months 
End of 
maintenance/observation 
phase for patients who 
do not progress. 

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Priority Biomarker 
Name 
Assay 
(CLIA: 
Y/N) 
Use in the Trial 
and Purpose 
Specimens 
Tested 
Collection Time Points Mandatory 
or 
Optional 
Assay Laboratory and Lab PI 
Blood-based Biomarkers 
2 
cfDNA 
Duplex 
Sequencing 
(DS) 
CLIA: N 
Exploratory 
Exploratory 
Endpoint 
Peripheral 
blood (Streck) 
Baseline 
Induction therapy phase: 
Days 14 and end of 
induction. 
Consolidation therapy 
phase: 
Post Cycle 1 and end of 
consolidation. 
M 
To be shipped to Radich 
laboratory, Fred Hutchinson 
Cancer Research Center 
Molecular Oncology Laboratory at 
Fred Hutchinson CancerResearch 
Center 
Jerald Radich 
jradich@fhcrc.org 
Maintenance therapy 
phase: 
Every 3 months 
3 
Immune cell 
subset 
analysis 
CyTOF 
CLIA: N 
Exploratory 
Correlate with 
clinical responses 
PMBCs from 
blood collected 
in heparin 
Baseline, end of 
consolidation, after 6 
months of treatment, 
after 1 year of treatment, 
and at relapse 
M 
MDACC CIMAC 
Dr. Ignacio I Wistuba 
iiwistuba@mdanderson.org 
Dr. Gheath Al-Atrash 
galatras@mdanderson.org   
Dr. Cara Haymaker 
CHaymaker@mdanderson.org 
4 
Olink 
Olink 
CLIA: N 
Exploratory 
Correlate with 
clinical responses 
Plasma from 
blood collected 
in heparin 
(same 
collection as 
for immune 
cell subset 
analysis) 
Baseline, end of 
consolidation, after 6 
months of treatment, 
after 1 year of treatment, 
and at relapse  
M 
Mt. Sinai CIMAC 
Sasha Gnjatic 
Sacha.gnjatic@mssm.edu 
5 
TCR-seq 
TCR-Seq 
 
CLIA: N 
Exploratory 
Correlate with 
clinical responses 
DNA from 
PBMCs from 
blood collected 
in EDTA or 
sodium heparin 
tubes 
Baseline, end of 
consolidation, after 6 
months of treatment, 
after 1 year of treatment, 
and at relapse 
M 
Adaptive Biotechnologies 
working with MDACC CIMAC 
Dr. Ignacio I Wistuba 
iiwistuba@mdanderson.org 
Dr. Gheath Al-Atrash 
galatras@mdanderson.org   
Dr. Cara Haymaker 

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CHaymaker@mdanderson.org 
Stool based biomarkers 
1 
Gut 
microbiome 
ch 
characterizati 
on 
16sRNA 
V4 region 
sequencing 
of bacterial 
genomic 
DNA 
• Exploratory 
Correlate with 
clinical response 
Stool 
(OMNIgene- 
GUT) 
• Baseline 
• Post Cycle 1 
Consolidation 
M 
MDACC 
CIMAC 
Gheath Al-Atrash 

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* CyTOF, WES, RNAseq, TCR and Olink analyses will be performed using cells and plasma from a single BM 
aspirate. 
5.9 Integral Laboratory Studies 
 
5.9.1 CBF gene alterations 
 
5.9.1.1 Specimen Receipt and Processing at Local Labs 
 
Samples should be received and processed as per standard operating procedures. 
 
5.9.1.2 
Site Performing Correlative Study 
This study will be conducted by local labs. 
5.9.2 FLT3 Mutations 
 
5.9.2.1 Specimen Receipt and Processing at 
Local Labs 
 
Samples should be received and processed as per standard operating procedures. 
 
5.9.2.2 
Site Performing Correlative Study 
This study will be conducted by local labs. 
5.9.3 MRD Status by Multicolor Flow Cytometry 
 
5.9.3.1 Specimen Receipt and Processing at the Department of Pathology and Laboratory 
Medicine Children’s Hospital Los Angeles 
Bone marrow aspirate will be processed as per laboratory standard operating procedures. 
 
5.9.3.2.    Site(s) Performing Correlative Study 
 
The study will be performed in the laboratory of Dr. Brent Wood at the Children’s Hospital Los 
Angeles 
 
6.0 Exploratory/Ancillary Correlative Studies 
 
6.1    MRD Status by Duplex Sequencing 
 
6.1.1 Specimen Receipt and Processing at the Department of Pathology and Laboratory 
Medicine Children’s Hospital Los Angeles 
6.1.2 
Bone marrow aspirate and blood will be processed as per laboratory standard operating 
procedures. 
 
6.1.2.1  Site(s) Performing Correlative Study 
  

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The study will be performed in the laboratory of Dr. Jerald Radich at the Fred Hutchinson 
Cancer Research Center. 
 
6.1.3 Multiplex Immunofluorescence (MIF) 
 
6.1.3.1 Specimen(s) Receipt and Processing at the EET Biobank 
 
The EET Biobank will receive and barcode FFPE core bone marrow biopsy blocks, and will 
store them at room temperature until distribution. 
 
When a request is received from CIMAC/NCI, the EET Biobank will cut slides from the blocks 
and ship the slides to MDACC CIMAC. 
 
 
6.1.3.2 Site(s) Performing Correlative Study 
 
This assay will be performed at MDACC CIMAC in the laboratory of Dr. Ignacio I Wistuba 
(Email: iiwistuba@mdanderson.org), Dr. Gheath Al-Atrash (Email: galatras@mdanderson.org), 
and Dr. Cara Haymaker (Email: CHaymaker@mdanderson.org) (MDACC CIMAC Co-PI’s).      
 
 
Mei Jiang 
Translational Molecular Pathology- TMP-IL-Pathology Lab 
UT MD Anderson Cancer Center  
Life Science Plaza Building  
2130 W. Holcombe Blvd 
Houston, TX 77030 
location: LSP9.3003 (shipping) 
 
 
6.1.4 Immune Cell Subset Analysis CYTOF 
 
6.1.4.1 Specimen(s) Receipt and Processing at the EET Biobank 
 
The EET Biobank will receive fresh BM aspirate collected in EDTA tubes and blood samples 
collected in heparin tubes. Upon receiving the bone marrow aspirate in EDTA tubes from the 
collection site, the EET Biobank will pool all samples from one timepoint together and prepare 
plasma and mononuclear cells following CIMAC guidelines. Blood collected at each time point 
will also be pooled and processed for plasma and mononuclear cells following CIMAC 
guidelines. Plasma aliquots will be stored in a -80°C freezer, and mononuclear cells will be stored 
in liquid nitrogen vapor phase. 
 
6.1.4.2 Site(s) Performing Correlative Study 
 
This study will be performed at the MDACC CIMAC under Dr. Ignacio I Wistuba 
(iiwistuba@mdanderson.org), Dr. Gheath Al-Atrash (Email: galatras@mdanderson.org), and 
Dr. Cara Haymaker (Email: CHaymaker@mdanderson.org) (MDACC CIMAC Co-PI’s). 

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UT MD Anderson Cancer Center  
Room 2SCR2.3219 
7435 Fannin St 
Houston, TX 77054 
c/o Karen Millerchip 
 
6.1.5 O-link Cytokine panel 
 
6.1.5.1 Specimen(s) Receipt and Processing at the EET Biobank 
 
The EET Biobank will receive fresh BM aspirate collected in EDTA tubes and blood samples 
collected in heparin tubes. Upon receiving the bone marrow aspirate in EDTA tubes from the 
collection site, the EET Biobank will pool all samples from one timepoint together and prepare 
plasma and mononuclear cells following CIMAC guidelines. Blood collected at each time point 
will also be pooled and processed for plasma and mononuclear cells following CIMAC 
guidelines. Plasma aliquots will be stored in a -80°C freezer, and mononuclear cells will be 
stored in liquid nitrogen vapor phase. The same blood samples collected in heparin tubes from 
the immune cell subset analysis will also be used for this assay. 
 
The EET Biobank will ship frozen plasma to the Mt. Sinai CIMAC. 
 
6.1.5.2 Site(s) Performing Correlative Study 
 
This study will be performed at the Mount Sinai CIMAC by Sacha Gnjatic. Email: 
Sacha.gnjatic@mssm.edu 
 
Hess Center for Science and Medicine 
5th floor, rooms 310/313 
Human Immune Monitoring Center (HIMC) 
Icahn School of Medicine at Mount Sinai 
1470 Madison Avenue 
New York, NY 10029 
 
6.1.6 Whole Exome Sequencing 
 
6.1.6.1 
Specimen(s) Receipt and Processing at the EET Biobank for Bone marrow aspirate 
 
Upon receiving the bone marrow aspirate in EDTA tubes from the collection site, the EET 
Biobank will pool all samples from one timepoint together and prepare plasma and 
mononuclear cells following CIMAC guidelines. Plasma aliquots will be stored in a -80°C 
freezer, and mononuclear cells will be stored in liquid nitrogen vapor phase. 
 
DNA and RNA will be co-extracted from bone marrow mononuclear cells. An aliquot of DNA will 
be used for this assay. The same blood samples collected in heparin tubes from the immune cell 
subset analysis can also be used for this assay if a bone marrow aspirate sample cannot be 
obtained. 

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6.1.6.2 
Specimen(s) Receipt and Processing at the EET Biobank for FFPE punch biopsies 
 
 The EET Biobank will extract germline DNA from the skin  punch biopsies upon receipt. The stock DNA tube will be 
stored in a  -80°C freezer. The EET Biobank will ship a DNA aliquot to MDACC CIMAC.  
6.1.6.3 
Site(s) Performing Correlative Study 
 
This study will be performed at the MDACC CIMAC under Dr. Ignacio I Wistuba 
(iiwistuba@mdanderson.org), Dr. Gheath Al-Atrash (Email: galatras@mdanderson.org), and 
Dr. Cara Haymaker (Email: CHaymaker@mdanderson.org) (MDACC CIMAC Co-PI’s). 
 
UT MD Anderson Cancer Center 
Room 4SCR5.2085 
1881 East Rd. 
Houston, TX 77054 
c/o Latasha Little,  
Srilekha Manali Rameshkumar  
 
6.1.7 RNA-Seq 
 
6.1.7.1 
Specimen(s) Receipt and Processing at the EET Biobank 
 
Upon receiving the bone marrow aspirate in EDTA tubes from the collection site, the EET Biobank 
will pool all samples from one timepoint together and prepare Plasma and mononuclear cells 
following CIMAC guidelines. Plasma aliquots will be stored in a -80°C freezer, and 
mononuclear cells will be stored in liquid nitrogen vapor phase.  
 
DNA and RNA will be co-extracted from bone marrow mononuclear cells. An aliquot of RNA 
will be used for this assay. The EET Biobank will ship this sample to MDACC CIMAC. 
 
 
 
 
 
6.1.7.2 
Site(s) Performing Correlative Study 
 
This study will be performed at the MDACC CIMAC under Dr. Ignacio I Wistuba 
(iiwistuba@mdanderson.org), Dr. Gheath Al-Atrash (Email: galatras@mdanderson.org), and 
Dr. Cara Haymaker (Email: CHaymaker@mdanderson.org) (MDACC CIMAC Co-PI’s). 
 
UT MD Anderson Cancer Center  
2130 W Holcombe Blvd. 
Life Science Plaza Suite 910, LSP9.3003 
Houston, TX77030-3306 

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c/o Dzifa Duose & Jessica Lan 
 
6.1.8 TCR-Seq 
6.1.8.1 
Specimen(s) Receipt and Processing at the EET Biobank 
 
Upon receiving the bone marrow aspirate in EDTA tubes from the collection site, the EET Biobank 
will pool all samples from one timepoint together and prepare Plasma and mononuclear cells 
following CIMAC guidelines. Plasma aliquots will be stored in a -80°C freezer, and mononuclear 
cells will be stored in liquid nitrogen vapor phase.  
 
Additionally, PBMCs from peripheral blood samples collected in EDTA or heparin tubes will also 
be used for this assay. 
 
Nucleic acids will be co-extracted from bone marrow mononuclear cells and peripheral blood 
mononuclear cells. An aliquot of DNA will be used for each assay (i.e., for TCRseq in bone 
marrow aspirate and for TCRseq in peripheral blood). 
 
6.1.8.2 
Site(s) Performing Correlative Study 
 
This study will be performed by Adapitve Biotechnologies working with the MDACC CIMAC 
under Dr. Ignacio I Wistuba (iiwistuba@mdanderson.org), Dr. Gheath Al-Atrash (Email: 
galatras@mdanderson.org), and Dr. Cara Haymaker (Email: CHaymaker@mdanderson.org) 
(MDACC CIMAC Co-PI’s).   
. 
 
Attn: BSM Department  
Adaptive Biotechnologies 
1165 Eastlake Ave East 
Seattle, WA 98109 
 
 
6.1.9 
Gut microbiome 
 
6.1.9.1   Specimen(s) receipt and processing at the EET Biobank 
 
Upon receipt of the sample in the OMNIgene-GUT kits, the stool sample will be processed, aliquoted, and 
stored in a -80°C freezer. 
The EET Biobank will ship this sample to the MDACC CIMAC lab.  
 
 
 
6.1.9.2    Site(s) Performing Correlative Study 
 

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The microbiome assay will be performed at MDACC CIMAC in the laboratory of Gheath Al-Atrash 
(Email: galatras@mdanderson.org). 
 
 
MD Anderson Cancer Center 
4SCRB5.2085, Unit 1954 
1881 East Rd. 
South Campus Research Building 3 
Houston, TX 77054 
c/o Chia-Chi (Tina) Chang 

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68 
 
 
7. TREATMENT PLAN 
 
7.1 Agent Administration 
 
Treatment will be administered on an inpatient and outpatient basis. Reported AEs and potential 
risks are described in Section 10. Appropriate dose modifications are described in Section 7. No 
investigational or commercial agents or therapies other than those described below may be 
administered with the intent to treat the patient's malignancy. 
 
7.1.1 Induction therapy phase 
 
Eligible patients will be randomized before proceeding to 7+3, or within one week of starting 7+3 in 1:1 
ratio, to 7+3 alone or 7+3+ Pembrolizumab (MK-3475). As shipment of pembrolizumab to site can only be 
ordered after randomization, every effort should be done to randomize the patient as soon as possible. See 
Section 9.1 for agent ordering information.  If patient is randomized after initiation of 7+3 therapy, the patient 
should continue to meet all eligibility criteria up to randomization date. Planned stratification factors include 
1) age (younger than 65 vs. 65 and older), 2) cytogenetics by FISH or metaphase karyotype (presence vs. 
absence of core binding factor inversions and translocations), and 3) t- AML or AML arising from 
prior/antecedent MDS (yes/no). All patients will receive cytarabine at 100 mg/m2/day continuous 
infusion on Days 1-7 and either idarubicin 12 mg/m2/day IV on Days 1-3 or daunorubicin 60 mg/m2/day 
IV on Days 1-3. A bone marrow biopsy on Day 14 (between Days 14 and 21) will be performed to assess 
for chemoablation. If patients have evidence of residual leukemia (defined as per institutional practice) in 
their bone marrow and following assessment by repeat transthoracic echocardiography (TTE) for 
assessment of ejection fraction, they will receive re-induction 5+2 chemotherapy, which means patients will 
receive cytarabine at 100 mg/m2/day continuous infusion on Days 1-5 and either idarubicin 12 mg/m2/day 
IV or daunorubicin 60 mg/m2/day IV on Days 1-2. Anthracycline dosing adjustments may need to be made, 
per institutional guidelines. Regardless of chemoablation status, on Day 8 of the induction chemotherapy 
and Q3W (±3 days) thereafter, half of the patients will receive Pembrolizumab (MK-3475) IV 
(intervention arm). The other half will not receive Pembrolizumab (MK-3475) (control arm). At time of 
count recovery, defined as platelet count more than 50,000 or neutrophil count more than 500, patients will 
undergo a bone marrow aspirate/biopsy in order to assess for response and MRD. If there is no count 
recovery, a bone marrow will have to be performed by day 42. If the bone marrow shows hypoplasia and 
patient remains without count recovery, a bone marrow will be performed q 2 weeks (+/- 3 days) till there is 
count recovery or evidence of persistent AML. If patients have a CR or a CRi, patients will continue with 
consolidation therapy. If patients do not achieve a CR or CRi, they will be taken off study. If 
Pembrolizumab is discontinued for any reason (after at least one dose of Pembrolizumab, the patient 
should continue on 7+3 or Re-Induction on study.  
 
 
 
 
 
 
 
 
 

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69 
 
 
Induction Therapy Regimen Description 
 
Agent 
Premedications 
; Precautions 
Dose* 
Route 
Schedule 
Cycle# 
Length 
Cytarabine 
Premedications/ 
100 mg/m2 
Continuous IV Days 1-7 
precautions as 
per standard 
local 
procedures. No 
Reconstituti 
on and 
diluents as 
specific 
premedications 
required. 
premedication 
per standard 
local 
procedures/ 
per package 
28-35 
days 
(4-5 
with antipyretic 
and 
antihistamine 
may be 
insert. 
Refer to 
Section 
weeks) 
considered. 
8.2.1 for 
additional 
information. 
Either Daunorubicin 
or Idarubicin 
Premedications/ 
precautions as 
per local 
Either 60 
mg/m2 
Daunorubici 
slow IV push 
over 10 to 15 
minutes into 
free-flowing 
side-port 
tubing of 
Days 1-3 
standard 
procedures. No 
specific 
premedications 
n or 12 
mg/m2 
Idarubicin 
on Days 1, 
a rapidly 
infusing IV 
solution of 
D5W or NS 
and infuse over 
15 to 30 
minutes, or per 
institutional 
standard for 
vesicant 
medications.  
`` 
required. 
premedication 
with antipyretic 
and 
2, and 3 
Reconstituti 
on and 
28-35 
antihistamine 
may be 
considered. 
diluents as 
per standard 
local 
procedures/ 
days 
(4-5 
weeks) 
per package 
insert. 
Refer to 

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70 
 
 
 
Sections 
8.2.2 and 
8.2.3 for 
additional 
information. 
 
Induction Therapy Regimen Description 
Agent 
Premedications 
; Precautions 
Dose* 
Route 
Schedule 
Cycle# 
Length 
Pembrolizumab 
(MK-3475) (For 
Intervention 
Premedication 
with antipyretic 
and 
200 mg 
See Section 
IV over 30 
minutes 
(between 25- 
Q3W (±3 
days) 
starting on 
28-35 
days 
(4-5 
weeks) 
Arm only) 
antihistamine 
may be 
considered. No 
premedication 
8.1 for 
instructions 
on the 
preparation 
40 minutes) 
using an  
infusion set 
containing a 
Day 8 
with steroids 
of the 
infusion 
solution. 
low-protein 
binding 0.2 to 
5 μm in-line 
filter. 
See Section 
8.1 for 
compatible 
infusion set 
materials 
including in- 
line filter. 
*Dose rounding per institutional guidelines is allowed. 
#If evidence of residual leukemia in bone marrow on Day 14 bone marrow, re-induction 5+2 
chemotherapy (cytarabine at 100 mg/m2/day continuous IV on Days 1-5 and either idarubicin 12 
mg/m2/day IV or daunorubicin 60 mg/m2/day IV on Days 1-2). Anthracycline dosing 
adjustments may need to be made per institutional guidelines 
 
7.1.2 Consolidation therapy phase 
 
Consolidation therapy will consist of up to four cycles of infusional HiDAC and Pembrolizumab 
(MK-3475) (intervention arm) vs. HiDAC alone (control arm). Patients <60 years of age will 
receive 3 g/m2 every 10-12 hours (Q12H) on Days 1, 3, and 5 (total 6 doses). Patients ≥60 
years of age will receive 1.5 g/m2 Q12H on Days 1, 3, and 5 (total 6 doses). Cycle 1 of 
consolidation therapy must start within 4 weeks of documenting remission status. Each cycle 
lasts ~28 days (up to 42 days). Patients in the intervention arm will continue to receive MK- 
3475 (pembrolizumab) Q3W (±3 days). Do not administer pembrolizumab concurrently with 
HIDAC chemotherapy Days 1-5 of each consolidation cycle. For consideration of timing of 
pembrolizumab, allow at least 3 hours after completion of chemotherapy prior to pembrolizumab 

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71 
 
 
administration. Administration window for pembrolizumab may be extended to allow for this 
timing as necessary. After Cycle 1 of HiDAC, bone marrow assessment will be performed at the 
time of count recovery (same rules apply as post induction bone marrow in case of lack of count 
recovery) for formal assessment of response and MRD. Patients, who remain in CR or CRi, will 
be allowed to proceed to up to 3 more cycles of HiDAC and Pembrolizumab (MK-3475) 
(intervention arm) vs. HiDAC alone (control arm). Patients, who are not in CR or CRi will be taken 
off study. . If Pembrolizumab (MK-3475) is discontinued for any reason (after at least one dose of 
Pembrolizumab), the patient should continue Consolidation therapy on study.  
 
 
Consolidation Therapy Regimen Description 
Agent 
Premedications 
; Precautions 
Dose* 
Route 
Schedule 
Cycle 
Length 
Pembrolizumab 
(MK-3475) (For 
Intervention Arm 
only) 
Premedication 
with antipyretic 
and 
antihistamine 
may be 
considered. No 
premedication 
with steroids 
200 mg 
See 
Section 8.1 
for 
instruction 
s on the 
preparation 
of the 
infusion 
solution. 
IV over 30 
minutes 
(between 25-40 
minutes) using  
an infusion set  
containing a  
low-protein  
binding 0.2 to 5 
μm in-line 
filter. 
See Section 8.1 
for compatible 
infusion set 
materials 
including in- 
line filter. 
Q3W (±3 
days) 
continuous 
from Day 8 
of 
Induction. 
For 
considerati 
on of 
timing of 
pembrolizu 
mab, allow 
at least 3 
hours after 
completion 
of 
chemothera 
py prior to 
pembrolizu 
mab 
administrat 
ion 
28 days 
(±7 days) 

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Cytarabine# 
Premedications/ 
precautions as 
per standard 
local 
procedures. No 
specific 
premedications 
required. 
premedication 
with antipyretic 
and 
antihistamine 
may be 
considered 
3 g/m2. 
For 
patients 
≥60 years, 
dose 
lowered to 
1.5 g/m2 
Q12H on 
Days 1, 3, 
and 5. 
Reconstitut 
ion and 
diluents as 
per 
standard 
local 
IV over 1-3 
hours every 10- 
12 hours 
Days 1, 3, 
and 5 
 
Consolidation Therapy Regimen Description 
Agent 
Premedications 
; Precautions 
Dose* 
Route 
Schedule 
Cycle 
Length 
procedures 
/ per 
package 
insert. 
Refer to 
Section 
8.2.1 for 
additional 
informatio 
n. 
*Dose rounding per institutional guidelines is allowed. 
 
7.1.3 Maintenance therapy phase 
 
Following count recovery from the last cycle of consolidation therapy, a bone marrow biopsy 
will be performed to assess for response and MRD. Patients, who remain in CR/CRi will 
continue on maintenance therapy while patients who do not remain in CR/CRi will be taken off 
study. Patients in CR/CRi will proceed with single-agent Pembrolizumab (MK-3475) 
(intervention arm) vs. no maintenance therapy (control arm) Q3W dosing schedule for up to 2 
years, calculated from first day of maintenance therapy, according to their original randomization 
(i.e., there will be no second randomization). For patients randomized to Arm B, Pembrolizumab 
(MK-3475) will be administered on Day 1 (+/- 3 days) of each maintenance cycle (each 
maintenance cycle is 21 days). Bone marrow samples will be obtained every 3 months while 

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patients are receiving maintenance or in observation to assess for MRD. Patients, who are 
deemed to require HSCT, can forgo any remaining protocol-defined consolidation or maintenance 
therapy and proceed with HSCT. Patients proceeding with HSCT will be followed 
for survival and SAEs through the first 100 days post-HSCT including assessment for occurrence 
of acute and chronic GVHD. 
 
Maintenance Therapy Regimen Description 
Agent 
Premedications 
; Precautions 
Dose 
Route 
Schedule 
Cycle 
Length 
pembrolizumab 
Premedication 
200 mg 
IV over 
Day 1(+/- 3 21 Days 
(MK-3475) (For 
Intervention Arm 
only) 
with antipyretic 
and 
antihistamine 
may be 
See Section 8.1 
for instructions 
on the 
30 
minutes 
(between 
25-40 
days) of 
each 21- 
day 
maintenanc 
considered. No 
premedication 
preparation of 
the infusion 
minutes) 
using an 
e cycle 
 
Maintenance Therapy Regimen Description 
Agent 
Premedications 
; Precautions 
Dose 
Route 
Schedule 
Cycle 
Length 
with steroids 
solution. 
infusion 
set 
containing 
a low- 
protein 
binding 
0.2 to 5 
μm in-line 
filter. 
See 
Section 
8.1 for 
compatibl 
e infusion 
set 
materials 
including 
in-line 
filter. 
 
7.1.4 CTEP IND Agent 
 
Trial treatment of Pembrolizumab (MK-3475) will be administered Q3W starting on Day 8 of 

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the first induction cycle of the study. Trial treatment may be administered up to 3 days before or 
after the scheduled day due to administrative reasons. 
 
Note: Dosing interruptions are permitted in the case of medical/surgical events or logistical 
reasons (i.e., elective surgery, unrelated medical events, patient vacation, holidays) not related to 
study therapy. Patients should be placed back on study therapy within 3 weeks of the scheduled 
interruption. If interruption is longer than 3 weeks, patient can resume study therapy with study chair 
approval. The reason for interruption should be documented in the patient’s study record. 
 
Pembrolizumab (MK-3475) will be administered as a dose of 200 mg using a 30-minute IV 
infusion. Infusion timing should be as close to 30 minutes as possible; however, a window of 
-5 minutes and +10 minutes is permitted (i.e., infusion time is 25-40 minutes). 
 
7.2 General Concomitant Medication and Supportive Care Guidelines 
 
 
The study team should check a frequently-updated medical reference for a list of drugs to avoid 
or minimize use of. Appendix C (Patient Clinical Trial Wallet Card) should be provided to 
patients. 
7.2.1 Pembrolizumab (MK-3475) Concomitant Medication 
 
Medications or vaccinations specifically prohibited in the exclusion criteria are not allowed 
during the ongoing trial. If there is a clinical indication for any medication or vaccination 
specifically prohibited during the trial, discontinuation from trial therapy or vaccination may be 
required. The investigator should discuss any questions regarding this with CTEP. The final 
decision on any supportive therapy or vaccination rests with the investigator and/or the patient's 
primary physician; however, the decision to continue the patient on trial therapy or vaccination 
schedule requires the mutual agreement of the Investigator, CTEP, and thepatient. 
 
Acceptable Concomitant Medications 
All treatments that the investigator considers necessary for a patient’s welfare may be 
administered at the discretion of the investigator in keeping with the community standards of 
medical care. All concomitant medication will be recorded on the case report form (CRF) 
including all prescription, over-the-counter (OTC), herbal supplements, and IV medications and 
fluids. If changes occur during the trial period, documentation of drug dosage, frequency, route, 
and date may also be included on the CRF. 
 
All concomitant medications received within 30 days before the first dose of trial treatment and 
30 days after the last dose of trial treatment should be recorded. Concomitant medications 
administered after 30 days after the last dose of trial treatment should be recorded for serious 
AEs (SAEs). 
 
Prohibited Concomitant Medications 
Patients are prohibited from receiving the following therapies during the Screening and 
Treatment Phase (including retreatment for post-complete response relapse) of this trial: 

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− 
Anti-leukemia Immunotherapy not specified in this protocol. 
− 
Anti-leukemia Chemotherapy not specified in this protocol. Note: If patient needs intrathecal 
chemotherapy after start of trial therapy on study as prophylaxis or for new development of CNS 
leukemia, patient is allowed to continue on study. 
− 
Investigational anti-leukemia agents other than Pembrolizumab (MK-3475). 
− 
Radiation therapy 
o Note: Radiation therapy to a symptomatic solitary lesion or to the brain may be 
considered on an exceptional case by case basis after consultation with study chair and 
CTEP. The patient must have clear measurable disease outside the radiated field. 
Administration of palliative radiation therapy will be considered clinical progression for 
the purposes of determining PFS. 
− 
Live vaccines within 30 days prior to the first dose of trial treatment and while 
participating in the trial. Examples of live vaccines include, but are not limited to, the 
following: measles, mumps, rubella, chicken pox, yellow fever, rabies, Bacillus 
Calmette–Guérin (BCG), and typhoid (oral) vaccine. Seasonal influenza vaccines for 
injection are generally killed virus vaccines and are allowed; however, intranasal 
influenza vaccines (e.g., Flu-Mist®) are live attenuated vaccines, and are not allowed. 
− 
Once on and during the treatment phase of the trial, high dose systemic steroid 
therapy should be avoided as much as possible but is not strictly prohibited. 
 
Patients who, in the assessment by the investigator, require the use of any of the aforementioned 
treatments for clinical management should be removed from the trial except if they are allowed to 
continue on study on case-by-case basis based on consultation with study chair and CTEP. Patients may 
receive other medications that the investigator deems to be medically necessary. 
 
The Exclusion Criteria describe other medications which are prohibited in this trial. 
There are no prohibited therapies during the Post-Treatment Follow-up Phase. 
 
7.2.2 Pembrolizumab Supportive Care Guidelines 
 
Patients should receive appropriate supportive care measures as deemed necessary by the treating 
investigator. Suggested supportive care measures for the management of AEs with potential 
immunologic etiology are also outlined in the table in Section 7.1.3. Where appropriate, these 
guidelines include the use of oral or IV treatment with corticosteroids as well as additional anti- 
inflammatory agents if symptoms do not improve with administration of corticosteroids. Note 
that several courses of steroid tapering may be necessary as symptoms may worsen when the 
steroid dose is decreased. For each disorder, attempts should be made to rule out other causes 
such as metastatic disease or bacterial or viral infection, which might require additional 
supportive care. The treatment guidelines are intended to be applied when the investigator 
determines the events to be related to Pembrolizumab. 
 
Note: If after the evaluation the event is determined not to be related, the investigator does not 
need to follow the treatment guidance (as outlined below). 
 
It may be necessary to perform conditional procedures such as bronchoscopy, endoscopy, or skin 
photography as part of the evaluation of the event. 

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7.2.3 Recognition and Treatment of Early Immune Related Events 
 
Subjects should receive appropriate supportive care measures as deemed necessary by the 
treating investigator. Suggested supportive care measures for the management of adverse events 
with potential immunologic etiology are outlined below. Where appropriate, these guidelines 
include the use of oral or intravenous treatment with corticosteroids as well as additional anti- 
inflammatory agents if symptoms do not improve with administration of corticosteroids. Note 
that several courses of steroid tapering may be necessary as symptoms may worsen when the 
steroid dose is decreased. For each disorder, attempts should be made to rule out other causes 
such as metastatic disease or bacterial or viral infection, which might require additional 
supportive care. The treatment guidelines are intended to be applied when the investigator 
determines the events to be related to MK3475 (pembrolizumab). Below represent guidelines 
and not required actions, but investigators are strongly encouraged to follow the below 
interventions. 
 
A baseline CT scan of the chest should be considered in all patients. 
 
It may be necessary to perform conditional procedures such as bronchoscopy, endoscopy, or skin 
photography as part of evaluation of the event. 
 
Pneumonitis: 
• 
For Grade 2 events, treat with systemic corticosteroids. When symptoms of Grade 2 
events improve to Grade 1 or less, steroid taper should be started and continued over no 
less than 4 weeks. 
• 
For Grade 3-4 events, immediately treat with intravenous steroids. Administer 
additional anti-inflammatory measures, as needed. Pembrolizumab should be 
permanently discontinued for grade 3 or 4 pneumonitis and persistent grade 2 
pneumonitis in the event of at least a possibly pembrolizumab-related 
pneumonitis. 
• 
Add prophylactic antibiotics for opportunistic infections in the case of prolonged steroid 
administration. 
 
Diarrhea/Colitis: 
Subjects should be carefully monitored for signs and symptoms of enterocolitis (such 
as diarrhea, abdominal pain, blood, or mucus in stool, with or without fever) and of 
bowel perforation (such as peritoneal signs and ileus). 
• 
All subjects who experience diarrhea/colitis should be advised to drink liberal quantities 
of clear fluids. If sufficient oral fluid intake is not feasible, fluid and electrolytes should 
be substituted via IV infusion. For Grade 2 or higher diarrhea, consider GI consultation 
and endoscopy to confirm or rule out colitis. 
• 
For Grade 2 diarrhea/colitis that persists greater than 3 days, administer oral 
corticosteroids. 
• 
For Grade 3 or 4 diarrhea/colitis that persists > 1 week, treat with intravenous steroids 
followed by high dose oral steroids. 
• 
When symptoms of Grade 2 events improve to Grade 1 or less, steroid taper should be 

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started and continued over no less than 4 weeks (Grade 3-4 events may require 
permanent discontinuation). 
 
Type 1 diabetes mellitus (if new onset, including diabetic ketoacidosis [DKA]) or ≥ Grade 3 
Hyperglycemia, if associated with ketosis (ketonuria) or metabolic acidosis (DKA) 
• 
For T1DM or Grade 3-4 Hyperglycemia 
o Insulin replacement therapy is recommended for Type I diabetes mellitus and for 
Grade 3-4 hyperglycemia associated with metabolic acidosis or ketonuria. 
o Evaluate patients with serum glucose and a metabolic panel, urine ketones, 
glycosylated hemoglobin, and C-peptide. 
 
Hypophysitis: 
• 
For Grade 2 events, treat with corticosteroids. When symptoms improve to Grade 1 or 
less, steroid taper should be started and continued over no less than 4 weeks. 
Replacement of appropriate hormones may be required as the steroid dose is tapered. 
• 
For Grade 3-4 events, treat with an initial dose of IV corticosteroids followed by oral 
corticosteroids. When symptoms improve to Grade 1 or less, steroid taper should be 
started and continued over no less than 4 weeks. Replacement of appropriate hormones 
may be required as the steroid dose is tapered. 
 
Hyperthyroidism or Hypothyroidism: 
Thyroid disorders can occur at any time during treatment. Monitor patients for changes in 
thyroid function (at the start of treatment, periodically during treatment, and as indicated based 
on clinical evaluation) and for clinical signs and symptoms of thyroid disorders. 
 
Grade 2 hyperthyroidism events (and Grade 2-4 hypothyroidism): 
In hyperthyroidism, non-selective beta-blockers (e.g. propranolol) are suggested as initial 
therapy. 
In hypothyroidism, thyroid hormone replacement therapy, with levothyroxine or liothyroinine, is 
indicated per standard of care. 
• Grade 3-4 hyperthyroidism 
o Treat with an initial dose of IV corticosteroid followed by oral corticosteroids. When 
symptoms improve to Grade 1 or less, steroid taper should be started and continued 
over no less than 4 weeks. 
o Replacement of appropriate hormones may be required as the steroid dose is tapered. 
Hepatic: 
• For Grade 2 events, monitor liver function tests more frequently until returned to baseline 
values (consider weekly). Treat with IV or oral corticosteroids 
• For Grade 3-4 events, treat with intravenous corticosteroids for 24 to 48 hours. 
• When symptoms of Grade 2 events improve to Grade 1 or less, a steroid taper should be 
started and continued over no less than 4 weeks (Grade 3-4 events may require 
permanent discontinuation). 
 
Renal Failure or Nephritis: 
• For Grade 2 events, treat with oral systemic corticosteroids. 

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• For Grade 3-4 events, treat with intravenous systemic corticosteroids. 
• When symptoms of Grade 2 events improve to Grade 1 or less, steroid taper should be 
started and continued over no less than 4 weeks (Grade 3-4 events may require 
permanent discontinuation). 
 
Management of Infusion Reactions: Signs and symptoms usually develop during or shortly after 
drug infusion and generally resolve completely within 24 hours of completion of infusion. 
 
Patients with grade 3-4 IRAEs that completely resolve may be allowed to continue on study on 
case-by-case basis based on consultation with study chair and CTEP. 
 
7.2.4 Concomitant treatments with cytarabine anthracycline chemotherapy 
 
During the induction cycles, G- and GM-CSF may be used only in case of prolonged neutropenia 
or neutropenic sepsis if non-blastic aplasia is obtained; there will be no such constraints in 
subsequent cycles. Use of growth factors during consolidation and maintenance is allowed but 
discouraged. 
 
Antibiotics and antifungals can be given according to institutional rules. For other drugs, refer to 
individual product US package inserts. Patients should receive all necessary supportive care, 
including blood product transfusions, erythropoietin, and pain medications. 
7.2.5 Tumor Lysis Syndrome Monitoring and Prophylaxis 
 
Patients with a low risk of developing tumor lysis syndrome (TLS) should be monitored for 
development of TLS and complications; normal hydration and no prophylaxis for hyperuricemia 
should be given except in cases of signs of metabolic changes, bulky and/or advanced disease 
and/or high proliferative disease, in which case allopurinol should be added. 
 
Patients with an intermediate risk of developing TLS should be monitored for TLS and 
complications, administered increased hydration (3 L/m2 per day) and administered allopurinol 
(100–300 mg, by mouth [PO], every 8 hours [Q8H], daily) without the need for alkalinization. 
 
In patients with high risk of developing TLS, frequent monitoring should be performed, 
increased hydration (3 L/m2 per day), unless evidence of renal insufficiency and oliguria, and 
rasburicase (0.1–0.2 mg/kg) for one dose and repeated only if clinically necessary. In patients 
with a prior history of glucose‐6‐phosphate dehydrogenase, rasburicase is contraindicated and 
allopurinol should be utilized instead ofrasburicase. 
 
Furthermore, management of hyperkalemia and/or hyperphosphatemia should be managed as per 
institutional routine. Lastly, patients who develop laboratory TLS who were originally classified as 
either low or intermediate risk, should receive rasburicase unless clinically contraindicated. 
 
7.3 Duration of Therapy 
 
In the absence of treatment delays due to AE(s), treatment may continue for up to 2 years of 

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maintenance therapy from first day of maintenance therapy or until one of the following criteria 
applies: 
 
• 
Disease progression 
• 
Once patient proceeds to HSCT 
• 
Intercurrent illness that prevents further administration of treatment. 
• 
AE(s) which require(s) treatment discontinuation (see also Section 7): 
o Any dosing interruption lasting >12 weeks with the following exceptions: Dosing 
interruptions >12 weeks that occur for non-drug-related reasons may be allowed if 
approved by the Principal Investigator. Prior to re-initiating treatment in a patient 
with a dosing interruption lasting >12 weeks, the Protocol Principal Investigator 
must be consulted. 
o Bone marrow and peripheral blood assessments should continue as per protocol 
even if dosing is interrupted. 
• 
Unacceptable AE(s) 
• 
Patient decides to withdraw from the study. 
• 
General or specific changes in the patient's condition render the patient unacceptable 
for further treatment in the judgment of the investigator. 
• 
Clinical progression 
• 
Severe, Significant, and persistent Patient non-compliance 
• 
Pregnancy 
o All women of child-bearing potential should be instructed to contact the 
investigator immediately if they suspect they might be pregnant (e.g., missed 
or late menstrual period) at any time during study participation. 
o The investigator must immediately notify CTEP in the event of a confirmed 
pregnancy in a patient participating in the study. 
• 
Termination of the study by sponsor 
• 
The drug manufacturer can no longer provide the study agent. 
 
The reason(s) for protocol therapy discontinuation, the reason(s) for study removal, and the 
corresponding dates must be documented in the Case Report Form (CRF). 
 
Patients with grade 3-4 IRAEs that completely resolve maybe allowed to continue on study on 
case-by-case basis based on consultation with study chair and CTEP. 
 
 
7.4 Duration of Follow-Up 
 
Patients will be followed up for survival and remission status for at least every 6 months for up 
to 5 years after the randomization or until death, whichever occurs first. Patients will be 
monitored for AE of Special Interest(s) post-transplant for the first 100 days, particularly 
GVHD and VOD, in both arms. We additionally recommend for the investigators that the 
patient not receive pembrolizumab within 30 days of receipt of transplant. Patients removed 
from study for unacceptable AE(s) will be followed until resolution or stabilization of the AE. 

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7.5 Criteria to Resume Treatment 
 
For non-autoimmune or inflammatory events, patients may resume treatment with study drug 
when the drug-related AE(s) resolve to ≤ grade 1 or baseline value, with the following 
exceptions: 
 
• Patients may resume treatment in the presence of grade 2 fatigue. 
• Patients with baseline grade 1 AST/ALT or total bilirubin who require dose delays for 
reasons other than a 2-grade shift in AST/ALT or total bilirubin may resume treatment in 
the presence of grade 2 AST/ALT OR total bilirubin. 
• Patients with combined grade 2 AST/ALT and total bilirubin values meeting study 
parameters outlined in Section 7.1 should have treatment permanently discontinued. 
• Non-drug-related toxicity including hepatic, pulmonary toxicity, diarrhea, or colitis, must 
have resolved to baseline before treatment is resumed. 
• Drug-related endocrinopathies (not including drug-related adrenal insufficiency or 
hypophysitis) adequately controlled with only physiologic hormone replacement may 
resume treatment after replacement correction and clinically stable regimen. 
 
If the criteria to resume treatment are met, the patient should restart treatment no sooner than the 
next scheduled time point per protocol. However, if the treatment is delayed past the next 
scheduled time point per protocol, the treatment should resume at the earliest convenient point 
that is within the 12-week delay period. 
 
If treatment is delayed >12 weeks, the patient must be permanently discontinued from study 
therapy, except as specified in Section 6.3 (Duration of Therapy). or except if they are allowed 
to continue on study on case-by-case basis based on consultation with study chair and CTEP. 
Patients with grade 3-4 IRAEs that completely resolve maybe allowed to continue on study on 
case-by-case basis based on consultation with study chair and CTEP. 
 
7.6 Contraception and Pregnancy 
 
                    7.6.1 Contraception 
 
Pembrolizumab (MK-3475) may have adverse effects on a fetus in utero. Furthermore, it is not 
known if pembrolizumab (MK- 3475) has transient adverse effects on the composition of 
sperm. 
 
Based on the mechanism of action and findings from animal reproduction studies, fetal harm 
may occur if cytarabine is administered during pregnancy. Risk to the fetus is decreased if 
treatment can be avoided during the first trimester; however, females of reproductive potential 
should avoid becoming pregnant during treatment and be advised of the potential risks. 
 
Idarubicin: Adverse events were observed in animal reproduction studies. Women of 
reproductive potential avoid pregnancy during treatment. 
 
Daunorubicin: Adverse events have been observed in animal reproduction studies. 

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Daunorubicin crosses the placenta. Women of reproductive potential should avoid pregnancy. 
 
For this trial, male patients will be considered to be of nonreproductive potential if they have 
azoospermia (whether due to having had a vasectomy or due to an underlying medical 
condition). 
 
Female patients will be considered of nonreproductive potential if they are either: 
 
1. Postmenopausal (defined as at least 12 months with no menses without an alternative 
medical cause; in women <45 years of age, a high follicle stimulating hormone (FSH) 
level in the postmenopausal range may be used to confirm a postmenopausal state in 
women not using hormonal contraception or hormonal replacement therapy. In the 
absence of 12 months of amenorrhea, a single FSH measurement is insufficient.); 
OR 
2. Have had a hysterectomy and/or bilateral oophorectomy, or bilateral salpingectomy at least 
6 weeks prior to screening. 
OR 
3. Has a congenital or acquired condition that prevents childbearing. 
 
Female and male patients with female partners of reproductive potential must agree to avoid becoming 
pregnant or impregnating a partner, respectively, while receiving study drug and for 120 days after 
the last dose of study drug by complying with one of the following: 
 
1. Practice abstinence† from heterosexual activity. 
OR 
2. Use (or have their partner use) adequate contraception during heterosexual activity. 
 
Acceptable methods of contraception are‡: 
 
Single method (1 of the following is acceptable): 
• intrauterine device (IUD) 
• vasectomy of a female patient’s male partner 
• contraceptive rod implanted into the skin. 
Combination method (requires use of 2 of the following): 
• diaphragm with spermicide (cannot be used in conjunction 
with cervical 
cap/spermicide) 
• cervical cap with spermicide (nulliparous women only) 
• contraceptive sponge (nulliparous women only) 
• male condom or female condom (cannot be used together) 
• hormonal contraceptive: oral contraceptive pill (estrogen/progestin pill or progestin- 
only pill), contraceptive skin patch, vaginal contraceptive ring, or subcutaneous 
contraceptive injection 
 
†Abstinence (relative to heterosexual activity) can be 
used as the sole method of 

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contraception if it is consistently employed as the patient’s preferred and usual lifestyle and 
if considered acceptable by local regulatory agencies and Ethics Review Committees 
(ERCs)/Institutional Review Boards (IRBs). Periodic abstinence (e.g., calendar, ovulation, 
sympto-thermal, post-ovulation methods, etc.) and withdrawal are not acceptable methods 
of contraception. 
‡If a contraceptive method listed above is restricted by local regulations/guidelines, then it 
does not qualify as an acceptable method of contraception for patients participating at sites 
in this country/region. 
 
Patients should be informed that taking the study medication may involve unknown risks to the 
fetus (unborn baby) if pregnancy were to occur during the study. In order to participate in the 
study, patients of childbearing potential must adhere to the contraception requirement (described 
above) from the day of study medication initiation (or 14 days prior to the initiation of study 
medication for oral contraception) throughout the study period up to 120 days after the last dose 
of trial therapy. If there is any question that a patient will not reliably comply with the 
requirements for contraception, that patient should not be entered into the study. 
 
7.6.2 Use in Pregnancy 
 
If a patient inadvertently becomes pregnant while on treatment with Pembrolizumab (MK-3475), 
the patient will immediately be removed from the study. The site will contact the patient at least 
monthly and document the patient’s status until the pregnancy has been completed or terminated. 
The outcome of the pregnancy will be reported without delay and within 24 hours if the outcome is a 
serious adverse experience (e.g., death, abortion, congenital anomaly, or other disabling or life- 
threatening complication to the mother or newborn). The study investigator will make every 
effort to obtain permission to follow the outcome of the pregnancy and report the condition of 
the fetus or newborn. If a male patient impregnates his female partner, the study personnel at the 
site must be informed immediately and the pregnancy reported and followed. 
 
7.6.3 Use in Nursing Women 
 
It is unknown whether Pembrolizumab (MK-3475) is excreted in human milk. Since many 
drugs are excreted in human milk, and because of the potential for serious adverse reactions in 
the nursing infant, patients who are breast-feeding are not eligible for enrollment. 
 
7.7 Treatment Up to 2 Years 
 
Treatment with Pembrolizumab (MK-3475) monotherapy will continue for up to 2 years, 
documented disease progression, unacceptable AE(s), intercurrent illness that prevents further 
administration of treatment, investigator’s decision to withdraw the patient, patient proceeding to 
HSCT, patient withdraws consent, pregnancy of the patient, noncompliance with trial treatment 
or procedure requirements, or administrative reasons. 
 
 
8 DOSING DELAYS/DOSE MODIFICATIONS 

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Dosing delays and dose modifications for 7+3 (along with added needed 5+2) and HiDAC 
therapy should follow institutional guidelines and/or treating threating physician’s opinion. 
 
8.1 Pembrolizumab Dose Modification and Supportive Care Guidelines for Drug-Related Adverse Events 
(revised 1-10-2023) 
 
8.1.1  Dose Modifications 
 
Adverse events (both nonserious and serious) associated with pembrolizumab exposure may represent an 
immunologic etiology. These AEs may occur shortly after the first dose or several months after the last dose of 
treatment. Pembrolizumab must be withheld for drug-related toxicities and severe or life-threatening AEs as the 
table in Section 1.1.3. 
Dosing interruptions are permitted in the case of medical/surgical events or logistical reasons not related to study 
therapy (e.g., elective surgery, unrelated medical events, patient vacation, and/or holidays). Patients should be 
placed back on study therapy within 3 weeks of the scheduled interruption. The reason for interruption should be 
documented in the patient's study record. 
 
8.1.2  Supportive Care Guidelines 
 
Patients should receive appropriate supportive care measures as deemed necessary by the treating investigator. 
Suggested supportive care measures for the management of AEs with potential immunologic etiology are also 
outlined in the table in Section 1.1.3. Where appropriate, these guidelines include the use of oral or IV treatment 
with corticosteroids as well as additional anti-inflammatory agents if symptoms do not improve with administration 
of corticosteroids. Note that several courses of steroid tapering may be necessary as symptoms may worsen when 
the steroid dose is decreased. For each disorder, attempts should be made to rule out other causes such as metastatic 
disease or bacterial or viral infection, which might require additional supportive care. The treatment guidelines are 
intended to be applied when the investigator determines the events to be related to pembrolizumab. 
 
Note: If after the evaluation the event is determined not to be related, the investigator does not need to follow the 
treatment guidance (as outlined below). 
 
It may be necessary to perform conditional procedures such as bronchoscopy, endoscopy, or skin photography as 
part of the evaluation of the event. 
 
8.1.3 Dose Modification and Toxicity Management for Immune-related Adverse Events Associated with  
Pembrolizumab  
 
AEs associated with pembrolizumab exposure may represent an immunologic etiology. These immune-related 
AEs (irAEs) may occur shortly after the first dose or several months after the last dose of pembrolizumab 
treatment and may affect more than one body system simultaneously. Therefore, early recognition and initiation 
of treatment is critical to reduce complications. Based on existing clinical study data, most irAEs were reversible 
and could be managed with interruptions of pembrolizumab, administration of corticosteroids and/or other 
supportive care. For suspected irAEs, ensure adequate evaluation to confirm etiology or exclude other causes. 

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Additional procedures or tests such as bronchoscopy, endoscopy, skin biopsy may be included as part of the 
evaluation. Based on the severity of irAEs, withhold or permanently discontinue pembrolizumab and administer 
corticosteroids. Pembrolizumab may cause severe or life-threatening infusion-reactions including severe 
hypersensitivity or anaphylaxis. Signs and symptoms usually develop during or shortly after drug infusion and 
generally resolve completely within 24 hours of completion of infusion. Dose modification and toxicity 
management guidelines for irAEs and infusion reactions associated with pembrolizumab are provided in the table 
below. 
 
Note that non-irAEs will be managed as appropriate, following clinical practice recommendations. 
 
Dose Modification and Toxicity Management Guidelines for Immune-related AEs and Infusion Reactions 
Associated with Pembrolizumab 
 
General instructions: 
1. For non-endocrine-related severe and life-threatening irAEs, investigators should consider the use of IV corticosteroids followed 
by oral steroids. Other immunosuppressive treatment should begin if the irAEs are not controlled by corticosteroids. Some non- 
endocrine irAEs do not require steroids. For example, celiac disease induced by pembrolizumab can be controlled by diet alone. 
2. For non-endocrine-related toxicities, pembrolizumab must be permanently discontinued if the irAE does not resolve or the 
corticosteroid dose is not ≤10 mg/day within 12 weeks of the last pembrolizumab treatment. 
3. Generally, when corticosteroids are used, investigators should begin a taper when the irAE is ≤Grade 1 and continue at least 
4 weeks. 
4. If pembrolizumab has been withheld due to a non-endocrine irAE, pembrolizumab may generally resume after the irAE has 
decreased to ≤Grade 1 after a corticosteroid taper. 
irAEs 
Toxicity grade (CTCAE 
V5.0) 
Action with 
pembrolizumab 
Corticosteroid and/or 
other therapies 
Monitoring and follow- 
up 
 
 
 
 
Pneumonitis 
Grade 2 
Withhold 
Administer corticosteroids 
(initial dose of 1 to 2 
mg/kg prednisone or 
equivalent) followed by 
taper. 
Add prophylactic antibiotics 
for opportunistic 
infections 
Monitor participants for 
signs and symptoms 
of pneumonitis. 
Evaluate participants 
with suspected 
pneumonitis with 
radiographic 
imaging and initiate 
corticosteroid 
treatment 
Recurrent Grade 2, 
Grade 3 or 4 
Permanently 
discontinue 
 
 
Diarrhea / Colitis 
Grade 2 or 3 
Withhold 
Administer corticosteroids 
(initial dose of 1 to 2 
mg/kg prednisone or 
equivalent) followed by 
taper 
Monitor participants for 
signs and symptoms 
of enterocolitis (i.e., 
diarrhea, abdominal 
pain, blood, or 
mucus in stool with 

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Recurrent Grade 3 or 
Grade 4 
Permanently 
discontinue 
Patients who do not respond 
to corticosteroids should 
be seen by a 
gastroenterologist for 
confirmation of the 
diagnosis and 
consideration of 
secondary immune 
suppression 
or without fever) 
and of bowel 
perforation (i.e. 
peritoneal signs and 
ileus) 
Specifically assess for 
celiac disease 
serologically and 
exclude Clostridium 
difficile infection. 
Participants with ≥Grade 
2 diarrhea 
suspecting 
enterocolitis should 
consider GI 
consultation and 
performing 
endoscopy to rule 
out enterocolitis and 
assess mucosal 
severity. 
Participants with 
diarrhea/colitis 
should be advised to 
drink liberal 
quantities of clear 
fluids. If sufficient 
oral fluid intake is 
not feasible, fluid 
and electrolytes 
should be 
substituted via IV 
Infusion. 
 
 
 
AST or ALT elevation or 
Increased Bilirubin 
Grade 2 a 
Withhold 
Administer corticosteroids 
(initial dose of 0.5 to 1 
mg/kg prednisone or 
equivalent) followed by 
taper 
Monitor with liver 
function tests 
(consider weekly or 
more frequently 
until liver enzyme 
value returned to 
baseline or is stable) 
Grade 3 b or 4 c 
Permanently 
discontinue 
Administer corticosteroids 
(initial dose of 1 to 2 
mg/kg prednisone or 
equivalent) followed by 
taper 

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Type 1 diabetes mellitus 
(T1DM) or 
Hyperglycemia 
Grade 1 or 2 
Continue 
Investigate for diabetes. 
In the absence of 
corticosteroids or 
diabetes medication 
non-adherence, any 
grade 
hyperglycemia may 
be an indication of 
beta-cell destruction 
and pembrolizumab- 
induced diabetes 
akin to type 1 
diabetes. This 
should be treated as 
a Grade 3 event. 
Given this risk, 
exercise caution in 
utilizing non-insulin 
hypoglycemic 
agents in this 
setting. After a 
thorough 
investigation of 
other potential 
causes, which may 
involve a referral to 
an endocrinologist, 
follow institutional 
guidelines. 
Monitor glucose control. 
New onset T1DM 
(evidence of -cell 
failure) or 
Grade 3 or 4 
hyperglycemia 
Withhold d 
Resume 
pembrolizumab when 
symptoms resolve and 
glucose levels are 
stable. 
Initiate treatment with 
insulin 
If patient is found to have 
diabetic ketoacidosis or 
hyperglycemic 
hyperosmolar syndrome, 
treat as per institutional 
guidelines with 
appropriate management 
and laboratory values 
(e.g. anion gap, ketones, 
blood pH, etc.) reported 
Monitor for glucose 
control. 
Strongly consider 
referral to 
endocrinologist 
Obtain C-peptide level 
paired with glucose, 
autoantibody levels 
(e.g. GAD65, islet 
cell autoantibodies), 
and hemoglobin 
A1C level 
 
Hypophysitis 
Grade 2 
Withhold 
Administer corticosteroids 
and initiate hormonal 
Monitor for signs and 
symptoms of 

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Grade 3 or 4 
Withhold or 
permanently 
discontinue d 
replacements as 
clinically indicated 
hypophysitis 
(including 
hypopituitarism and 
adrenal 
insufficiency) 
Provide adrenal 
insufficiency 
precautions 
including 
indications for stress 
dose steroids and 
medical alert 
jewelry. 
Strongly consider 
referral to 
endocrinologist 
 
 
 
Hyperthyroidism 
Grade 2 
Consider withholding. 
Resume 
pembrolizumab when 
symptoms are 
controlled, and thyroid 
function is improving. 
Treat with nonselective beta- 
blockers (e.g., 
propranolol) or 
thionamides as 
appropriate 
Initiate treatment with anti- 
thyroid drug such as 
methimazole or 
carbimazole as needed 
Monitor for signs and 
symptoms of thyroid 
disorders. 
Strongly consider 
referral to 
endocrinologist 
Grade 3 or 4 
Withhold or 
permanently 
discontinue d 
 
 
Hypothyroidism 
Grade 2, 3 or 4 
Continue 
Initiate thyroid replacement 
hormones (e.g., 
levothyroxine or 
liothyronine) per 
standard of care 
Monitor for signs and 
symptoms of thyroid 
disorders 
Nephritis: 
grading according to 
increased creatinine or 
acute kidney injury 
Grade 2 
Withhold 
Administer corticosteroids 
(prednisone 
1 to 2 mg/kg or 
equivalent) followed by 
taper 
Monitor changes of renal 
function 
Strongly consider 
referral to 
nephrologist 
Grade 3 or 4 
Permanently 
discontinue 
 
 
 
 
 
Cardiac Events 
(including myocarditis, 
pericarditis, arrhythmias, 
impaired ventricular 
function, vasculitis) 
Asymptomatic cardiac 
enzyme elevation with 
clinical suspicion of 
myocarditis (previously 
CTCAE v4.0 Grade 1), or 
Grade 1 
Withhold 
Based on severity of AE 
administer 
corticosteroids 
Ensure adequate 
evaluation to 
confirm etiology 
and/or exclude other 
causes. 
Strongly consider 
referral to 
cardiologist and 
cardiac MRI 
Consider 
endomyocardial 
biopsy 
If event resolves to 
Grade 1 or better, 
taper corticosteroids 
over ≥1 month 

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Grade 2, 3 or 4 
Permanently 
discontinue 
Initiate treatment with 
corticosteroids 
equivalent to 1-2 
mg/kg/day IV 
methylprednisolone and 
convert to 1-2 
mg/kg/day oral 
prednisone or equivalent 
upon improvement. 
If event does not improve 
within 48 hours after 
initiating 
corticosteroids, consider 
adding an 
immunosuppressive 
agent. 
Initiate treatment per 
institutional guidelines 
and consider 
antiarrhythmic drugs, 
temporary pacemaker, 
extracorporeal 
membrane oxygenation 
(ECMO), ventricular 
assist device (VAD), or 
pericardiocentesis as 
appropriate 
Ensure adequate 
evaluation to 
confirm etiology 
and/or exclude other 
causes. 
Strongly consider 
referral to 
cardiologist and 
cardiac MRI 
Consider 
endomyocardial 
biopsy 
If event resolves to 
Grade 1 or better, 
taper corticosteroids 
over ≥1 month 
 
 
 
 
Exfoliative Dermatologic 
Conditions 
Suspected SJS, TEN, or 
DRESS 
Withhold 
Based on severity of AE 
administer 
corticosteroids 
Ensure adequate 
evaluation to 
confirm etiology or 
exclude other 
causes. 
Strongly consider 
referral to 
dermatologist 
Consider skin biopsy for 
evaluation of 
etiology. 
Confirmed SJS, TEN, or 
DRESS 
Permanently 
discontinue 
 
 
 
All Other irAEs 
Persistent Grade 2 
Withhold 
Based on severity of AE 
administer 
corticosteroids 
Ensure adequate 
evaluation to 
confirm etiology or 
exclude other causes 
Grade 3 
Withhold or 
discontinue based on 
the event e 
Recurrent Grade 3 or 
Grade 4 
Permanently 
discontinue 

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Infusion-Related Reactions 
 
Infusion Reactions 
NCI CTCAE Grade 
Treatment 
Premedication at subsequent 
dosing 
Mild reaction; 
infusion interruption 
not indicated; 
intervention not 
indicated 
Grade 1 
Increase monitoring of vital signs as medically 
indicated until the participant is deemed medically 
stable in the opinion of the investigator. 
None 
Requires therapy or 
infusion interruption 
but responds 
promptly to 
symptomatic 
treatment (e.g., 
antihistamines, 
NSAIDs, narcotics, 
IV fluids); 
prophylactic 
medications indicated 
for ≤24 hrs. 
Grade 2 
• 
Stop Infusion. 
• 
Additional appropriate medical therapy 
may include but is not limited to: 
• 
IV fluids 
• 
Antihistamines 
• 
NSAIDs 
• 
Acetaminophen 
• 
Narcotics 
• 
Increase monitoring of vital signs as 
medically indicated until the participant is 
deemed medically stable in the opinion of 
the investigator. 
• 
If symptoms resolve within 1 hour of 
stopping drug infusion, the infusion may be 
restarted at 50% of the original infusion 
rate (e.g. from 100 mL/hr. to 50 mL/hr.). 
Otherwise dosing will be held until 
symptoms resolve and the participant 
should be premedicated for the next 
scheduled dose. 
Participants who develop Grade 2 toxicity 
despite adequate premedication should be 
permanently discontinued from further study 
drug treatment 
Participant may be premedicated 
1.5h (± 30 minutes) prior to 
infusion of study intervention with: 
Diphenhydramine 50 mg PO (or 
equivalent dose of antihistamine). 
Acetaminophen 500-1000 mg PO 
(or equivalent dose of analgesic). 

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Infusion Reactions 
NCI CTCAE Grade 
Treatment 
Premedication at subsequent 
dosing 
Prolonged (i.e., not 
rapidly responsive to 
symptomatic 
medication and/or 
brief interruption of 
infusion); recurrence 
of symptoms 
following initial 
improvement; 
hospitalization 
indicated for other 
clinical sequelae 
(e.g., renal 
impairment, 
pulmonary infiltrates) 
Grade 3 
• 
Stop Infusion. 
• 
Additional appropriate medical therapy 
may include but is not limited to: 
• 
Epinephrine** 
• 
IV fluids 
• 
Antihistamines 
• 
NSAIDs 
• 
Acetaminophen 
• 
Narcotics 
• 
Oxygen 
• 
Pressors 
• 
Corticosteroids (e.g. methylprednisolone 2 
mg/kg/day or dexamethasone 10 mg every 
6 hours) 
• 
Increase monitoring of vital signs as 
medically indicated until the participant is 
deemed medically stable in the opinion of 
the investigator. 
• 
Hospitalization may be indicated. 
**In cases of anaphylaxis, epinephrine should be 
used immediately. 
Participant is permanently discontinued from further 
study drug treatment. 
No subsequent dosing. 
Life-threatening; 
pressor or ventilator 
support indicated 
Grade 4 
Admit participant to intensive care unit (ICU) and 
initiate hemodynamic monitoring, mechanical 
ventilation, and/or IV fluids and vasopressors as 
needed. Monitor other organ function closely. 
Manage constitutional symptoms and organ 
toxicities as per institutional practice. 
Follow Grade 3 recommendations as applicable. 
No subsequent dosing. 

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Infusion Reactions 
NCI CTCAE Grade 
Treatment 
Premedication at subsequent 
dosing 
AE(s)=adverse event(s); ALT= alanine aminotransferase; AST=aspartate aminotransferase; CTCAE=Common Terminology Criteria for Adverse 
Events; DRESS=Drug Rash with Eosinophilia and Systemic Symptom; ECMO=extracorporeal membrane oxygenation; GI=gastrointestinal; 
ICU=intensive care unit; IO=immuno-oncology; ir=immune related; IV=intravenous; MRI=magnetic resonance imaging; PO=per os; SJS=Stevens- 
Johnson Syndrome; T1DM=type 1 diabetes mellitus; TEN=Toxic Epidermal Necrolysis; ULN=upper limit of normal; VAD=ventricular assist device. 
Note: Non-irAE will be managed as appropriate, following clinical practice recommendations. 
a AST/ALT: >3.0 to 5.0 x ULN if baseline normal; >3.0 to 5.0 x baseline, if baseline abnormal; 
bilirubin:>1.5 to 3.0 x ULN if baseline normal; >1.5 to 3.0 x baseline if baseline abnormal 
b AST/ALT: >5.0 to 20.0 x ULN, if baseline normal; >5.0 to 20.0 x baseline, if baseline abnormal; bilirubin:>3.0 to 10.0 x ULN if baseline 
normal; >3.0 to 10.0 x baseline if baseline abnormal 
c AST/ALT: >20.0 x ULN, if baseline normal; >20.0 x baseline, if baseline abnormal; 
bilirubin: >10.0 x ULN if baseline normal; >10.0 x baseline if baseline abnormal 
d The decision to withhold or permanently discontinue pembrolizumab is at the discretion of the investigator or treating physician. If 
control achieved or ≤Grade 2, pembrolizumab may be resumed. 
e Events that require discontinuation include but are not limited to: encephalitis and other clinically important irAEs (e.g. vasculitis and 
sclerosing cholangitis). 
 
Appropriate resuscitation equipment should be available at the bedside and a physician readily available during the period of drug 
administration. For further information, please refer to the Common Terminology Criteria for Adverse Events v5.0 (CTCAE) at 
http://ctep.cancer.gov. 
 
Neurological Toxicities 
 
Event 
Management 
Immune-mediated 
neuropathy, Grade 1 
• 
Continue pembrolizumab. 
• 
Investigate etiology. 
• 
Any cranial nerve disorder (including facial paresis) should be managed as per Grade 2 
management guidelines below. 
Immune-mediated 
neuropathy, including 
facial paresis, Grade 2 
• 
Withhold pembrolizumab for up to 12 weeks after event onset. a 
• 
Investigate etiology and refer patient to neurologist. 
• 
Initiate treatment as per institutional guidelines. 
• 
For general immune-mediated neuropathy: 
o If event resolves to Grade 1 or better, resume pembrolizumab. b 
o If event does not resolve to Grade 1 or better while withholding pembrolizumab, 
permanently discontinue pembrolizumab. c 
• 
For facial paresis: 
o If event resolves fully, resume pembrolizumab. b 
o If event does not resolve fully while withholding pembrolizumab, permanently 
discontinue pembrolizumab. c 
Immune-mediated 
neuropathy, including 
facial paresis, Grade 3 or 4 
• 
Permanently discontinue pembrolizumab. c 
• 
Refer patient to neurologist. 
• 
Initiate treatment as per institutional guidelines. 
Myasthenia gravis and 
Guillain-Barré syndrome 
(any grade) 
• 
Permanently discontinue pembrolizumab. c 
• 
Refer patient to neurologist. 
• 
Initiate treatment as per institutional guidelines. 
• 
Consider initiation of corticosteroids equivalent to 1−2 mg/kg/day oral or IV prednisone. 
a Pembrolizumab may be withheld for a longer period of time (i.e., 12 weeks after event onset) to allow for corticosteroids (if 

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initiated) to be reduced to the equivalent of 10 mg/day oral prednisone. The acceptable length of the extended period of time 
must be based on an assessment of benefit−risk by the investigator and in alignment with the protocol requirements for the 
duration of treatment and documented by the investigator. 
b If corticosteroids have been initiated, they must be tapered over 1 month to the equivalent of 10 mg/day oral prednisone 
before pembrolizumab can be resumed. 
c Resumption of pembrolizumab may be considered in patients who are deriving benefit and have fully recovered from the 
immune-mediated event. The decision to re-challenge patients with pembrolizumab should be based on investigator's 
assessment of benefit−risk and documented by the investigator (or an appropriate delegate). 
 
Event 
Management 
Immune-mediated myelitis, Grade 1 
• 
Continue pembrolizumab unless symptoms worsen or do not improve. 
• 
Investigate etiology and refer patient to a neurologist. 
Immune-mediated myelitis, Grade 2 
• 
Permanently discontinue pembrolizumab. 
• 
Investigate etiology and refer patient to a neurologist. 
• 
Rule out infection. 
• 
Initiate treatment with corticosteroids equivalent to 1-2 mg/kg/day oral 
prednisone. 
Immune-mediated myelitis, Grade 3 
or 4 
• 
Permanently discontinue pembrolizumab. 
• 
Refer patient to a neurologist. 
• 
Initiate treatment as per institutional guidelines. 
 
Event 
Management 
Immune-mediated 
meningoencephalitis, all 
grades 
• 
Permanently discontinue pembrolizumab. a 
• 
Refer patient to neurologist. 
• 
Initiate treatment with corticosteroids equivalent to 1−2 mg/kg/day IV 
methylprednisolone and convert to 1−2 mg/kg/day oral prednisone or equivalent 
upon improvement. 
• 
If event does not improve within 48 hours after initiating corticosteroids, consider 
adding an immunosuppressive agent. 
• 
If event resolves to Grade 1 or better, taper corticosteroids over 1 month. 
a Resumption of pembrolizumab may be considered in patients who are deriving benefit and have fully recovered from the 
immune-mediated event. The decision to re-challenge patients with pembrolizumab should be based on investigator's 
assessment of benefit−risk and documented by the investigator (or an appropriate delegate). 
 
8.1.4 
Non-immune-related Adverse Events 
 
Additionally, Pembrolizumab (MK-3475) will be withheld for other drug-related grade 4 
hematologic toxicities (except for during the induction and consolidation phase), non- 
hematological toxicity ≥ grade 3 including laboratory abnormalities that are judged as possibly 
or potentially related to Pembrolizumab (MK-3475), and severe or life-threatening AEs. The 
table below includes dose modification guidelines for other toxicities that do not appear to be 
irAEs. 
 
Dose Modification Guidelines for Non-IRAE-Related Adverse Events 

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Toxicity 
Grade 
Hold 
Treatment 
(Y/N) 
Timing for 
restarting 
treatment 
Dose/Schedule for 
restarting 
treatment 
Discontinue 
Subject 
Hematological Toxicity 
1, 2, 3 
No 
N/A 
N/A 
N/A* 
4 
Yes 
Toxicity 
resolves to 
grade 0-1 or 
baseline 
May increase the 
dosing interval by 1 
week 
Toxicity does not 
resolve within 12 
weeks of last 
infusion 
Permanent 
discontinuation 
should be 
considered for any 
severe or life- 
threatening event 
Non-hematological 
toxicity 
NOTE: Exception to be 
treated similar to 
grade 1 toxicity 
• Grade 2 alopecia 
• Grade 2 fatigue 
For additional information 
regarding Adverse Events 
with a potential Immune- 
Etiology reference Section 
7.1.1. 
1 
No 
N/A 
N/A 
N/A 
2 
Consider 
withholding 
for 
persistent 
symptoms 
Toxicity 
resolves to 
grade 0-1 or 
baseline 
Clinical AE resolves 
within 4 weeks: 
Same dose and 
schedule 
Clinical AE does 
not resolve within 4 
weeks: May 
increase the dosing 
interval by 1 week 
for each 
occurrence.  
Toxicity does not 
resolve within 12 
weeks of last 
infusion 
3 
Yes 
Toxicity 
resolves to 
grade 0-1 or 
baseline 
May increase the 
dosing interval by 1 
week for each 
occurrence 
Toxicity does not 
resolve within 12 
weeks of last 
infusion 
 
Toxicity 
Grade 
Hold 
Treatment 
(Y/N) 
Timing for 
restarting 
treatment 
Dose/Schedule for 
restarting 
treatment 
Discontinue 
Subject 
4 
Yes 
N/A 
N/A 
Subject must be 
discontinued 
*Note: As severe cytopenias during induction and consolidation phase of AML therapy are expected due to 
Intensive chemotherapy and the underlying disease, Pembrolizumab (MK-3475) will not be held for 
hematologic toxicity during these parts of the protocol therapy except in situations where it is felt causing or 
contributing to the hematologic toxicity. 
 
In case toxicity does not resolve to grade 0-1 within 12 weeks after last infusion, protocol 
treatment should be discontinued. With Protocol Principal Investigator agreement, subjects with 
a laboratory AE still at grade 2 after 12 weeks may continue treatment in the trial only if 
asymptomatic and controlled. Patients who experience a recurrence of the same severe or life - 
threatening event at the same grade or greater with re-challenge of Pembrolizumab (MK-3475) 
should be discontinued from protocol treatment. 
 
 

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8.2        Commercial Agents 
Below are proposed guidelines for adjustments, but investigators should determine the most 
appropriate course of action based on the specifics of the situation and circumstances for each 
individual patient. 
 
8.2.1   Cytarabine 
 
Hepatic Impairment 
Dosage should be modified depending on clinical response and degree of hepatic impairment, 
but no quantitative recommendations are available. 
 
Renal Impairment 
CrCl >60 mL/min: no dosage adjustment needed. 
CrCl ≤60 mL/min: increased risk of cerebellar and cerebral toxicity with high dose regimens. a 
recommended dosage adjustment is as follows: for a serum creatinine of 1.5–1.9 mg/dL or 
increase of serum creatinine from baseline of 0.5–1.2 mg/dL during treatment, reduce the dose of 
cytarabine to 1 g/m2/dose. for a serum creatinine ≥2 mg/dL or if the change in baseline serum 
creatinine was >1.2 mg/dL, reduce the dose of cytarabine to 100 mg/m2/day continuous 
IV infusion. 
 
8.2.2 
Daunorubicin 
 
Hepatic Impairment 
Total bilirubin 1.2–3 mg/dL: REDUCE RECOMMENDED DOSE BY50%. 
Total bilirubin >3–5 mg/dL: reduce recommended dose by 75%. 
Total bilirubin >5 mg/dL: omit dose. 
 
Renal Impairment 
It is recommended in patients with a serum creatinine >3 mg/dL to administer 50% of the dose. 
 
8.2.3 Idarubicin 
 
Hepatic Impairment 
Total bilirubin 2.5–5 mg/dL: reduce recommended dose by 50%. 
Total bilirubin >5 mg/dL: do not administer. 
 
Renal Impairment 
Dosage adjustment is recommended in patients with a serum creatinine greater than 2.5 mg/dL, 
but no quantitative recommendations are available. 
 
 
9 PHARMACEUTICAL INFORMATION 
 
A list of the AEs and potential risks associated with the investigational or commercial agents 
administered in this study can be found in Section 10.1. 

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9.1 CTEP IND Agent 
 
9.1.1 Pembrolizumab (MK-3475) NSC # 776864 
 
Other Names: Pembrolizumab, SCH 900475, KEYTRUDA® 
 
Classification: Anti-PD-1 MAb 
Molecular Weight: 148.9-149.5 KDa 
CAS Number: 1374853-91-4 
Mode of Action: The programmed cell death 1 (PD-1) receptor is an inhibitory receptor 
expressed by T-cells. When bound to either of its ligands, PD-L1 or PD-L2, activated PD-1 
negatively regulates T-cell activation and effector function. The pathway may be engaged by 
tumor cells to suppress immune control. Pembrolizumab (MK-3475) blocks the negative immune 
regulatory signaling by binding to the PD-1 receptor, inhibiting the interaction between PD-1 
and its ligands. 
 
Description: Pembrolizumab (MK-3475) is a humanized MAb of the IgG4/kappa isotype. 
 
How Supplied: Pembrolizumab (MK-3475) is supplied by Merck & Co., Inc. and distributed 
by the Pharmaceutical Management Branch, CTEP/DCTD/NCI. Pembrolizumab (MK-3475) 
injection is a sterile, preservative-free, clear to slightly opalescent, colorless to slightly yellow 
solution for intravenous use. Each vial contains 100 mg of pembrolizumab (MK-3475) in 4 mL 
of solution. Each 1 mL of solution contains 25 mg of pembrolizumab (MK-3475) and is 
formulated in: L-histidine (1.55 mg), polysorbate 80 (0.2 mg), sucrose (70 mg), and Water for 
Injection, USP. 
 
Preparation: Pembrolizumab (MK-3475) solution for infusion must be diluted prior to 
administration. Do not shake the vials. Do not use if opaque or extraneous particulate matter other 
than translucent to white proteinaceous particles is observed. Do not use if discolored. To prepare 
the infusion solution add the dose volume of Pembrolizumab (MK-3475) to an infusion bag 
containing 0.9% Sodium Chloride Injection, USP or 5% Dextrose Injection, USP. Gently invert the 
bag 10-15 times to mix the solution. The final concentration must be between 1 mg/mL to 10 
mg/mL. 
 
Compatible IV bag materials: PVC plasticized with DEHP, non-PVC (polyolefin), EVA, or PE 
lined polyolefin. 
 
Storage: Store intact vials between 2°C - 8°C (36°F - 46°F). Do not freeze. Protect from light by 
storing in the original box. 
 
If a storage temperature excursion is identified, promptly return Pembrolizumab (MK-3475) to 
between 2-8°C and quarantine the supplies. Provide a detailed report of the excursion (including 

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documentation of temperature monitoring and duration of the excursion) to 
PMBAfterHours@mail.nih.gov for determination of suitability. 
 
Stability: Refer to the package label for expiration. 
 
Administer prepared solutions immediately after preparation. If not administered immediately, prepared 
solutions may be stored refrigerated for up to 24 hours. Pembrolizumab (MK-3475) solutions may be stored at 
room temperature for a cumulative time of up to 6 hours. This includes room temperature storage of liquid drug 
product solution in vials, room temperature storage of infusion solution in the IV bag, and the duration of 
infusion. 
 
Route of Administration: IV infusion only. Do not administer as an IV push or bolus injection. 
 
Method of Administration: Infuse over approximately 30 minutes (range: 25 - 40 minutes) using 
an infusion set containing a low-protein binding 0.2 to 5 µm in-line filter made of polyethersulfone 
or polysulfone. Infusion rate should not exceed 6.7 mL/min. A central line is not required; 
however, if a subject has a central venous catheter in place, it is recommended that it be used for the 
infusion. Do not co-administer other drugs through the same infusion line. Following the infusion, 
flush the IV line with normal saline. 
 
Compatible infusion set materials: PVC plasticized with DEHP or DEHT, PVC and tri-(2- 
ethylhexyl) trimellitate, polyethylene lined PVC, polyurethane, or polybutadiene 
 
Patient Care Implications: Refer to the protocol for information on evaluation and 
management of potential immune-related AEs. 
 
Availability 
 
Pembrolizumab (MK-3475) is an investigational agent supplied to investigators by the Division 
of Cancer Treatment and Diagnosis (DCTD), NCI. 
 
Pembrolizumab (MK-3475) is provided to the NCI under a Collaborative Agreement between the 
Pharmaceutical Collaborator and the DCTD, NCI (see Section 13.4). 
 
9.1.2 Agent Ordering and Agent Accountability 
 
9.1.2.1 NCI-supplied agents may be requested by eligible participating 
Investigators (or their authorized designee) at each participating 
institution.The CTEP-assigned protocol number must be used for 
ordering all CTEP-supplied investigational agents. The eligible 
participating investigators at each participating institution must be 
registered with CTEP, DCTD through an annual submission of FDA 
Form 1572 (Statement of Investigator), NCI Biosketch, Agent 
Shipment Form, and Financial Disclosure Form (FDF). If there are 
several participating investigators at one institution, CTEP- supplied 
investigational agents for the study should be ordered under the name 

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of one lead participating investigator at that institution. 
Sites may order initial agent supplies when a subject has been randomized. 
 
Submit agent requests through the AURORA PMB Online Agent Order Processing 
application. Access to AURORA requires the establishment of a CTEP Identity and 
Access Management (IAM) account and the maintenance of an “active” account 
status, a “current” password, and active person registration status. For questions 
about drug orders, transfers, returns, or accountability, call or email PMB any time. 
Refer to the PMB’s AURORA website for specific policies and guidelines related 
to agent management;  https://ctepcore.nci.nih.gov/aurora. 
 
 
 
9.1.2.2 Agent Inventory Records – The investigator, or a responsible party 
designated by the investigator, must maintain a careful record of the 
receipt, dispensing and final disposition of all agents received from 
the PMB using the appropriate NCI Investigational Agent (Drug) 
Accountability Record (DARF) available on the CTEP forms page. 
Store and maintain separate NCI Investigational Agent 
Accountability Records for each agent, strength, formulation and 
ordering investigator on this protocol. 
9.1.2.3   Material Safety Data Sheets –  
The current versions of the material safety data sheets (MSDS or SDS) for PMB-
distributed agents will be accessible to site investigators and research staff through the 
PMB AURORA application. Questions about MSDS access may be directed to the 
PMB at PMBAfterHours@mail.nih.gov or by using the dialog function in AURORA 
to communicate with PMB staff. 
9.1.2.4 Product Quality Complaint (PQC) 
A product quality complaint is defined as any suspicion of a 
product defect related to a potential quality issue during 
manufacturing, packaging, release testing, stability monitoring, 
dose preparation, storage or distribution of the product, or delivery 
system.  Not all PQCs involve a study subject.  Lot or batch 
numbers are of high significance and need to be provided where 
and when possible. PQC must be reported to the PMB as soon as 
the PQC is identified.  Report PQC to PMB at 
PMBAfterHours@mail.nih.gov or by using the dialog function in 
AURORA to communicate with PMB staff. 
9.1.3 Investigator Brochure Availability 
 
The current versions of the IBs for the agents will be accessible to site investigators and research 
staff through the AURORA PMB  application. Access to AURORA requires the establishment 
of a CTEP IAM account and the maintenance of an “active” account status, a “current” password 
and active person registration status. Questions about IB access may be directed to the PMB IB 
Coordinator via email. 
 

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9.1.4 Useful Links and Contacts 
 
• CTEP Forms, Templates, Documents: http://ctep.cancer.gov/forms/ 
• NCI CTEP Investigator Registration: RCRHelpDesk@nih.gov 
• PMB policies and guidelines: http://ctep.cancer.gov/branches/pmb/agent_management.htm 
• 
PMB AURORA application: 
https://ctepcore.nci.nih.gov/aurora. 
• CTEP Identity and Access Management (IAM) account: 
https://ctepcore.nci.nih.gov/iam/ 
• CTEP IAM account help: ctepreghelp@ctep.nci.nih.gov 
• IB Coordinator: IBCoordinator@mail.nih.gov 
• PMB email: PMBAfterHours@mail.nih.gov 
• 
PMB phone and hours of service: (240) 276-6575 Monday through Friday between 8:30 am 
and 4:30 pm (ET) 
 
9.2 
Commercial Agents 
 
9.2.1. Cytarabine NSC # 63878 
 
Other Names : ara-C 
Chemical Name : 4-amino-1-β-Darabinofuranosyl-2(1H)-pyrimidinone 
 
Classification: antineoplastic 
Molecular Weight: 243.217 g/mol 
CAS Number: 63878 
Mode of Action: Cytarabine inhibits DNA synthesis. Cytarabine gains entry into cells by a 
carrier process, and then must be converted to its active compound, aracytidine triphosphate. 
Cytarabine is a pyrimidine analog and is incorporated into DNA; however, the primary action is 
inhibition of DNA polymerase resulting in decreased DNA synthesis and repair. The degree of 
cytotoxicity correlates linearly with incorporation into DNA; therefore, incorporation into the 
DNA is responsible for drug activity and toxicity. Cytarabine is specific for the S phase of the 
cell cycle (blocks progression from the G1 to the S phase). 
 
Side Effects : Refer to drug package insert for additional information. 
 
Side effects include: 
• Cardiovascular: Angina pectoris, chest pain, local thrombophlebitis, pericarditis 
• Central nervous system: Aseptic meningitis, cerebral dysfunction, dizziness, headache, 
neuritis, neurotoxicity, paralysis (intrathecal and IV combination therapy), reversible 
posterior leukoencephalopathy syndrome 
• Dermatologic: Acute generalized exanthematous pustulosis, alopecia, dermal ulcer, 
ephelis, pruritus, skin rash, urticaria 

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• Endocrine & metabolic: Hyperuricemia 
• Gastrointestinal: Abdominal pain, anal fissure, anorexia, diarrhea, esophageal ulcer, 
esophagitis, increased serum amylase, increased serum lipase, intestinal necrosis, 
mucositis, nausea, pancreatitis, sore throat, toxic megacolon, vomiting 
• Genitourinary: Urinary retention 
• Hematologic & oncologic: Anemia, bone marrow depression, hemorrhage, leukopenia, 
megaloblastic anemia, neutropenia (onset: 1 to 7 days; nadir [biphasic]: 7 to 9 days a nd 
at 15 to 24 days; recovery [biphasic]: 9 to 12 days and at 24 to 34 days), 
reticulocytopenia, thrombocytopenia (onset: 5 days; nadir: 12 to 15 days; recovery 15 to 
25 days) 
• Hepatic: Hepatic insufficiency, hepatic sinusoidal obstruction syndrome (formerly 
known as hepatic veno-occlusive disease), increased serum transaminases (acute), 
jaundice 
• Hypersensitivity: Allergic edema, anaphylaxis 
• Infection: Sepsis 
• Local: Cellulitis at injection site, inflammation at injection site (SC injection), local 
inflammation (anus), pain at injection site (SC injection) 
• Neuromuscular & skeletal: Rhabdomyolysis 
• Ophthalmic: Conjunctivitis 
• Renal: Renal insufficiency 
• Respiratory: Acute respiratory distress, dyspnea, interstitial pneumonitis 
• Miscellaneous: Drug toxicity (cytarabine syndrome; chest pain, conjunctivitis, fever, 
maculopapular rash, malaise, myalgia, ostealgia), fever 
 
Availability: Commercially available. 
 
Product Description: Cytarabine is an odorless, white to off-white, crystalline powder which is 
freely soluble in water and slightly soluble in alcohol and in chloroform. Cytarabine is 
commercially available. 
 
Preparation: Clear colorless solution. Refer to standard local procedure or the package insert 
for standard preparation instructions. 
 
Storage and Stability: From a microbiological point of view, the product should be used 
immediately. If not used immediately, in-use storage times and conditions are the responsibility 
of the user and would normally not be longer than 24 hours at 2-8°C, unless dilution has taken 
place in controlled and validated aseptic conditions. 
 
Route of Administration: Continuous IV, per institutional guidelines. 
Additional information can be found in the package insert for cytarabine. 
9.2.2 Daunorubicin NSC # 82151 
Other Names : Cerubidin 

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Classification: 
antineoplastic Molecular 
Weight: 527.52 g/mol 
CAS Number: 82151 
Mode of Action: Daunorubicin inhibits DNA and RNA synthesis by intercalation between 
DNA base pairs and by steric obstruction. Daunomycin intercalates at points of local uncoiling 
of the double helix. Although the exact mechanism is unclear, it appears that direct binding to 
DNA (intercalation) and inhibition of DNA repair (topoisomerase II inhibition) result in 
blockade of DNA and RNA synthesis and fragmentation of DNA. 
Side Effects : Refer to drug package insert for additional information. 
Side effects include: 
• >10%: 
o Cardiovascular: Cardiac failure (dose-related, may be delayed for 7 to 8 years 
after treatment), ECG abnormality (transient, generally asymptomatic and self- 
limiting; includes atrial premature contractions, ST segment changes on ECG, 
supraventricular tachycardia, ventricular premature contractions) 
o Dermatologic: Alopecia (reversible) 
o Gastrointestinal: Nausea (mild), stomatitis, vomiting (mild) 
o Genitourinary: Red urine discoloration 
o Hematologic & oncologic: Bone marrow depression (onset: 7 days; nadir: 10 to 
14 days; recovery: 21 to 28 days; primarily leukopenia; anemia, 
thrombocytopenia) 
o Miscellaneous: Radiation recall phenomenon 
• 1% to 10%: 
o Dermatologic: Discoloration of sweat 
o Endocrine & metabolic: Hyperuricemia 
o Gastrointestinal: Abdominal pain, diarrhea, discoloration of saliva, 
gastrointestinal ulcer 
o Local: Post-injection flare 
o Ophthalmic: Discoloration of tears 
Availability: Commercially available 
Product Description: Daunorubicin hydrochloride is the hydrochloride salt of an anthracycline 
cytotoxic antibiotic produced by a strain of Streptomyces coeruleorubidus. It is provided as a 
deep red sterile liquid in vials for intravenous administration only. Its molecular formula is 
C27H29NO10•HCl with a molecular weight of 563.99. It is a hygroscopic crystalline powder. 
The pH of a 5 mg/mL aqueous solution is 4 to 5. Daunorubicin is commercially available. 
 
Preparation: Deep red liquid. Refer to the package insert for standard preparation instructions. 

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Storage and Stability: Stored at 25°C (77°F); excursions permitted to 15°C–30°C (59°F–86°F). 
Needs to be protected from moisture. 
 
Route of Administration: slow IV push over 10 to 15 minutes into the tubing of a rapidly 
infusing IV solution of 1L D5W or NS or and infuse over 15 to 30 minutes), or as per standard 
institutional practice. 
 
Additional information can be found in the package insert for daunorubicin. 
 
9.2.3 Idarubicin NSC # 256439 
Other Names : Idamycin 
Classification: antineoplastic 
Molecular Weight: 497.494 g/mol 
CAS Number: 256439 
Mode of Action: Idarubicin inhibits DNA and RNA synthesis by intercalation between DNA 
base pairs and by steric obstruction. Although the exact mechanism is unclear, it appears that 
direct binding to DNA (intercalation) and inhibition of DNA repair (topoisomerase II inhibition) 
result in blockade of DNA and RNA synthesis and fragmentation of DNA. 
Side Effects : Refer to drug package insert for additional information. 
Side effects include: 
• >10%: 
o Cardiovascular: Cardiac failure (dose-related), ECG abnormalities (transient; 
includes atrial premature contractions, S-T wave changes, supraventricular 
tachycardia, ventricular premature contractions; generally asymptomatic and self- 
limiting) 
o Central nervous system: Headache 
o Dermatologic: Alopecia (25% to 30%), skin rash (11%), urticaria 
o Gastrointestinal: Vomiting (30% to 60%), gastrointestinal hemorrhage (30%), 
diarrhea (9% to 22%), stomatitis (11%), nausea 
o Genitourinary: Urine discoloration (darker yellow) 
o Hematologic & oncologic: Anemia (effects are generally less severe with oral 
dosing), bone marrow suppression (nadir: 10 to 15 days; recovery: 21 to 28 days, 
primarily leukopenia, effects are generally less severe with oral dosing), 
thrombocytopenia (effects are generally less severe with oral dosing) 
o Hepatic: Increased serum bilirubin (≤44%), increased serum transaminases 
(≤44%) 
o Miscellaneous: Radiation recall phenomenon 
• 1% to 10%: 

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o Central nervous system: Peripheral neuropathy, seizure 
Availability: Commercially available. 
 
Product Description: Idarubicin hydrochloride for injection, USP is a sterile, semi-synthetic 
antineoplastic anthracycline for intravenous use. Chemically, idarubicin hydrochloride is 5,12- 
Naphthacenedione, 9-acetyl-7-[(3-amino-2,3,6-trideoxy-α-L-lyxohexopyranosyl)oxy]-7,8,9,10- 
tetrahydro-6,9,11-trihydroxyhydrochloride, (7S-cis). Idarubicin is commercially available. 
 
Preparation: 20 mg vials are reconstituted with 20 ml of water. Refer to the package insert for 
standard preparation instructions. 
 
Storage and Stability: Stored at controlled room temperature, 15° to 30°C (59° to 86°F) and 
protected from light. 
 
Route of Administration: slow IV push over 10 to 15 minutes into the tubing of a rapidly 
infusing IV solution of 1L D5W or NS or and infuse over 15 to 30 minutes), or as per standard 
institutional practice. 
Potential Drug Interactions: Refer to drug package insert for additional information. 
Additional information can be found in the package insert for idarubicin. 
 
10 STATISTICAL CONSIDERATIONS 
 
10.1 Study Design/Endpoints 
 
This is an open label, randomized, phase 2 study. The study will consist of an induction, 
consolidation, and maintenance phase of therapy. A total of 50 patients will be included (25 
patients in the intervention arm and 25 patients in the control arm). 
 
Induction chemotherapy for AML has been demonstrated to result in MRD negative CR (not just 
CR) in about 50% of patients treated (Freeman et al., 2013). Few large data sets of MRD 
negative CR exist across different subpopulations. We hypothesize that the addition of MK- 
3475 (pembrolizumab) to induction chemotherapy will lead to a 25% improvement of MRD 
negative CR/CRi after induction and one cycle of consolidation therapy compared to 7+3 and 
one cycle of consolidation chemotherapy alone. We expect a response rate of 75% in the 
intervention arm and 50% in the control arm for patients completing their respective 
treatments. Using intention-to-treat analysis, in open label study, with 25 patients in the 
intervention arm and 25 patients in the control arm and a significance level of 0.1,  we have 
approximately 71% power to detect an MRD-negative CR/CRi response rate difference of 25% 
using a one-sided Z-test for two proportions. 
 
Early safety schedule change: After the first 6 patients have been enrolled on the experimental  
arm, a safety review committee comprised of the study chair, the PIs of any patient who was  
enrolled in the experimental arm of the trial, and a representative from CTEP will review the  

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toxicity data to identify any concerning drug-related safety signals before enrolling further  
patients on the experimental arm. We will temporarily hold enrollment in the study if two or 
more patients of the first evaluable six patients in pembrolizumab (MK-3475) arm develop  
pembrolizumab-related severe toxicity during evaluability period. The evaluability period includes  
the first 35 days after pembrolizumab first dose, till hematologic recovery, or till starting  
consolidation therapy, whichever occurs first, as defined in section 9.5.1. To be considered  
evaluable for purposes of the early safety rule, the patient must have received at least one  
pembrolizumab dose. If the true toxicity rate is 50% or greater, this rule will stop the trial  
with probability at least 0.89. Similarly, if the true toxicity rate is 33%, this stopping rule will  
end the trial with probability 0.65. In the event this occurs, the study would resume with another  
6-patient cohort in which pembrolizumab dosing will start on Day 21 instead of Day 8.  
The patient has to receive at least one dose of pembrolizumab to be considered evaluable for  
safety assessment for purposes of early stopping safety rule. If the altered schedule still results  
in unacceptable toxicity, we will discuss alternative approaches with CTEP. No enrollment  
may take place until this discussion has occurred and a suitable plan has been developed. 
Toxicity analysis: The first six enrolled patients will be assessed for severe toxicity in the 
pembrolizumab arm. In the event that severe toxicity occurs in 2 or more of the first 6 patients, 
the study would resume with another 6-patient cohort in which pembrolizumab dosing will 
start on Day 21 instead of Day 8. If the altered schedule still results in unacceptable toxicity, we 
will discuss alternative approaches with CTEP. A formal toxicity analysis will be conducted after 
25 efficacy-evaluable patients in each study arm.  
 
Follow up: For patients who neither relapse nor die, the DOR will be censored on the date of 
their last evaluable disease assessment. Median RFS and DOR will be estimated with Kaplan- 
Meier curves with 95% confidence interval calculated based on the Brookmeyer-Crowley 
method. Long-term patient survival and disease status information will be collected for up to 
three years from first dose. An analysis of the survival and RFS data will be conducted based on 
the five-year follow up from date of randomization. Patients alive at the end of the follow-up 
period or lost to follow-up will be censored at the last date the patient is known to be alive. 
Continuous variables will be expressed by means, standard deviations and 95% confidence 
intervals. Frequencies will be computed for discrete data. Bonferroni’s correction will be used 
for multiple comparisons, if any, to protect the overall type I error rate. 
 
10.1.1 Primary endpoint 
 
MRD-negative CR/CRi response rate at time of count recovery after first cycle of consolidation 
therapy with chemotherapy + Pembrolizumab (MK-3475) vs. 7+3 and one cycle of 
consolidation chemotherapy alone. 
 
10.1.2 MRD assessment by multicolor flow cytometry (MFC) as an integral biomarker 
 
The current state of the art in AML MRD by flow cytometry can detect 0.1% for ~95% of 
patients and 0.01% for a major subset of those (~40-50%) but is heavily dependent on the 
immunophenotype of the leukemia and nature of the background population. The sensitivity of 
the assay varies depending on those two variables, so the threshold at present is not uniformly 

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0.01% for all patients. Importantly, the primary futility and efficacy analyses will compare MRD 
negative CR/CRi rates between the two arms, and if the MRD by flow can detect below this 
limit of 0.01% in only 40% of patients, then 60% of patients might not contribute to study 
primary endpoint differential effect. Given the limitations of the current technology, an MRD 
quantified below <0.1% may still be consistent with residual leukemia. We therefore have 
decided to use this cutoff to distinguish MRD-positive from MRD-negative patients as this 
threshold was found relevant in most published studies to date and has been included in the 
official consensus document on minimal residual disease by the European Leukemia Net MRD 
Working Party. Thus, cutoff levels below 0.1% (e.g., <0.01%) may define patients with 
particularly good outcome. 
 
10.2 Sample Size/Accrual Rate 
 
A total of 50 patients (25 per arm) are planned to be enrolled in the intent-to-treat analysis, which 
will evaluate a 25% difference in the rate of MRD-negative CR/CRi with ~71% power and 
an overall, one-sided alpha level of 0.1. All patients being randomized and starting induction 
therapy should be included in the primary analysis. 
 
 
 
 
 
PLANNED ENROLLMENT REPORT 
 
Racial Categories 
Ethnic Categories 
Total 
Not Hispanic or Latino 
Hispanic or Latino 
Female 
Male 
Female 
Male 
American Indian/ Alaska 
Native 
0 
0 
0 
0 
0 
Asian 
1 
1 
0 
0 
2 
Native Hawaiian or 
Other Pacific Islander 
1 
1 
0 
0 
2 
Black or African 
American 
1 
1 
1 
1 
4 
White 
15 
20 
1 
2 
38 
More Than One Race 
1 
1 
1 
1 
4 
Total 
19 
24 
3 
4 
50 
PHS 398 / PHS 2590 (Rev. 08/12 Approved Through 8/31/2015) 
OMB No.0925-0001/0002 

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10.3  Stratification Factors 
 
 
Planned stratification factors include age (younger than 65 vs. 65 and older), cytogenetics by 
FISH or metaphase karyotype (presence vs. absence of core binding factor inversions and 
translocations), and t-AML or AML arising from prior/antecedent MDS (yes/no). Dose 
escalation and MTD determination are not parts of this study. 
 
10.4    Analysis of Secondary and Exploratory Endpoints 
 
10.4.1 Secondary Endpoints 
 
10.4.1.1 Efficacy Endpoints: 
 
• Assess the rate of CR/CRi as defined per the ELN 2017 (see Appendix D) response 
criteria at time of count recovery after induction therapy with chemotherapy + 
Pembrolizumab (MK-3475). 
 
• Assess MRD negativity at Day 14. 
• Assess the rate of MRD-negative CR at end of induction therapy. 
 
• Assess the rate of MRD-negative CR at end of consolidation. 
 
• Assess rates of complete remission with partial recovery count (CRh) and Hematologic 
improvement (HI) to red blood cells and platelets. 
 
• Assess the percentage of patients with MRD-CR at time of count recovery after first 
cycle of consolidation therapy using a MRD cut-off of 0.01%. 
 
• Assess EFS, RFS, calculated as the time from initial treatment to either disease relapse or 
death. 
 
• Assess the DOR (defined as the time from first CR to the date of the first documented 
relapse or death, whichever occurs first) and OS. 
 
10.4.1.2 Safety Endpoints: 
 
• Assess safety endpoints including proportion of patients who develop severe toxicity. 
 
10.4.2 Exploratory Endpoints 
 
10.4.2.1 MRD assessment by DS as an exploratory biomarker 
 
All diagnostic and remission samples will undergo DNA extraction at the Fred Hutchinson 

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Cancer Research Center. De-identified DNA samples will be shipped to TwinStrand for DS at 
an average molecular depth of 20,000x. Samples will initially be investigator-blinded as to any 
patient information. Library preparation, sequencing, and analysis will be performed with 
TwinStrand’s optimized workflow, and TwinStrand’s bioinformatics core will perform all 
analyses related to assay output. 
 
We anticipate two potential issues using DS: 
 
(1) MRD positive cases without relapse (“false positives”): 
It is possible that our assay will not always be able to distinguish true MRD from clonal 
hematopoiesis of indeterminate potential (CHIP), and misclassification of CHIP as MRD 
would yield “false positive” results. While some mutations in AML genes (e.g., KRAS, 
FLT3) are pathognomonic for disease, others (e.g., DNMT3A) are potentially related to 
CHIP and can persist in remission without indicating imminent relapse and can even be 
detected in healthy older individuals. Rather than simply ignoring these mutations, we 
will use our dataset to see if they can provide prognostic information. If our model does 
not adequately differentiate between MRD and CHIP based on a high proportion of false 
positive cases, we will consider alternative strategies, including having our Fred Hutch 
collaborators perform flow sorting to isolate lymphoid cells, with the theory that 
mutations present in both lymphoid and myeloid cells are very likely to be CHIP-related. 
It should be noted that any sufficiently sensitive MRD assay faces with the same problem 
of distinguishing biological background from true disease, and this issue is not unique to 
our novel DS approach. Rather than ignoring the “CHIP problem”, however, we will 
harness the power of a large dataset plus the unprecedented sensitivity of the DS platform 
to understand how best to analyze all genomic data, CHIP-related and not, to provide 
prognostic information. 
 
(2) MRD negative cases that relapse (“false negatives”): 
One reason for residual disease to be undetectable in bone marrow is hemodilution, or 
contamination with peripheral blood. We will verify whether hemodilution is present on 
pathology reports. However, we should also consider the possibility that our assay either 
does not capture or is insufficiently sensitive for the particular persistent clone(s). 
Insufficient quantity of DNA is unlikely to lead to false negatives: ~1 μg is needed for 
DS, and we would expect to isolate dozens of micrograms from each sample. In all 
“false negative” cases, we will perform our DS panel on the patient’s marrow sample 
from diagnosis. For any mutations present at diagnosis, we would plan to repeat 
sequencing to a greater depth on remission marrows just for this subset. Practically, we 
would be able to sequence to a depth of 50,000x or 100,000x, which would provide 80% 
power to detect alleles with frequency of 0.003% (1 in 30,000) or 0.0017% (1 in 60,000) 
respectively. While we have designed our panel to have one-size-fits-all utility for the 
vast majority of AML patients, if we find that deeper sequencing reduces false negatives, 
we could either routinely sequence the entire panel more deeply or, most cost-effectively, 
simply use only a small subset of the probes to interrogate mutations known to be 
relevant for a given patient based on diagnosis. It is also possible that rare patients will 
have mutations that are not picked up in our panel. Our panel, which is relatively small, 

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does not include several larger AML-associated rearrangements. Many commercial 
products based on RT-PCR are able to detect these rearrangements extremely accurately. 
For the rare samples where these are the only markers of disease, we could supplement 
our panel with specific RT-PCR for these fusion products. 
 
10.4.2.1.1 Comparing DS and MFC for MRD detection as an exploratory biomarker 
 
In order to compare both approaches, DS will be performed on these samples at a molecular 
depth of 20,000x and at the same time the samples will also perform 10-color MFC, although 
researchers will be blinded to MFC results. The primary analysis will involve comparison in 
performance of MRD detected by DS vs. MFC patients. McNemar’s Chi-squared test for paired 
samples (regular DS vs. MFC and ultra-deep DS vs. MFC) will be used to compare the MRD 
measures. 
 
10.4.2.1.2 Analyzing circulating free DNA (cfDNA) as an exploratory biomarker 
We will leverage the high sensitivity and accuracy of DS to detect and quantitate low level 
mutations in circulating free DNA (cfDNA) which will allow for examining the prognostic 
value of not only circulating tumor DNA (ctDNA) but also specific genes and mutation allele 
frequencies. Blood samples will be collected at baseline and pre-defined time points after 
treatment. 
 
The ability to accurately assess ctDNA-based MRD would 1) allow the development and 
application of DS strategy for ctDNA-based longitudinal MRD monitoring in acute myeloid 
leukemia. While MRD is known to be a powerful tool to predict relapse, shortcomings in 
sensitivity and accuracy of conventional MRD techniques such as MFC limits MRD detection 
and risk assessment. In this regard, the unprecedented sensitivity and accuracy of DS would 
allow for very low-level detection of MRD and potentially more accurately predict relapse. 
Second, DS-based ctDNA MRD detection serves as a proof-of-principle study supporting the 
utility of ctDNA as a clinical biomarker of relapse in hematologic malignancies. ctDNA 
represents one of the most sensitive and noninvasive biomarkers in solid cancers. However, 
there far fewer studies exploring its utility in hematologic malignancies. Third, if shown to be 
an accurate predictor of relapse, ctDNA MRD detection may potentially act as a sensitive and 
accurate surrogate marker of long-term response in future clinical trials. Finally, ctDNA MRD 
detection will allow us to monitor disease progression and understand patterns of relapse in 
the standard and I/O with standard therapy settings, especially addressing if these are different 
in the setting of I/O directed therapy. Further, ctDNA monitoring can provide insights into 
clonal tumor hierarchy and capture subclonal dynamics. 
 
The best cell for monitoring MRD is unclear. In chronic myeloid leukemia (CML) studies 
have shown that peripheral blood monitoring is comparable to bone marrow, and therefore 
ELN and NCCN guidelines recommend a bone marrow only at diagnosis, with interval 
monitoring of MRD performed on peripheral blood. Assessment of the bone marrow in the 
MRD state may be limited by special factors (the biopsy needle misses the residual AML), or 
dilution (the tube sent for MRD is usually the last one). Thus, peripheral blood may have an 
advantage of being a collection site for many small clusters of MRD residing in the marrow. 
AML derived ctDNA may be an advantage over both marrow and peripheral assessment, as it 

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assays discarded DNA from AML cells residing in marrow niches, but not circulating. ctDNA 
constitutes only a fraction of the total cfDNA pool in the plasma and, therefore, it is very 
likely that ctDNA occurs at very low allele frequencies. However, ctDNA is an excellent 
template for sensitive DNA sequencing techniques, as the ctDNA is “pre-sheared” to a 
uniform fragment size of ~175 bp due to Caspase activity. Not having to ultrasonically 
fragment the ctDNA, as opposed to normal genomic DNA, improves conversion efficiency 
several hundred percent. We will leverage the high sensitivity and accuracy of DS to detect 
and quantitate low level mutations in cfDNA which will allow for examining the prognostic 
value of not only ctDNA but also specific genes and mutation allele frequencies. Blood 
samples will be collected at baseline and pre-defined time points after treatment. 
 
To compare cfDNA to both DS-based and MFC serial assessment for relapse prediction, 
separate multivariate models will be developed for each method. Relapse will be modeled as a 
time-to-event endpoint and Cox regression will be used to assess the association of ctDNA 
based MRD with this outcome. In the SWOG study, S0106 for newly diagnosed adult AML, 
MRD status by MFC was a strong predictor of survival, yet MRD predicted relapse-free 
survival with a poor c-statistic of 0.58.1 We hypothesize that DS will lead to an improvement 
in prediction accuracy of relapse-risk (c-statistic 0.85). Assuming MRD-negativity (among 
CR/CRi) ratios of 1, 3, 0.3, and 0.45 for intensive chemo, intensive chemo + Pem, Aza + Ven, 
and Aza + Ven + Pem arms, respectively, and a correlation coefficient (ῥ) between MFC and 
DS assays of 0.75 (based on MFC and NGS comparison data),29 a minimum total sample size 
of 16, 24, 20, and 18 evaluable patients with CR/CRi would provide 80% power to detect a 
between-arm differencing based on a comparison of two proportional hazards survival curves 
with one-sided type-I error of 10% without correction for the multiplicity of endpoints: TTR; 
DFS; and OS. 
 
 
10.4.2.2 Assessment of immune cell subsets and its correlation with the response to combine 
chemotherapy and anti PD-1 directed therapy 
 
We will possibly have limited number of cells in the BM aspirate. Therefore, we will analyze 
the immune profile combining a high-throughput technique that will allow us to study more than 
20 markers in a single cell analysis using mass cytometry (CyTOF). These techniques will 
provide information about the architecture of the tumor microenvironment (TME) (i.e., BM) and 
the distribution of markers of interest detected with CyTOF. CyTOF-based single cell analysis is 
a new technology that combines flow cytometry with metal-conjugated antibodies detected by 
mass spectrometry permitting higher order multiplexing (up to 100 molecules) on a single cell 
analysis (Bendall et al., 2011). Additionally, after fixation and permeabilization, cells will be 
stained with anti-human Ki-67-151Eu (B56; BD Pharmingen Ab conjugated with lanthanide 
MaxPar Europium Chloride 151Eu using the MaxPar X8 Ab labeling kit) for 30 min at room 
temperature. Cells will be acquired on CyTOF 2 instrument (DVS; Fluidigm Sciences). All data 
will be analyzed and graphs generated using the DVS Cytobank software (Cytobank). 
 
We will assess these immune cell markers at different time points of therapy: At baseline (prior 

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to application of 7+3 chemotherapy), at the end of consolidation, after 6 months and 1 year of 
treatment, and at relapse. We also plan to measure CD47 levels on leukemic blasts prior to and 
after chemotherapy and pembrolizumab application to see whether CD47 expression levels 
correlate with responders vs. non-responders and whether expression levels change at different 
times of therapy. Statistical analyses of the frequency of CD8+, CD4+, Foxp3 Tregs, CD8+/Foxp3+ 
Tregs, TCM/TEMRA, TEM/TEMRA, the percentage of Ki67 and GzmB in PD-1+, Eomes+ CD8 T-cells 
to compare changes over time from baseline to several time-points will be performed by using 
mixed effects modeling with a Benjamini-Hochberg correction to control for false discovery 
rates. 
 
 
10.4.2.3  PD-1 and PD-L1 expression 
 
An association of clinical response with the expression of PD-L1 AML BM cells will be 
assessed by a Pearson Chi-square test on a 2×2 table of frequencies. The dependent variable will 
be defined as response (yes vs. no), and QIF categories (negative vs. positive) will be the 
independent variables. We will also monitor the dynamic change of PD-L1 expression over the 
course of treatment and its correlation with clinical response. Longitudinal measurements of PD- 
L1 will be examined using mixed-effects modeling. 
 
We will assess immune cell markers at different time points of therapy: at baseline (prior to 
application of 7+3 chemotherapy), at the end of consolidation, after 6 months and 1 year of 
treatment, and at relapse. We also plan to measure CD47 levels on leukemic blasts prior to and 
after chemotherapy and Pembrolizumab (MK-3475) administration to see whether CD47 
expression levels correlate with responders vs. non-responders and whether expression levels 
change at different times of therapy. Statistical analyses of the frequency of CD8+, CD4+, FoxP3 
Tregs, CD8+/FoxP3+ Tregs, TCM/TEMRA, TEM/TEMRA, the percentage of Ki67 and GzmB in PD- 
1+, Eomes+ CD8 T-cells to compare changes over time from baseline to several time-points will be 
performed by using mixed effects modeling with a Benjamini-Hochberg correction to control for 
false discovery rates. 
 
10.4.2.4  Olink Assay 
 
Inflammation is a key underlying factor for the pathophysiology of a wide range of diseases, 
including malignancies. The Olink inflammation biomarker panel provides a high-throughput, 
multiplex immunoassay enabling analysis of 92 inflammation-related protein biomarkers across 
96 samples simultaneously. The objective is to investigate protein signatures associated with 
response and efficacy. 
 
10.4.2.5  WES 
 
The determination of the mutation load, a total number of nonsynonymous point mutations, by 
whole-exome sequencing (WES) was shown to be useful in predicting the treatment responses to 
cancer immunotherapy (Lyu, 2018). Tumor mutational burden (TMB), along with PD-L1 

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expression, is a useful biomarker for immune checkpoint blockade selection across certain 
cancer types and TMB determination correlated with WES (Chan, 2019). We will use massively 
parallel sequencing technology to sequence the genomic DNA of tumor cells (leukemic bone 
marrow) and normal cells (germline) obtained from patients with AML at baseline. Mutational 
load by WES will be correlated with clinic-pathological parameters such as response to 
treatment, survival and immune infiltrating profile, and T cell repertoire diversity and clonality. 
 
10.4.2.6   RNA-seq 
 
The MD Anderson CIMAC uses Agilent RNA isolation products for RNAseq. RNA will be 
prepared using suitable purification system depending on sample source (fresh or FFPE). RNA 
integrity of FFPE RNA will be assessed using either the Agilent 4200 TapeStation and High 
Sensitivity RNA ScreenTape or the Agilent 2100 Bioanalyzer and RNA 6000 Pico Chip. Either 
method will employ the region analysis method to determine the percentage of RNA in the 
sample that is >200 nt for each sample to be processed. It is necessary to have RNA 
molecules >200 nt for efficient library construction and this value for each sample will 
determine appropriate conditions at various steps in the workflow. 
 
10.4.2.7  TCR-seq 
 
One mechanism by which checkpoint inhibitors are thought to elicit their effects by broadening 
the TCR repertoire. We will perform high-throughput sequencing of the TCR Vb CDR3 regions 
on ﬂow cytometrically sorted T-cell subsets to assess the effect of immunotherapy on the 
diversity of the T-cell repertoire and assess for correlation to clinical outcomes. TCR sequencing 
analysis may be performed using DNA from tumor tissues as well as PBMC. Briefly, 500 ng 
tumor DNA or 3-6 µg PBMC DNA will be subjected to high throughput TCR Vβ CDR3 
sequencing on an Illumina HiSeq sequencer with at least 5-fold coverage by ImmunoSEQ™ 
sequencing (Adaptive Biotechnologies, Seattle, WA). TCR diversity and clonality (defined as 1- 
(entropy)/log2(#) of productive unique sequences, where the entropy term takes into account the 
varying clone frequency) will be calculated using a software by Adaptive Technologies. T-cell 
repertoire diversity and clonality will be correlated with clinic-pathological parameters such as 
response to treatment, survival, and immune infiltrating profile, as well as genomic profiles (total 
mutation burden, non-synonymous mutation burden, predicted neoantigen burden, clonal 
mutation burden and clonal predicted neoantigen burden). TCR profile generated from 
treatment-refractory tumors at the time of disease progression will be compared to data from pre- 
treatment tumor samples to explore the TCR repertoire evolution of these tumors under 
therapeutic pressure. The dynamic changes of TCR from PMBC, when longitudinal blood 
samples are available, will be correlated to response to immune checkpoint blockade or 
chemotherapy and survival. 
 
10.4.2.8 Gut microbiome 
There is increasing evidence that dynamic changes within the microbiome can affect both immune and cancer cells. 
Imbalance in microbial communities promotes chronic inflammation and predisposes to cancer (Garett, 2015). Our 
collaborators have shown that microbiome is also involved in the metabolism of drugs and could explain person-to- 
person variability and responsiveness to therapy (Alexander et al, 2017, Zimmermann et al, 2019). Unlike in several 

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solid tumors wherein, immune-check point blockade is used alone or in combination or with cytotoxic agents, the 
drug synergism to treat AML is unique. Majority of these combinations results in mucositis which helps microbes 
breach gastro-intestinal (GI) epithelial barrier. It is plausible that with each cycle of treatment, exposure of immune 
cells to time -prevalent oral and GI microbiome is subject to temporal variance. Extrapolating preclinical data, 
taxonomically unique microbes could further determine priming of antigen presenting cell interaction with immune- 
effector cells that ultimately determine clinical phenotype. Based on our current understanding on the mechanism 
of action of PD-1 inhibitors, our overall hypothesis is that gastro-intestinal microbiome holds salutary prognostic 
and therapeutic promise in patients with AML. Our preliminary hypothesis is that gut microbiome modulates clinical 
responses and toxicity in patients with AML receiving Check Point Inhibitors (CPI). To this end our proposal will 
test the longitudinal relationships between the taxonomy and diversity of gut microbiome and metabolite on clinical 
outcomes and toxicity. 
 
 
Analysis of microbiome communities will be performed in R (http://www.R-project.org), using phyloseq27 to 
calculate α- and β- diversity metrics. The Shannon Diversity Index (SDI) will be used for α-diversity (variance 
within a particular sample; a measure of bacterial diversity) metric calculations, and weighted and unweighted 
UniFrac for β-diversity distances (characterization of differences in microbial community composition; a measure 
of temporal stability of the microbial community structure).28 Temporal variability will be determined using 
coefficients of variation (CV) [a measure of stability of species diversity over time] of the SDI and unweighted 
and weighted UniFrac distances per patient, with higher values indicative of more variable community 
composition. Pairwise differences in temporal variability across body sites will be made using Mann–Whitney U 
test, whereas pairwise differences among response groups performed using Student’s t-test. Linear correlations 
between CVs will be determined using Pearson’s r correlation. The population cohort will be divided into quartiles 
based on CV of the weighted UniFrac distance values or SDI where the first quartile is defined as stable, second 
and third as average, and fourth as variable. Measurements of intra-patient temporal variability and patient stability 
categories will be analyzed for their correlations with genera abundances. Groups of patients will be analyzed to 
determine if patients with subsequent clinical outcomes had significantly different levels of microbiome temporal 
variability. Potential clinical drivers of microbiome temporal instability will be determined using multivariable 
regression analyses. Untargeted metabolite analyses from stool samples with mass spectroscopy will be performed. 
 
The primary outcome of interest is to longitudinally characterize microbial shifts in patients with AML receiving 
PD-1 inhibitors. For this analysis standard of care will be compared with the experimental arm which includes 
chemotherapy + Pembrolizumab (MK-3475) combination. Secondary outcomes of interest will be a measure of 
both efficacy and toxicity which will include variables like rates of CR/CRi, progression free survival (PFS), 
overall survival (OS), cumulative incidence of graft versus host disease (GVHD)/non-relapse mortality (NRM). 
Association between microbial diversity on the percentage of patients with minimal residual disease (MRD) 
negative complete remission (CR) (MRD-CR) as measured by flow cytometry at the end of first consolidation 
cycle will also be explored. 
 
 
Exploratory outcomes: 
Correlation between microbial/metabolome changes with immune-checkpoint expression and kinetics of immune 
cell subset recovery and programming in the standard of care and experimental arm will be evaluated. As a marker 
of mucosal immunity, changes in immune cell content within stool samples in patients that experience colitis or 
GI-GVHD will be compared to suitable controls. 

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Part 1: Assess whether baseline biomarkers or the serial measurements of biomarkers are associated with 
response outcomes. 
 
A univariate logistic regression will be selected to assess baseline biomarkers associated with response outcomes. 
The dynamic changes of PD-L1/ PD-1 expressions, concentration of cytokine, RNA seq/TCRseq, gut microbiome, 
etc. will be monitored. The measurements of biomarkers in changes over time from baseline to several time-points 
will be performed by using generalized linear mixed effects modeling with a Benjamini-Hochberg correction 
(Benjamini et al., 1995) to control for false discovery rate. 
 
Part 2: Assess whether baseline biomarkers or the serial measurements of biomarkers are associated with 
survival outcomes. 
The Kaplan-Meier method and log-rank test will be used to estimate the distribution of survival between/among 
different marker strata. Univariate or Multivariate cox proportional hazard models will be employed to explore the 
significance of biomarkers on survival outcomes, while adjusting for the potential prognostic factors. The interaction 
effects between treatment and biomarkers also will be evaluated. Serial measurements of biomarkers will be estimated 
at baseline, end of induction, post-consolidation cycle1 and 4, every 3 months during maintenance, one year, and end 
of treatment when applicable. Landmark analysis (Benjamini et al., 1995, Dafni et al., 2011) or joint modeling (Huang 
et al., 2016) will be used to assess serial measurements of biomarkers dynamical impacts on survival outcomes, where 
appropriate. 
 
Part 3: Compare the biomarkers effects between treatment arms, intensive chemo + pembro vs. intensive chemo 
only. 
The associations between treatment arms and baseline biomarkers will be evaluated using Chi-squared test/ Fisher’s 
exact test, ANOVA, and the Mann-Whitney U tests as appropriate. Trajectory trends of the changes in markers’ 
values or status across the measurement time will be explored using generalized linear mixed models. The bar plots 
and trajectory time plots will visually show the differences over time between treatment arms. The associations 
between markers and the demographic/prognostic factors will also be assessed using the similar statistical methods. 
 
Part 4: Evaluate the correlations between biomarkers. 
The correlations between biomarkers will be evaluated using Pearson/Spearman rank-order correlation coefficients, 
Chi-squared/Fisher’s exact tests, and Wilcoxon rank sum / Kruskal-Wallis tests as appropriate. A scatter plot, 
boxplot, and mosaic plot will also be generated for visualization. The multiplicity of the endpoints will be adjusted 
using the correction of Benjamini and Hochberg (Benjamini et al., 1995). 
 
10.5 
For phase 2 protocols only: Reporting and Exclusions 
 
10.5.1 Evaluation of Toxicity 
 
All patients will be evaluable for toxicity from the time of their first treatment with 
Pembrolizumab (MK-3475). 
 
Severe pembrolizumab-related toxicity is defined as the occurrence of any of the below 
AEs that are attributed to pembrolizumab. The evaluability period includes the first 35 
days after pembrolizumab first dose, till hematologic recovery, or till starting 
consolidation therapy, whichever occurs first.

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10.5.1.1 Any grade 3 or 4 non-hematologic toxicity, that is deemed related to Pembrolizumab 
(MK-3475), with the following exceptions: A) Transient laboratory abnormalities that 
can be treated or resolve to grade 2 or less within 7 days. B) grade 3 expected and 
known drug-related autoimmune events (such as pneumonitis, enterocolitis, hepatitis, 
hypophysitis, and skin rash) that resolve within 4 weeks of steroid therapy to grade 1. 
 
10.5.1.2 Grade 4 thrombocytopenia or neutropenia associated with treatment-associated aplasia 
lasting >42 days from the last dose of study drug (pembrolizumab). Given that severe 
neutropenia and thrombocytopenia are features of AML and commonly encountered in 
this patient population, they will not be used to define unacceptable toxicity except if 
associated with prolonged treatment-associated aplasia AND not associated with active 
disease. 
 
10.5.1.1.3 Patients with grade 3 and 4 immune related AEs (with exception of events that 
resolve with steroids to grade 1 or less and do not require continued steroid therapy 
beyond 4 weeks). 
 
10.5.2 
Evaluation of Response 
 
All patients who were randomized must be assessed for response to treatment, even if there are 
major protocol treatment deviations or if they are ineligible. Each patient will be assigned one of 
the accepted ELN2017 response categories (see Appendix D). [Note: By arbitrary convention, 
category 9 usually designates the “unknown” status of any type of data in a clinical database.] 
 
All patients who were randomized (with the possible exception of those who received no study 
medication) should be included in the main analysis of the response rate. Patients who do not 
achieve CR/CRi after induction and first consolidation will be considered to have a treatment 
failure. Thus, an incorrect treatment schedule or drug administration does not result in exclusion 
from the analysis of the response rate. 
 
All conclusions should be based on all eligible patients. Sub-analyses may then be performed on the 
basis of a subset of patients, excluding those for whom major protocol deviations have been identified 
(e.g., early death due to other reasons, early discontinuation of treatment, major 
protocol violations, etc.). However, these sub-analyses may not serve as the basis for drawing 
conclusions concerning treatment efficacy, and the reasons for excluding patients from the 
analysis should be clearly reported. The 95% confidence intervals should also be provided. 
 
10.6 Data Safety Monitoring Board 
 
The conduct of this study will be overseen by the ETCTN DSMB. The DSMB will be 
responsible for recommendations to the Principal Investigator and NCI regarding possible trial 
closure and/or early reporting of the study. The study team (with the exception of the study 
statistician) will not have access to the summary outcome data until released by the DSMB. 

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11 
ADVERSE EVENTS: LIST AND REPORTINGREQUIREMENTS 
 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial. The 
following list of AEs (Section 10.1) and the characteristics of an observed AE (Sections 10.2 and 
10.3) will determine whether the event requires expedited reporting via the CTEP Adverse Event 
Reporting System (CTEP-AERS) in addition to routine reporting. 
 
11.1 Comprehensive Adverse Events and Potential Risks List (CAEPR) 
 
11.1.1 CAEPRs for CTEP IND Agent 
 
11.1.1.1 
Comprehensive Adverse Events and Potential Risks list (CAEPR) 
for 
MK-3475 (pembrolizumab, NSC 776864) 
 
The Comprehensive Adverse Events and Potential Risks list (CAEPR) provides a single list of reported and/or potential adverse 
events (AE) associated with an agent using a uniform presentation of events by body system. In addition to the comprehensive list,  
a subset, the Specific Protocol Exceptions to Expedited Reporting (SPEER), appears in a separate column and is identified with  
bold and italicized text. This subset of AEs (SPEER) is a list of events that are protocol specific exceptions to expedited reporting to 
NCI (except as noted below).  Refer to the 'CTEP, NCI Guidelines: Adverse Event Reporting Requirements' 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/aeguidelines.pdf for further clarification. Frequency is 
provided based on 3793 patients. Below is the CAEPR for Pembrolizumab (MK-3475). 
 
NOTE: Report AEs on the SPEER ONLY IF they exceed the grade noted in parentheses next to the AE in the SPEER.  If 
this CAEPR is part of a combination protocol using multiple investigational agents and has an AE listed on different  
SPEERs, use the lower of the grades to determine if expedited reporting is required. 
. 
Version 2.8, August 14, 20241 
 
 
 Adverse Events with Possible  
 Relationship to Pembrolizumab (MK-3475) 
 (CTCAE 5.0 Term) 
[n= 3793] 
 
 
 Specific Protocol 
Exceptions to Expedited 
Reporting (SPEER)  
 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
 
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS 
  
 
Anemia2 
 
  
 
 
Blood and lymphatic system 
disorders - Other (immune 
thrombocytopenic purpura)2 
  
 
 
Blood and lymphatic system 
disorders - Other (autoimmune 
hemolytic anemia)2 
  
 
Lymph node pain2 
 
  
CARDIAC DISORDERS 
  
 
 
Myocarditis2 
  
 
 
Pericarditis2 
  
ENDOCRINE DISORDERS 
  
 
Adrenal insufficiency2 
 
  
 
 
 Endocrine disorders - Other  
  

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 Adverse Events with Possible  
 Relationship to Pembrolizumab (MK-3475) 
 (CTCAE 5.0 Term) 
[n= 3793] 
 
 
 Specific Protocol 
Exceptions to Expedited 
Reporting (SPEER)  
 
 (hypoparathyroidism)2 
 
 Endocrine disorders - Other   
 (thyroiditis)2 
 
  
 
Hyperthyroidism2 
 
  
 
Hypophysitis2 
 
  
 
Hypopituitarism2 
 
  
 
Hypothyroidism2 
 
  
EYE DISORDERS 
  
 
 
Eye disorders - Other (Vogt-
Koyanagi-Harada syndrome) 
  
 
 
Uveitis2 
  
GASTROINTESTINAL DISORDERS 
  
 
Abdominal pain 
 
  
 
Colitis2 
 
  
 
Diarrhea2 
 
 Diarrhea2 (Gr 2) 
 
 
Enterocolitis2 
  
 
 
Gastritis2 
  
 
 
Gastrointestinal disorders - 
Other (exocrine pancreatic 
insufficiency) 
  
 
Mucositis oral2 
 
  
 
Nausea 
 
 Nausea (Gr 2) 
 
Pancreatitis2 
 
  
 
Small intestinal mucositis2 
 
  
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS 
  
 
Chills 
 
  
Fatigue 
 
 
 Fatigue (Gr 2) 
 
Fever2 
 
  
HEPATOBILIARY DISORDERS 
  
 
Hepatobiliary disorders - Other 
(autoimmune hepatitis)2 
 
  
 
 
Hepatobiliary disorders - Other 
(sclerosing cholangitis) 
  
IMMUNE SYSTEM DISORDERS 
  
 
 
Anaphylaxis2 
  
 
 
Cytokine release syndrome2 
  
 
 
Immune system disorders - 
Other (acute graft-versus-host-
disease)2,3 
  
 
 
Immune system disorders - 
Other (hemophagocytic 
lymphohistiocytosis)2 
  
 
Immune system disorders - 
Other (sarcoidosis)2 
 
  
 
 
Serum sickness2 
  
INFECTIONS AND INFESTATIONS 
  
 
 
Myelitis2 
  
INJURY, POISONING AND PROCEDURAL COMPLICATIONS 
  
 
Infusion related reaction 
 
  

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 Adverse Events with Possible  
 Relationship to Pembrolizumab (MK-3475) 
 (CTCAE 5.0 Term) 
[n= 3793] 
 
 
 Specific Protocol 
Exceptions to Expedited 
Reporting (SPEER)  
 
INVESTIGATIONS 
  
 
Alanine aminotransferase 
increased2 
 
  
 
Alkaline phosphatase increased  
  
 
Aspartate aminotransferase 
increased2 
 
  
 
Blood bilirubin increased 
 
  
 
 
GGT increased 
  
 
 
Lipase increased 
  
 
 
Serum amylase increased 
  
METABOLISM AND NUTRITION DISORDERS 
  
 
Anorexia 
 
  
 
Hyponatremia 
 
  
 
 
Metabolism and nutrition 
disorders - Other (diabetic 
ketoacidosis)2 
  
 
 
Metabolism and nutrition 
disorders - Other (type 1 
diabetes mellitus)2 
  
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS 
  
 
Arthralgia2 
 
 Arthralgia 2 (Gr 2) 
 
Arthritis2 
 
  
 
Back pain 
 
  
 
Joint range of motion decreased  
  
 
Myalgia2 
 
  
 
Myositis2 
 
  
NERVOUS SYSTEM DISORDERS 
  
 
 
Guillain-Barre syndrome2 
  
 
 
Myasthenia gravis 
  
 
 
Nervous system disorders - 
Other (autoimmune 
neuropathy)2 
  
 
 
Nervous system disorders - 
Other (demyelination)2 
  
 
 
Nervous system disorders - 
Other (myasthenic syndrome)2 
  
 
 
Nervous system disorders - 
Other (nerve paresis)2 
  
 
 
Nervous system disorders - 
Other (neuromyopathy)2 
  
 
 
Nervous system disorders - 
Other (non-infectious 
encephalitis)2 
  
 
 
Nervous system disorders - 
Other (non-infectious 
meningitis)2 
  
 
 
 Nervous system disorders -   
 Other (non-infectious myelitis) 
  
 
 
Nervous system disorders - 
Other (optic neuritis) 
  
 
 
Nervous system disorders - 
  

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 Adverse Events with Possible  
 Relationship to Pembrolizumab (MK-3475) 
 (CTCAE 5.0 Term) 
[n= 3793] 
 
 
 Specific Protocol 
Exceptions to Expedited 
Reporting (SPEER)  
 
Other (polyneuropathy)2 
 
 
Paresthesia 
  
 
 
Peripheral motor neuropathy2 
  
RENAL AND URINARY DISORDERS 
  
 
 
Acute kidney injury 
  
 
 
Renal and urinary disorders - 
Other (autoimmune nephritis)2 
  
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS 
  
 
 
Pneumonitis2 
  
SKIN AND SUBCUTANEOUS TISSUE DISORDERS 
  
 
Bullous dermatitis2 
 
  
 
 
Erythema multiforme2 
  
 
Erythroderma 
 
  
 
 
Palmar-plantar 
erythrodysesthesia syndrome 
  
 
Pruritus2 
 
 Pruritus2 (Gr 2) 
 
Rash acneiform2 
 
  
 
Rash maculo-papular2 
 
 Rash maculo-papular2 (Gr 2) 
 
 
Skin and subcutaneous tissue 
disorders - Other (Drug reaction 
with eosinophilia with systemic 
symptoms [DRESS])2 
  
 
Skin and subcutaneous tissue 
disorders - Other (dermatitis)2 
 
  
 
Skin hypopigmentation2 
 
  
 
 
Stevens-Johnson syndrome2 
  
 
 
Toxic epidermal necrolysis2 
  
 
Urticaria2 
 
  
VASCULAR DISORDERS 
  
 
 
Vasculitis2 
  
 
1This table will be updated as the toxicity profile of the agent is revised.  Updates will be distributed to all Principal Investigators at the 
time of revision.  The current version can be obtained by contacting PIO@CTEP.NCI.NIH.GOV. 
Your name, the name of the investigator, the protocol and the agent should be included in the e-mail. 
 
2Immune-mediated adverse reactions have been reported in patients receiving Pembrolizumab (MK-3475).  Adverse events 
potentially related to Pembrolizumab (MK-3475) may be manifestations of immune-mediated adverse events. In clinical trials, most 
immune-mediated adverse reactions were reversible and managed with interruptions of Pembrolizumab (MK-3475), administration of 
corticosteroids and supportive care. 
 
3Acute graft-versus-host disease has been observed in patients treated with Pembrolizumab (MK-3475) who received hematopoeitic 
stem cell transplants. 
 
 
Adverse events reported on Pembrolizumab (MK-3475) trials, but for which there is insufficient evidence to suggest that 
there was a reasonable possibility that Pembrolizumab (MK-3475) caused the adverse event: 
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS - Blood and lymphatic system disorders - Other (pancytopenia); Disseminated 
intravascular coagulation 

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CARDIAC DISORDERS - Atrial fibrillation; Cardiac arrest; Chest pain - cardiac; Heart failure; Myocardial infarction; Pericardial 
effusion; Pericardial tamponade; Ventricular arrhythmia 
EYE DISORDERS - Eye pain 
GASTROINTESTINAL DISORDERS - Abdominal distension; Ascites; Constipation; Duodenal hemorrhage; Dysphagia; 
Gastrointestinal disorders - Other (intussusception); Gastrointestinal disorders - Other (diverticulitis); Gastrointestinal disorders - 
Other (intestinal obstruction); Oral pain; Rectal hemorrhage; Small intestinal perforation; Upper gastrointestinal hemorrhage; 
Vomiting 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS - Edema face; Edema limbs; Facial pain; Gait disturbance; 
General disorders and administration site conditions - Other (general physical health deterioration); Generalized edema; Malaise; 
Non-cardiac chest pain; Pain 
INVESTIGATIONS - CPK increased; Cholesterol high; Creatinine increased; Fibrinogen decreased; Lymphocyte count decreased; 
Neutrophil count decreased; Platelet count decreased; Weight loss; White blood cell decreased 
METABOLISM AND NUTRITION DISORDERS - Dehydration; Hypercalcemia; Hyperglycemia; Hyperkalemia; Hypertriglyceridemia; 
Hyperuricemia; Hypoalbuminemia; Hypokalemia; Hypophosphatemia; Metabolism and nutrition disorders - Other (failure to thrive); 
Tumor lysis syndrome 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS - Bone pain; Generalized muscle weakness; Joint effusion2; 
Musculoskeletal and connective tissue disorder - Other (groin pain); Pain in extremity 
NERVOUS SYSTEM DISORDERS - Aphonia; Depressed level of consciousness; Dysarthria; Edema cerebral; Encephalopathy; 
Headache; Hydrocephalus; Lethargy; Meningismus; Nervous system disorders - Other (brainstem herniation); Seizure; Syncope; 
Tremor 
PSYCHIATRIC DISORDERS - Agitation; Confusion 
RENAL AND URINARY DISORDERS - Nephrotic syndrome; Proteinuria; Renal and urinary disorders - Other (hydronephrosis); 
Urinary incontinence; Urinary tract pain 
REPRODUCTIVE SYSTEM AND BREAST DISORDERS - Pelvic pain 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS - Cough; Dyspnea; Hypoxia; Laryngeal inflammation; Pleural 
effusion; Pleuritic pain2; Pneumothorax; Respiratory failure 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS - Alopecia; Dry skin; Skin and subcutaneous tissue disorders - Other (drug 
eruption) 
VASCULAR DISORDERS - Hypertension; Peripheral ischemia; Thromboembolic event 
 
 
Note: Pembrolizumab (MK-3475) in combination with other agents could cause an exacerbation of any adverse event currently 
known to be caused by the other agent, or the combination may result in events never previously associated with either agent. 
 
 
 
 
 
11.1.2 Common Adverse Events for Commercial Agents 
 
11.1.2.1 Cytarabine 
Common AEs (occurring in greater than 30%) for patients taking cytarabine: 
Headache 
Low blood counts 
Nausea and vomiting 
Mouth sores 
Increases in blood tests measuring liver function. 
 
Less common side effects (occurring in about 10-29%) for patients receiving 
cytarabine: 
 

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Diarrhea 
Loss of appetite 
Skin rash, redness, and 
itching Flu-like symptoms 
Pain, redness, and skin peeling of the palms of hands and soles of feet (hand-foot 
syndrome) 
Temporary hair loss 
Eye pain, tearing, sensitivity to light and blurred vision with high-dose therapy 
Please refer to the package insert for cytarabine for a comprehensive list of AEs. 
11.1.2.2 Daunorubicin 
Common AEs (occurring in greater than 30%) for patients taking daunorubicin: 
Pain along the site where the medication was given. 
Urine may appear red, red-brown, orange or pink from the color of the medication. 
Low blood counts 
Nausea or vomiting 
Mouth sores 
Hair loss on the scalp or elsewhere on the body 
Less common (occurring in 10-29%) for patients taking daunorubicin: 
Diarrhea. 
Problems with fertility 
Darkening of skin at the site of previous radiation therapy 
Darkening, discoloration of nail beds 
 
Please refer to the package insert for daunorubicin for a comprehensive list of AEs. 
 
11.1.2.3 Idarubicin 
 
Common AEs for idarubicin include: 
Common (occurring in greater than 30%) for patients taking Idarubicin: 
Pain along the site where the medication was given. 
Low blood counts 
Urine may appear red, red-brown, orange or pink from the color of the medication 
Nausea or vomiting. 
Mouth sores. 
Hair loss on the scalp or elsewhere on the body 
Diarrhea/abdominal cramps 
 

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Less common side effects (occurring in about 10-29%) of patients receiving 
Idarubicin: 
 
Fever 
Headache 
Nail thickening, nail banding 
Discoloration of the skin or nails. 
Darkening of the skin where previous radiation treatment has been given. (radiation 
recall). 
Hand -foot syndrome (Palmar-plantar erythrodysesthesia or PPE) 
Loss of fertility 
Heart rhythm abnormalities 
Increases in blood tests measuring liver function. 
 
Please refer to the package insert for idarubicin for a comprehensive list of AEs. 
 
11.1.3 Unknown or Potential Risks Associated with the Use of Pembrolizumab (MK-
3475) in Combination with AML Therapy 
Immune check point blockade studies have demonstrated the feasibility and safety of combining 
immunotherapy with intensive chemo induction feasible and safe in younger AML patients 
(Ravandi et al., 2019). In a single-arm phase II part of a phase I/II study, 44 patients aged 18–60 
years (>60 years if eligible for intensive chemotherapy) with newly diagnosed AML (n=42) or high- 
risk myelodysplastic syndrome (n=2) treatment included a 1.5 g/m2, 24-hour infusion of cytarabine 
daily on Days 1–4 (three days only for patients >60 years), alongside 12 mg/m2 daily on Days 1–3 
of idarubicin. Nivolumab was given on Day 24 at a dose of 3 mg/kg which was repeated every two 
weeks for a year in responders. AEs are detailed in the table below. 
 
AEs regardless of causality 
 
Grade 1-2, n (%) 
Grade 3, n (%) 
Grade 4, n (%) 
Nausea 
1 (2) 
1 (2) 
0 
Diarrhea 
3 (7) 
7 (16) 
0 
Mucositis or stomatitis 
1 (2) 
0 
0 
Muscle weakness 
0 
1 (2) 
0 
Syncope 
0 
1 (2) 
0 
Elevated transaminases 
3 (5) 
1 (2) 
0 
Elevated bilirubin 
0 
1 (2) 
0 
Febrile Neutropenia 
1 (2) 
13 (30) 
1 (2) 
Rash 
1 (2) 
2 (5) 
0 
Pneumonitis 
1 (2) 
0 
0 
Colitis 
1 (2) 
1 (2) 
1 (2) 
Pancreatitis 
1 (2) 
1 (2) 
0 
Cholecystitis 
0 
1 (2) 
0 
Thrombosis or embolism 
1 (2) 
0 
0 
 

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There were no nivolumab treatment-associated deaths and post-transplant severe GVHD was not 
significantly increased and manageable. 
 
11.2 
Adverse Event Characteristics 
 
• CTCAE term (AE description) and grade: The descriptions and grading scales found 
in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
5.0 will be utilized for AE reporting. All appropriate treatment areas should have access 
to a copy of the CTCAE version 5.0. A copy of the CTCAE version 5.0 can be 
downloaded from the CTEP website 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
 
For expedited reporting purposes only: 
- AEs for the agent that are bold and italicized in the CAEPR (i.e., those listed in the 
SPEER column, Section 10.1) should be reported through CTEP-AERS only if the 
grade is above the grade provided in the SPEER. 
 
- 
Attribution of the AE: 
- 
Definite – The AE is clearly related to the study treatment. 
- 
Probable – The AE is likely related to the study treatment. 
- 
Possible – The AE may be related to the study treatment. 
- 
Unlikely – The AE is doubtfully related to the study treatment. 
- 
Unrelated – The AE is clearly NOT related to the study treatment. 
 
11.3 
Expedited Adverse Event Reporting 
 
11.3.1 Rave-CTEP-AERS Integration 
 
The Cancer Therapy Evaluation Program Adverse Event Reporting System (CTEP- 
AERS) integration enables evaluation of post-baseline AEs entered in Rave to determine 
whether they require expedited reporting, and facilitates entry in CTEP-AERS for those 
AEs requiring expedited reporting. 
 
All AEs that occur after baseline are collected in Medidata Rave using the Adverse Event 
form, which is available for entry at each treatment or reporting period, and used to 
collect AEs that start during the period or persist from the previous reporting period. The 
Clinical Research Associate (CRA) will enter AEs that occur prior to the start of 
treatment on a baseline form that is not included in the Rave-CTEP-AERS integration. 
AEs that occur prior to enrollment must begin and end on the baseline Adverse Event 
form and should not be included on the standard Adverse Events form that is available at 
treatment unless there has been an increase in grade. 
 
Prior to sending AEs through the rules evaluation process, site staff should verify the 
following on the Adverse Event form in Rave: 
 

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• The reporting period (course/cycle) is correct, and 
• AEs are recorded and complete (no missing fields) and the form is query-free 
(fields added to the form during study build do not need to be query-free for the 
integration call with CTEP-AERS to be a success). 
 
The CRA reports AEs in Rave at the time the Investigator learns of the event. If the CRA 
modifies an AE, it must be re-submitted for rules evaluation. 
 
Upon completion of AE entry in Medidata Rave, the CRA submits the AE for rules 
evaluation by completing the Expedited Reporting Evaluation form. Both NCI and 
protocol-specific reporting rules evaluate the AEs submitted for expedited reporting. A 
report is initiated in CTEP-AERS using information entered in Medidata Rave for AEs 
that meet reporting requirements. The CRA completes the report by accessing CTEP- 
AERS via a direct link on the Medidata Rave Expedited Reporting Evaluation form. 
 
In the rare occurrence that Internet connectivity is lost, a 24-hour notification is to be 
made to CTEP by telephone at 301-897-7497. Once Internet connectivity is restored, the 
24-hour notification that was phoned in must be entered immediately into CTEP-AERS 
using the deep link from Medidata Rave. 
 
Additional information about the CTEP-AERS integration is available on the CTSU 
website: 
 
• Study specific documents: Protocols > Documents > Education and Promotion, 
and 
• Expedited Safety Reporting Rules Evaluation user guide: Resources > CTSU 
Operations Information > User Guides. 
 
NCI requirements for SAE reporting are available on the CTEP website: 
 
• NCI Guidelines for Investigators: Adverse Event Reporting Requirements is 
available at 
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/aeguid 
elines.pdf. 
 
11.3.2 Distribution of Adverse Event Reports 
 
CTEP-AERS is programmed for automatic electronic distribution of reports to the following 
individuals: Principal Investigator and Adverse Event Coordinator(s) (if applicable) of the 
Corresponding Organization or Lead Organization, the local treating physician, and the Reporter 
and Submitter. CTEP-AERS provides a copy feature for other e-mail recipients. 
 
11.3.3 Expedited Reporting Guidelines 
 
Use the NCI protocol number and the protocol-specific patient ID assigned during trial 

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registration on all reports. 
 
Note: A death on study requires both routine and expedited reporting, regardless of causality as 
long as the death occurred within 30 days after the last administration of the investigational 
agent. Attribution to treatment or other cause must be provided. 
 
Death due to progressive disease should be reported as Grade 5 “Disease progression” in the 
system organ class (SOC) “General disorders and administration site conditions.” Evidence that 
the death was a manifestation of underlying disease (e.g., radiological changes suggesting tumor 
growth or progression: clinical deterioration associated with a disease process) should be 
submitted. 
 
Late Phase 2 and Phase 3 Studies: Expedited Reporting Requirements for Adverse 
Events that Occur on Studies under an IND/IDE within 30 Days of the Last 
Administration of the Investigational Agent/Intervention1, 2 
 
 
 
FDA REPORTING REQUIREMENTS FOR SERIOUS ADVERSE EVENTS (21 CFR Part 312) 
NOTE:  Investigators MUST immediately report to the sponsor (NCI) ANY SAEs, whether or not they are 
considered related to the investigational agent(s)/intervention (21 CFR 312.64). 
An AE is considered serious if it results in ANY of the following outcomes: 
1) 
Death 
2) 
A life-threatening AE 
3) 
An AE that results in inpatient hospitalization or prolongation of existing hospitalization for ≥ 24 
hours 
4) 
A persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions 
5) 
A congenital anomaly/birth defect. 
6) 
Important Medical Events (IME) that may not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon medical judgment, they may 
jeopardize the patient or subject and may require medical or surgical intervention to prevent one of 
the outcomes listed in this definition. (FDA, 21 CFR 312.32; ICH E2A and ICH E6). 
 
ALL SAEs that meet the above criteria MUST be immediately reported to the NCI via CTEP-AERS 
within the timeframes detailed in the table below. 
Grade 1-3 Timeframes 
Grade 4-5 Timeframes 
24-Hour notification, 10 Calendar Days 
24-Hour notification, 5 Calendar Days 
NOTE:   Protocol-specific exceptions to expedited reporting of SAEs are found in the Specific 
Protocol Exceptions to Expedited Reporting (SPEER) portion of the CAEPR. 
Expedited AE reporting timeframes are defined as: 
o 
“24-Hour notification, 5 Calendar Days” - The SAE must initially be reported via CTEP-
AERS within 24 hours of learning of the SAE, followed by a complete expedited report 
within 5 calendar days of the initial 24-hour report. 
o 
“24-Hour notification, 10 Calendar Days” - The SAE must initially be reported via CTEP-
AERS within 24 hours of learning of the SAE, followed by a complete expedited report 
within 10 calendar days of the initial 24-hour report. 

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1SAEs that occur more than 30 days after the last administration of investigational agent/intervention 
and have an attribution of possible, probable, or definite require reporting as follows: 
Expedited 24-Hour notifications are required for all SAEs followed by a complete report 
• 
Within 5 calendar days for Grade 4-5 SAEs 
• 
Within 10 calendar days for Grade 1-3 SAEs 
2For studies using nuclear medicine or molecular imaging IND agents (NM, SPECT, or PET), the 
SAE reporting period is limited to 10 radioactive half-lives, rounded UP to the nearest whole day, 
after the agent/intervention was last administered.  Footnote “1” above applies after this reporting 
period. 
 
Effective Date: August 30, 2024 
 
 
 
 
 
11.4 
Routine Adverse Event Reporting 
 
All Adverse Events must be reported in routine study data submissions. AEs reported 
expeditiously through CTEP-AERS must also be reported in routine study data 
submissions. 
 
Adverse event data collection and reporting, which are required as part of every clinical trial, are 
done to ensure the safety of patients enrolled in the studies as well as those who will enroll in 

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future studies using similar agents. AEs are reported in a routine manner at scheduled times 
during the trial using Medidata Rave. For this trial the Adverse Event CRF is used for routine 
AE reporting in Rave. 
 
11.5 
Pregnancy 
 
Although not an adverse event in and of itself, pregnancy as well as its outcome must be 
documented via CTEP-AERS. In addition, the Pregnancy Information Form included within 
the NCI Guidelines for Adverse Event Reporting Requirements must be completed and 
submitted to CTEP. Any pregnancy occurring in a patient or patient’s partner from the time of 
consent to 90 days after the last dose of study drug must be reported and then followed for 
outcome. Newborn infants should be followed until 30 days old. Please see the “NCI 
Guidelines for Investigators: Adverse Event Reporting Requirements for DCTD (CTEP and CIP) 
and DCP INDs and IDEs” (at http://ctep.cancer.gov/protocolDevelopment/adverse_effects.htm) 
for more details on how to report pregnancy and its outcome to CTEP. 
 
11.6 
Secondary Malignancy 
 
A secondary malignancy is a cancer caused by treatment for a previous malignancy (e.g., 
treatment with investigational agent/intervention, radiation or chemotherapy). A secondary 
malignancy is not considered a metastasis of the initial neoplasm. 
 
CTEP requires all secondary malignancies that occur following treatment with an agent under an 
NCI IND/IDE be reported expeditiously via CTEP-AERS. Three options are available to 
describe the event: 
 
• 
Leukemia secondary to oncology chemotherapy (e.g., AML) 
• 
Myelodysplastic syndrome (MDS) 
• 
Treatment-related secondary malignancy 
 
Any malignancy possibly related to cancer treatment (including AML/MDS) should also be 
reported via the routine reporting mechanisms outlined in each protocol. 
 
11.7 
Second Malignancy 
 
A second malignancy is one unrelated to the treatment of a prior malignancy (and is NOT a 
metastasis from the initial malignancy). Second malignancies require ONLY routine AE 
reporting unless otherwise specified. 

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12. STUDY CALENDAR 
 
Screening evaluations are to be conducted within 21 days prior to start of protocol therapy. Blood tests will be taken at every clinic 
visit for 1 year (if applicable, drawn when routine blood samples are taken). In the event that the patient’s condition is deteriorating, 
laboratory evaluations should be repeated within 48 hours prior to initiation of the next cycle of therapy. 
 
Induction Phase 
Consolidation Phasea 
Mainten 
ance / 
Observat 
ion 
Phase  
£ 
After 6 
Months of 
Treatment 
After 1 
Year of 
Treatm 
ent 
End of 
Mainte 
nance / 
Observ 
ationb 
Post- 
treatme 
nt 
Follow 
-Upc 
Cycle 1 
Cycles 2 a, 3, 4 
Screening 
y 
D1 
D 
2 
D 
3 
D 
4 
D 
5 
D 
6 
D7 
D 
8 
D9 
D 
14 
D 
28 
D 
1 
D 
3 
D 
5 
D 
28 
D 
1 
D 
3 
D 
5 
D 
28 
Cytarabined 
X 
X 
X 
X 
X 
X 
X 
Xe 
X 
X 
X 
X 
X 
X 
Daunorubicin or 
Idarubicind 
X 
X 
X 
Xe 
Pembrolizumab 
(MK-3475) 
f 
Every 3 weeks starting on Day 8 of the Induction phase 
Informed 
Consent 
X 
Eligibility 
Criteria 
X 
Medical History, 
Demographics 
X 
Prior and 
Concomitant 
Medication 
Review 
X 
X 
Survival Status 
X 
EKGw 
X 
Physical Exam 
X 
X 
X 
Xn 
X 
X 
X 
X 
X 
Vital signs 
X 
X 
X 
Xn 
X 
X 
X 
X 
X 
Height 
X 
Weight 
X 
Performance 
Statusg 
X 
Xn 
X 
X 
X 
X 
Adverse event 
evaluation 
X 
X 
Comprehensive 
Chemistry Panelh,v 
X 
X 
X 
X 
X 
X 
X 
X 
Xu 
Xu 
X 
Xn 
X 
X X 
X 
Xv 
X 

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Induction Phase 
Consolidation Phasea 
Mainten 
ance / 
Observat 
ion 
Phase 
£ 
After 6 
Months of 
Treatment
(+/- 14 
days) 
After 1 
Year of 
Treatm 
ent 
(+/- 14 
days) 
End of 
Mainte 
nance / 
Observ 
ationb 
Post- 
treatme 
nt 
Follow 
-Upc 
Cycle 1 
Cycles 2 a, 3, 4 
Screening 
y 
D1 
D 
2 
D 
3 
D 
4 
D 
5 
D 
6 
D7 
D 
8 
D9 
D 
14 
D 
28 
D 
1 
D 
3 
D 
5 
D 
28 
β 
D 
1 
D 
3 
D 
5 
D 
28  
β 
CBC with 
Differentiali,v 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Xn 
X 
X X 
X 
Xv 
X 
X 
Pregnancy Testj 
– Urine or Serum 
β-HCG 
X 
Urinalysis 
X 
X 
X 
Xn 
X 
X 
X 
X 
TSH; T3, T4k 
X 
X 
X 
X 
PT, PTT, 
fibrinogen 
 
X 
    X 
 
 
Bone marrowl 
Aspiration 
X 
Xm 
Xn 
X 
Xo 
Xp 
X 
X 
Xs 
Bone marrow 
biopsy 
X 
Xn 
X 
Xo 
X 
X 
Xs 
Stool 
X 
Xz 
Research Blood 
Draws 
X 
Xm 
Xn 
X 
Xo 
Xq 
Xr 
Xr 
Xs 
Skin Punch 
Biopsy  α 
X 
Transthoracic 
ECHO (TTE) or 
MUGA 
X 
Xt 
CT chest 
Xx 
a If the patient does not have a CR/CRi before starting this part of the study, the patient should go off study. Patients <65 may complete 3-4 cycles of consolidation therapy. Patients ≥65 years old 
may complete 1-2 cycles of consolidation therapy. 
b End of treatment visit will be immediately following final dose of Pembrolizumab (MK-3475). 
c After completing study treatment, clinical follow-up assessments are required every 6 months from date of therapy discontinuation. Common clinical practice should be monitored by 
institutional practice (i.e., every 3 months though not required by protocol), but assessment results are only required every 6 months (+/- 14 days). All participants should be followed for 
survival to 3 years after end of treatment. For patients who achieve undetectable MRD during study treatment, central MRD assessments should continue in addition to general clinical 
follow. 
d Cytarabine should be dosed as appropriate for each phase of the study. Daunorubicin (60 mg/m2/day) or Idarubicin (12 mg/m2/day) 
e If patient has residual leukemia at the time of bone marrow aspiration, the patient may receive a 5+2 administration of these drugs. Adjust anthracycline dose as per institutional standards for 
Re- Induction. 
f Only for patients randomized to Arm B at 200 mg. Pembrolizumab (MK-3475) will be administered on Day 8 of induction and then Q3 weeks (+/- 3 days). Do not administer pembrolizumab 
concurrently with HIDAC chemotherapy Days 1-5 of each consolidation cycle. For consideration of timing of pembrolizumab, allow at least 3 hours after completion of chemotherapy prior to 
pembrolizumab administration. Administration window for pembrolizumab may be extended to allow for this timing as necessary. Each cycle of Maintenance is 21 days with pembrolizumab given 
on day 1 of each cycle up to 2 years. 
g Note: Performance status evaluations are based on a 4-week cycle. At minimum, performance status should be evaluated at the beginning of every cycle. 
h Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, LDH, phosphorus, potassium, total protein, SGOT [AST], SGPT [ALT], sodium. 

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128 
 
 
i CBCs (with differential and platelet count), which includes WBC, ANC, platelets, Hgb, and Hct required for protocol therapy must be done <24 hours prior to the treatment cycle. 
j For women of childbearing potential, a serum or urine pregnancy test must be conducted within 72 hours prior to first dose of study treatment on both Arm A and Arm B. If the urine test is 
positive or cannot be confirmed as negative, a serum pregnancy test will be required. 
k. TSH should be performed at baseline and <72 hours prior to each dose of Pembrolizumab (MK-3475). Additional T3 and T4 tests should be performed as clinically indicated, if TSH is abnormal, if symptoms are suggestive 
of thyroid dysfunction. 
l. Bone marrow aspirate is required to be submitted for central MRD testing as outline in Section 5. Submit to Dr. Brent Wood’s lab at Children’s Hospital Los Angeles. Results will be forwarded 
to the submitting institution within 1-2 business days of receipt of the bone marrow aspirate samples. Local bone marrow biopsy (aspirate) must be performed at the indicated time points and 
should follow the institutional standard of care. 
m To be collected between Day 14 and Day 21. 
n To be collected between Day 28 and Day 42. 
o To be collected at the end of consolidation therapy only. 
p Bone marrow aspirations every 3 months during maintenance/observation phase are optional and should only be collected when part of standard of care. 
q To be collected every 3 months during maintenance phase. 
r 
Blood collection for the immune cell subset analysis and Olink assay only. 
s Bone marrow aspiration and blood should be collected for MRD status in patients who have not relapse. For patients who have relapsed, bone marrow core biopsies, bone marrow aspirate, and 
blood should be collected. Blood collection for cfDNA will be obtained at the same time-points as blood collection for MRD status. 
t. Repeat TTE should be performed before 5+2 reinduction. 
u PT, PTT, and fibrinogen will be additionally collected on days 8 and 9 (pre- and post-) in the pembrolizumab arm. 
v CBC and CMP will be obtained within 3 days of receipt of each pembrolizumab dose in the pembrolizumab arm and every 3 weeks during treatment and 
every 6 (+/- 1 week) weeks during observation for patients in the control arm.  
w ECGs performed after screening will be done as medically indicated. 
x CT chest should be considered as a part of screening (optional) 
y Screening period is up to 21 days 
z At time of Post-Consolidation Cycle 1 bone marrow collection (+/- 3 days) 
α Refer to Section 5.1 Summary Table for Specimen Collection and Appendix F Skin Punch Biopsy Verification Form  
β (+/- 3) days  
£ Observation visits in Maintenance on control arm may be extended to 6 weeks (+/- 1 week).   
 
 

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 13            MEASUREMENT OF EFFECT 
 
Responses in AML patients will be assessed using ELN 2017 response criteria (Dohner et al., 
2017) given shortcomings of the International Working Group 2003 criteria in the era of MRD 
assessment (Cheson et al., 2003; Bloomsfield et al., 2018). 
 
13.1  Definitions 
 
Evaluable for toxicity. All patients will be evaluable for toxicity from the time of their first 
treatment with 7+3 with or without Pembrolizumab (MK-3475) 
 
Evaluable for objective response. Only those patients who have measurable disease present at 
baseline, have received at least one cycle of therapy, and have had their disease reevaluated will 
be considered evaluable for response. These patients will have their response classified 
according to the definitions stated below. (Note: Patients who exhibit objective disease 
progression prior to the end of Cycle 1 will also be considered evaluable). All patients starting 
induction therapy should be included in the primary analysis with drop-outs considered as not 
being MRD- for the analysis of rates. 
 
13.2  Hematologic Response 
 
13.2.1 Complete Response (CR) without minimal residual disease (CRMRD−): If studied 
pretreatment, CR with negativity for a genetic marker by RT-qPCR, or CR with 
negativity by MFC 
 
13.2.2 Complete remission (CR): Bone marrow blasts <5%; absence of circulating blasts and 
blasts with Auer rods; absence of extramedullary disease; ANC ≥1.0 × 109/L (1000/µL); 
platelet count ≥100 × 109/L (100 000/µL); MRD+ or unknown 
 
13.2.3 CR with incomplete count recovery (CRi): All CR criteria except for residual neutropenia 
(<1.0 × 109/L [1000/µL]) or thrombocytopenia (<100 × 109/L [100,000/µL] 
 
13.2.4 CR with partial recovery count (CRh): All CR criteria with neutrophil 
count ≥0.5 × 109/L and platelet count ≥50 × 109/L. CRh is being recorded as a separate response 
criteria from other responses (for example patient could have CRi by traditional criteria but also 
CRh). 
 
 
13.2.5 Hematologic improvement: Erythroid response (E) (pretreatment Hgb <11 g/dL) Hgb 
increase by ≥1.5 g/dL, Platelet response (P) (pretreatment platelets <100 × 109/L) 
Absolute increase of ≥30 × 109/L for patients starting with >20 × 109/L platelets Increase 
from <20 × 109/L to >20 × 109/L and by at least 100%. 

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13.2.6 Morphologic leukemia-free state (MLFS): Bone marrow blasts <5%; absence of 
blasts with Auer rods; absence of extramedullary disease; no hematologic recovery 
required. (Marrow should not merely be “aplastic”; at least 200 cells should be 
enumerated or cellularity should be at least 10%) 
 
13.2.7 Partial remission (PR) All hematologic criteria of CR; decrease of bone marrow blast 
percentage to 5% to 25%; and decrease of pretreatment bone marrow blast percentage by 
at least 50% 
 
 
Peripheral Blood Counts: 
● 
Neutrophil count ≥1.0 × 109/L. 
● 
Platelet count ≥100 × 109/L. 
● 
Reduced hemoglobin concentration or hematocrit has no bearing on remission status. 
● 
Leukemic blasts must not be present in the peripheral blood. 
 
Bone Marrow Aspirate and Biopsy: 
● Cellularity of bone marrow biopsy must be >20% with maturation of all cell lines. 
● <5% blasts 
● Auer rods must not be detectable. 
 
Extramedullary leukemia, such as CNS or soft tissue involvement, must not be present. 
 
13.2.8 Stable disease: Absence of CRMRD−, CR, CRi, PR, MLFS; and criteria for PD not met. 
 
Note: stable disease would not count as an adequate response in terms of response assessment for 
study purposes. However, some patients with stable disease can also achieve hematologic 
improvements such as transfusion independence without reduction in blasts, and some patients on 
immunotherapy arm might need longer time than 6 months to respond or could have more stable 
disease that translates into longer survival in this randomized study. Furthermore, there are very few 
options available for most patients after azacitidine-venetoclax and therefore it is imperative to 
ensure patients can have the opportunity to achieve maximal benefit from this therapy. We therefore 
will allow the treating investigator to keep patients on treatment with stable disease at their discretion 
if they judge the patient to be deriving clinical benefit on study. 
 
13.2.9  Progressive disease (PD): Evidence for an increase in bone marrow blast 
percentage and/or increase of absolute blast counts in the blood. 
● 
>50% increase in marrow blasts over baseline (a minimum 15%  point increase is 
required in cases with <30% blasts at baseline; or persistent marrow blast percentage of 
>70% over at least 3 mo; without at least a 100% improvement in ANC to an absolute level 
(>0.5 × 109/L [500/µL], and/or platelet count to >50 × 109/L [50 000/µL] non-transfused) 
● 
>50% increase in peripheral blasts (WBC × % blasts) to >25 × 109/L 
(>25 000/μL) (in the absence of differentiation syndrome) 
● 
New extramedullary disease Some protocols may allow transient addition of 
hydroxyurea to lower blast counts. 

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“Progressive disease” is usually accompanied by a decline in ANC and platelets and 
increased transfusion requirement and decline in performance status or increase in 
symptoms. 
 
13.2.9.1 MRD Response 
 
Refer to section 
9.1.1 
13.3     Other Response Parameters 
 
• EFS or RFS will be calculated as the time from initial treatment to either disease relapse or 
death. Duration of response of remission duration will be calculated as the time from first 
CR to the date of the first documented relapse or death, whichever occurs first. OS will be 
calculated from time from initial treatment to death. 
 
 
14 STUDY OVERSIGHT AND DATA REPORTING / REGULATORY 
REQUIREMENTS 
 
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 10 
(Adverse Events: List and Reporting Requirements). 
 
14.1 Study Oversight 
 
This protocol is monitored at several levels, as described in this section. The Protocol Principal 
Investigator is responsible for monitoring the conduct and progress of the clinical trial, including 
the ongoing review of accrual, patient-specific clinical and laboratory data, and routine and 
serious adverse events; reporting of expedited adverse events; and accumulation of reported 
adverse events from other trials testing the same drug(s). The Protocol Principal Investigator and 
statistician have access to the data at all times through the CTMS web-based reporting portal. 
 
All Study Investigators at participating sites who register/enroll patients on a given protocol are 
responsible for timely submission of data via Medidata Rave and timely reporting of adverse 
events for that particular study. This includes timely review of data collected on the electronic 
CRFs submitted via Medidata Rave. 
 
All studies are also reviewed in accordance with the enrolling institution’s data safety monitoring 
plan. 
 
14.2 Data Reporting 
 
Medidata Rave is a clinical data management system being used for data collection for this 
trial/study. Access to the trial in Rave is controlled through the CTEP-IAM system and role 
assignments. To access Rave via iMedidata: 

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• 
Site staff will need to be registered with CTEP and have a valid and active CTEP-IAM 
account, and 
• 
Assigned one of the following Rave roles on the relevant Lead Protocol Organization 
(LPO) or Participating Organization roster at the enrolling site: Rave CRA, Rave Read 
Only, Rave CRA (LabAdmin), Rave SLA, or Rave Investigator. Refer to 
https://ctep.cancer.gov/investigatorResources/default.htm for registration types and 
documentation required. 
o To hold Rave CRA or Rave CRA (Lab Admin) role, site staff must hold a 
minimum of an AP registration type, 
o To hold Rave Investigator role, the individual must be registered as an NPIVR or 
IVR, and 
o To hold Rave Read Only role, site staff must hold an Associates (A) registration 
type. 
 
If the study has a DTL, individuals requiring write access to Rave must also be assigned the 
appropriate Rave tasks on the DTL. 
 
Upon initial site registration approval for the study in Regulatory Support System (RSS), all 
persons with Rave roles assigned on the appropriate roster will be sent a study invitation e-mail 
from iMedidata. To accept the invitation, site staff must log in to the Select Login 
(https://login.imedidata.com/selectlogin) using their CTEP-IAM username and password, and 
click on the accept link in the upper right-corner of the iMedidata page. Site staff will not be 
able to access the study in Rave until all required Medidata and study specific trainings are 
completed. Trainings will be in the form of electronic learnings (eLearnings),and can be 
accessed by clicking on the link in the upper right pane of the iMedidata screen. If an eLearning 
is required and has not yet been taken, the link to the eLearning will appear under the study name 
in iMedidata instead of the Rave EDC link; once the successful completion of the eLearning has 
been recorded, access to the study in Rave will be granted, and a Rave EDC link will display 
under the study name. 
 
Site staff that have not previously activated their iMedidata/Rave account at the time of initial 
site registration approval for the study in RSS will also receive a separate invitation from 
iMedidata to activate their account. Account activation instructions are located on the CTSU 
website in the Rave section under the Rave resource materials (Medidata Account Activation and 
Study Invitation Acceptance). Additional information on iMedidata/Rave is available on the 
CTSU members’ website in the Data Management > Rave section at www.ctsu.org/RAVE/ or by 
contacting the CTSU Help Desk at 1-888-823-5923 or by e-mail at ctsucontact@westat.com. 
 
14.2.1 Method 
 
This study will be monitored by the Clinical Trials Monitoring Service (CTMS). Data will be 
submitted to CTMS at least once every two weeks via Medidata Rave (or other modality if 
approved by CTEP). Information on CTMS reporting is available at: 
http://www.theradex.com/clinicalTechnologies/?National-Cancer-Institute-NCI-11. 
On-site 
audits will be conducted on an 18-36 month basis as part of routine cancer center site visits. 

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133 
 
 
More frequent audits may be conducted if warranted by accrual or due to concerns regarding 
data quality or timely submission. For CTMS monitored studies, after users have activated their 
accounts, please contact the Theradex Help Desk at (609) 619-7862 or by email at 
CTMSSupport@theradex.com for additional support with Rave and completion of CRFs. 
 
14.2.2 Responsibility for Data Submission 
 
For ETCTN trials, it is the responsibility of the PI(s) at the site to ensure that all investigators at 
the ETCTN Sites understand the procedures for data submission for each ETCTN protocol and 
that protocol specified data are submitted accurately and in a timely manner to the CTMS via the 
electronic data capture system, Medidata Rave. 
 
Data are to be submitted via Medidata Rave to CTMS on a real-time basis, but no less than once 
every 2 weeks. The timeliness of data submissions and timeliness in resolving data queries will be 
tracked by CTMS. Metrics for timeliness will be followed and assessed on a quarterly basis. For the 
purpose of Institutional Performance Monitoring, data will be considered delinquent if it is greater 
than 4 weeks past due. 
 
Data from Medidata Rave and CTEP-AERS is reviewed by the CTMS on an ongoing basis as 
data is received. Queries will be issued by CTMS directly within Rave. The queries will appear 
on the Task Summary Tab within Rave for the CRA at the ETCTN to resolve. Monthly web- 
based reports are posted for review by the Drug Monitors in the IDB, CTEP. Onsite audits will 
be conducted by the CTMS to ensure compliance with regulatory requirements, GCP, and NCI 
policies and procedures with the overarching goal of ensuring the integrity of data generated 
from NCI-sponsored clinical trials, as described in the ETCTN Program Guidelines, which may 
be found on the CTEP 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/adverse_events.htm) and 
CTSU websites. 
 
CTMS will utilize a core set of eCRFs that are Cancer Data Standards Registry and Repository 
(caDSR) compliant (http://cbiit.nci.nih.gov/ncip/biomedical-informatics- 
resources/interoperability-and-semantics/metadata-and-models). Customized eCRFs will be 
included when appropriate to meet unique study requirements. The PI is encouraged to review 
the eCRFs, working closely with CTMS to ensure prospectively that all required items are 
appropriately captured in the eCRFs prior to study activation. CTMS will prepare the eCRFs 
with built-in edit checks to the extent possible to promote data integrity. 
 
CDUS data submissions for ETCTN trials activated after March 1, 2014, will be carried out by 
the CTMS contractor, Theradex. CDUS submissions are performed by Theradex on a monthly 
basis. The trial’s lead institution is responsible for timely submission to CTMS via Rave, as 
above. 
 
Further information on data submission procedures can be found in the ETCTN Program 
Guidelines 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/adverse_events.htm). 

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134 
 
 
14.3 Data Quality Portal 
 
The Data Quality Portal (DQP) provides a central location for site staff to manage 
unanswered queries and form delinquencies, monitor data quality and timeliness, 
generate reports, and review metrics. 
 
The DQP is located on the CTSU members’ website under Data Management. The Rave 
Home section displays a table providing summary counts of Total Delinquencies and 
Total Queries. DQP Queries, DQP Delinquent Forms, and the DQP Reports modules are 
available to access details and reports of unanswered queries, delinquent forms, and 
timeliness reports. Review the DQP modules on a regular basis to manage specified 
queries and delinquent forms. 
 
The DQP is accessible by site staff that are rostered to a site and have access to the CTSU 
website. Staff that have Rave study access can access the Rave study data using a direct 
link on the DQP. 
 
To learn more about DQP use and access, click on the Help icon displayed on the Rave 
Home, DQP Queries, and DQP Delinquent Forms modules. 
 
Note: Some Rave protocols may not have delinquent form details or reports specified on 
the DQP. A protocol must have the Calendar functionality implemented in Rave by the 
Lead Protocol Organization (LPO) for delinquent form details and reports to be available 
on the DQP. Site staff should contact the LPO Data Manager for their protocol regarding 
questions about Rave Calendaring functionality. 
 
14.4 Collaborative Agreements Language 
 
The agent(s) supplied by CTEP, DCTD, NCI used in this protocol is/are provided to the NCI 
under a Collaborative Agreement (CRADA, CTA, CSA) between the Pharmaceutical 
Company(ies) (hereinafter referred to as “Collaborator(s)”) and the NCI Division of Cancer 
Treatment and Diagnosis. Therefore, the following obligations/guidelines, in addition to the 
provisions in the “Intellectual Property Option to Collaborator” 
(http://ctep.cancer.gov/industryCollaborations2/intellectual_property.htm) contained within the 
terms of award, apply to the use of the Agent(s) in this study: 
 
14.4.1 Agent(s) may not be used for any purpose outside the scope of this protocol, 
nor can Agent(s) be transferred or licensed to any party not participating in the 
clinical study. Collaborator(s) data for Agent(s) are confidential and proprietary 
to Collaborator(s) and shall be maintained as such by the investigators. The 
protocol documents for studies utilizing Agents contain confidential 
information and should not be shared or distributed without the permission of 
the NCI. If a copy of this protocol is requested by a patient or patient’s family 
member participating on the study, the individual should sign a confidentiality 
agreement. A suitable model agreement can be downloaded from: 
http://ctep.cancer.gov. 

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135 
 
 
 
14.4.2 For a clinical protocol where there is an investigational Agent used in combination with 
(an)other Agent(s), each the subject of different Collaborative Agreements, the access to and 
use of data by each Collaborator shall be as follows (data pertaining to such combination use 
shall hereinafter be referred to as "Multi-Party Data”): 
 
14.4.2.1 
NCI will provide all Collaborators with prior written notice 
regarding the existence and nature of any agreements governing their 
collaboration with NCI, the design of the proposed combination 
protocol, and the existence of any obligations that would tend to restrict 
NCI's participation in the proposed combination protocol. 
 
14.4.2.2 
Each Collaborator shall agree to permit use of the Multi-Party Data 
from the clinical trial by any other Collaborator solely to the extent 
necessary to allow said other Collaborator to develop, obtain regulatory 
approval or commercialize its own Agent. 
 
14.4.2.3 
Any Collaborator having the right to use the Multi-Party Data 
from these trials must agree in writing prior to the commencement of 
the trials that it will use the Multi-Party Data solely for development, 
regulatory aproval, and commercialization of its own Agent. 
 
14.4.3 Clinical Trial Data and Results and Raw Data developed under a Collaborative 
Agreement will be made available to Collaborator(s), the NCI, and the FDA, as 
appropriate and unless additional disclosure is required by law or court order as 
described in the IP Option to Collaborator 
(http://ctep.cancer.gov/industryCollaborations2/intellectual_property.htm). 
Additionally, all Clinical Data and Results and Raw Data will be collected, used 
and disclosed consistent with all applicable federal statutes and regulations for the 
protection of human subjects, including, if applicable, the Standards for Privacy of 
Individually Identifiable Health Information set forth in 45 C.F.R. Part 164. 
 
14.4.4 When a Collaborator wishes to initiate a data request, the request should first be sent 
to the NCI, who will then notify the appropriate investigators (Group Chair for 
Cooperative Group studies, or PI for other studies) of Collaborator's wish to contact 
them. 
 
14.4.5 Any data provided to Collaborator(s) for Phase 3 studies must be in accordance 
with the guidelines and policies of the responsible Data Monitoring Committee 
(DMC), if there is a DMC for this clinical trial. 
 
14.4.6 Any manuscripts reporting the results of this clinical trial must be provided to CTEP 
by the Group office for Cooperative Group studies or by the principal investigator for 
non- Cooperative Group studies for immediate delivery to Collaborator(s) for advisory 
review and comment prior to submission for publication. Collaborator(s) will have 
30 days from the date of receipt for review. Collaborator shall have the right to 

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136 
 
 
request that publication be delayed for up to an additional 30 days in order to ensure 
that Collaborator’s confidential and proprietary data, in addition to Collaborator(s)’s 
intellectual property rights, are protected. Copies of abstracts must be provided to 
CTEP for forwarding to Collaborator(s) for courtesy review as soon as possible and 
preferably at least three (3) days prior to submission, but in any case, prior to 
presentation at the meeting or publication in the proceedings. Press releases and 
other media presentations must also be forwarded to CTEP prior to release. 
Copies of any manuscript, abstract and/or press release/ media presentation should be sent to: 
Email: ncicteppubs@mail.nih.gov 
The Regulatory Affairs Branch will then distribute them to Collaborator(s). No publication, 
manuscript or other form of public disclosure shall contain any of Collaborator’s confidential/ 
proprietary information. 
 
14.5 Genomic Data Sharing Plan 
 
The investigators and statistician and/or bioinformaticians for a study will have access to all data 
on mutations and variants stored in the Theradex Data Base and the GDC. This information will 
be sequestered from access throughout the study until it is analyzed for purposes of reporting and 
publishing of the study results. As specified in the CRADA for the agents used in the clinical 
study, the pharmaceutical collaborator will have at least 6 months, longer if needed for a 
regulatory filing, to review the data and or receive copies of the data once the study is completed 
and analyzed, or sooner, if specified for purposes of generating Intellectual Property. Once these 
timeframes have been exceeded, the data will be available through a Data Access Committee 
(DAC) in the GDC following NCI and Collaborator review of the proposals. 
 
14.6 Incidental/Secondary Findings Disclosure Procedure 
 
Given the potential clinical implications conferred by detecting a germline and/or somatic 
mutation in one of the proven cancer susceptibility genes, this protocol will use the following 
disclosure procedure, consistent with the recommendations of the American College of Medical 
and Genomics (ACMG) (Green et al., 2013 and Kalia et al., 2016): 
 
The NCI Molecular Characterization Laboratory will review the mutations/variants once at the 
time of initial specimen evaluation according to the most recent version of the ACMG guidance 
on variants. The NCI Molecular Characterization Laboratory will not re-review all specimens 
received if a new version of the ACMG guidance is published after the initial review. 
 
For each participant with a pathogenic or likely pathogenic germline and/or somatic variant 
detected in the WES of blood (as defined in the ACMG guidance), the NCI Molecular 
Characterization Laboratory will report to the Program Director or Scientific Officer the UPID 
and variant(s) identified. The Program Director or Scientific Officer will contact Theradex to 
obtain the name of the protocol, investigator treating the patient, and the Principal Investigator of 
the grant. The treating physician will be contacted by phone and in writing to ask the patient 
whether he or she is interested in learning more about the finding. 

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137 
 
 
 
If the patient wants to know more, the physician should contact the Program Director for more 
information about the mutation/variant. The treating physician and a medical genetics counselor 
should meet with the patient to discuss the importance and meaning of the finding, but not the 
finding itself, and notify the patient that this research finding must be confirmed by Sanger 
sequencing at the patient’s/patient insurer’s expense in a Clinical Laboratory Improvement 
Amendments (CLIA)-approved laboratory. The treating physician and genetic counselor should 
inform the patient of the confirmed result and its meaning and significance to the patient. If 
desired, the patient may elect to undergo genetic counseling and confirmatory CLIA-approved 
clinical testing on his or her own. Neither the research laboratory nor the National Cancer 
Institute will be responsible for the costs incurred for any confirmatory genetic testing or 
counseling. 
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39:74-88. 

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APPENDIX A 
PERFORMANCE STATUS CRITERIA 
 
ECOG Performance Status Scale 
Grade 
Descriptions 
0 
Normal activity. Fully active, able 
to carry on all pre-disease 
performance without restriction. 
1 
Symptoms, but ambulatory. 
Restricted in physically strenuous 
activity, but ambulatory and able 
to carry out work of a light or 
sedentary nature (e.g., light 
housework, office work). 
2 
In bed <50% of the time. 
Ambulatory and capable of all 
self-care, but unable to carry out 
any work activities. Up and about 
more than 50% of waking hours. 
3 
In bed >50% of the time. Capable of 
only limited self-care, confined to 
bed or chair more than 50% of 
waking hours. 
4 
100% bedridden. Completely 
disabled. Cannot carry on any 
self-care. Totally confined to bed 
or chair. 
5 
Dead. 

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APPENDIX B FORMULA TO ESTIMATE RENAL FUNCTION USING SERUM 
CREATININE 
 
Formulas to estimate renal function using serum creatinine provided by the NCI’s Investigational 
Drug Steering Committee (IDSC) Pharmacological Task Force in table below. 
 
1. Estimated glomerular filtration rate (eGFR) using the Chronic Kidney Disease Epidemiology  
Collaboration (CKD-EPI) (Levey et al., 2009). 
Formulae: 
Race and 
Serum Creatinine (SCr), 
Equation 
Sex 
µmol/L (mg/dL) 
Black 
Female 
≤62 (≤0.7) 
GFR = 166 × (SCr/0.7)−0.329 ×(0.993)Age 
>62 (>0.7) 
GFR = 166 × (SCr/0.7)−1.209 ×(0.993)Age 
Male 
≤80 (≤0.9) 
GFR = 163 × (SCr/0.9)−0.411 ×(0.993)Age 
>80 (>0.9) 
GFR = 163 × (SCr/0.9)−1.209 ×(0.993)Age 
White or 
other 
Female 
≤62 (≤0.7) 
GFR = 144 × (SCr/0.7)−0.329 ×(0.993)Age 
>62 (>0.7) 
GFR = 144 × (SCr/0.7)−1.209 ×(0.993)Age 
Male 
≤80 (≤0.9) 
GFR = 141 × (SCr/0.9)−0.411 ×(0.993)Age 
>80 (>0.9) 
GFR = 141 × (SCr/0.9)−1.209 ×(0.993)Age 
SCr in mg/dL; Output is in mL/min/1.73 m2 and needs no further conversions. 
2. eGFR using the Modification of Diet in Renal Disease (MDRD) Study (Levey et al., 2006). 
175 x SCr–1.154 × age–0.203 × 0.742 (if female) × 1.212 (if black) 
Output is in mL/min/1.73 m2 and needs no further conversions. 
3. Estimated creatinine clearance (ClCr) by the Cockcroft-Gault (C-G) equation (Cockcroft and  
Gault, 1976). 
 
Followed by conversion to a value normalized to 1.73 m2 with the patient’s body surface area 
(BSA). 
 
References 
1. Levey, A.S., L.A. Stevens, C.H. Schmid, et al. (2009). A new equation to estimate 
glomerular filtration rate. Ann Inter Med. 150:604-612. 
2. Levey, A.S., J. Coresh, T. Greene, et al. (2006). Using standardized serum creatinine 
values in the modification of diet in renal disease study equation for estimating 
glomerular filtration rate. Ann Intern Med. 145:247-254. 
3. Cockcroft, D.W. and M.H. Gault. (1976). Prediction of creatinine clearance from serum 
creatinine. Nephron. 16:31-41. 

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APPENDIX C 
PATIENT CLINICAL TRIAL WALLET CARD 
 
 
CLINICAL TRIAL WALLET CARD 
Show this card to all of your 
healthcare providers andkeep 
it with you in case you go to 
the emergencyroom. 
Patient Name: 
Diagnosis: 
Study Doctor: 
Study Doctor Phone #: 
NCI Trial #: 10300 
Study Drug(S): Pembrolizumab (MK-3475), 
cytarabine, and either daunorubicin or 
idarubicin 
For more information: 1-800-4-CANCER 
cancer.gov | clinicaltrials.gov 

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APPENDIX D 
RESPONSE CRITERIA (PER ELN 2017 
RECOMMENDATIONS) 
 
Response criteria in AML 
Response 
 
CR without minimal residual disease (CRMRD−) 
If studied pretreatment, CR with 
negativity for a genetic marker by RT-qPCR, or CR with negativity by MFC 
 
Complete remission (CR): Bone marrow blasts <5%; absence of circulating blasts and blasts 
with Auer rods; absence of extramedullary disease; ANC ≥1.0 × 109/L (1000/µL); platelet count 
≥100 × 109/L (100 000/µL); MRD+ or unknown 
 
CR with incomplete count recovery (CRi): All CR criteria except for residual neutropenia (<1.0 
× 109/L [1000/µL]) or thrombocytopenia (<100 × 109/L [100 000/µL]) 
 
CR with incomplete hematologic recovery (CRh): All CR criteria with neutrophil count (>0.5 × 
109/L [1000/µL]) and platelet count (>50 × 109/L [100 000/µL]) 
 
 
Morphologic leukemia-free state (MLFS): Bone marrow blasts <5%; absence of blasts with Auer 
rods; absence of extramedullary disease; no hematologic recovery required. (Marrow should not 
merely be “aplastic”; at least 200 cells should be enumerated or cellularity should be at least 
10%) 
 
Partial remission (PR) All hematologic criteria of CR; decrease of bone marrow blast percentage 
to 5% to 25%; and decrease of pretreatment bone marrow blast percentage by at least 50% 
 
Treatment failure 
Primary refractory disease: No CR or CRi after 2 courses of intensive induction treatment; 
excluding patients with death in aplasia or death due to indeterminate cause 
 
Death in aplasia: Deaths occurring ≥7 d following completion of initial treatment while 
cytopenic; with an aplastic or hypoplastic bone marrow obtained within 7 d of death, without 
evidence of persistent leukemia 
 
Death from indeterminate cause: Deaths occurring before completion of therapy, or <7 d 
following its completion; or deaths occurring ≥7 d following completion of initial therapy with 
no blasts in the blood, but no bone marrow examination available 
 
Response criteria for clinical trials 
 
Stable disease: Absence of CRMRD−, CR, CRi, PR, MLFS; and criteria for PD not met Period 
of stable disease should last at least 3 months 
 
Progressive disease (PD): Evidence for an increase in bone marrow blast percentage and/or 

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increase of absolute blast counts in the blood  
• >50% increase in marrow blasts over baseline (a minimum 15% point increase is required in 
cases with <30% blasts at baseline; or persistent marrow blast percentage of >70% over at least 3 
mo; without at least a 100% improvement in ANC to an absolute level (>0.5 × 109/L [500/µL], 
and/or platelet count to >50 × 109/L [50 000/µL] nontransfused) 
• >50% increase in peripheral blasts (WBC × % blasts) to >25 × 109/L (>25 000/μL) (in the 
absence of differentiation syndrome) 
• New extramedullary disease 
Some protocols may allow transient addition of hydroxyurea to 
lower blast counts 
“Progressive disease” is usually accompanied by a decline in ANC and platelets and increased 
transfusion requirement and decline in performance status or increase in symptoms 
 
Relapse 
 
Hematologic relapse (after CRMRD−, CR, CRi) 
Bone marrow blasts ≥5%; or 
reappearance of blasts in the blood; or development of extramedullary disease 
 
Molecular relapse (after CRMRD−) 
If studied pretreatment, reoccurrence of MRD as 
assessed by RT-qPCR or by MFC 

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APPENDIX E 
BONE MARROW CORE BIOPSY SAMPLES 
o Tissue must be fixed in neutral-buffered formalin (no acid-basedproducts). 
o For collection sites shipping samples in Ethanol, formalin fixed tissue will be 
transferred to 70% ethanol at room temperature for up to 72 hours before processing 
(Steps 3 to 13, Table 1) is completed at the Biorepository. 
o The tissue will be processed on an automated tissue processor following Steps 3 to 
12 as suggested in Table 1 so long as total time from ethanol to embedding (in gray) 
exceeds 4 hours. 
o Do not use a microwave processor. 
o The tissue will be embedded in paraffin (Step 13, Table 1). 
 
Table 1. Main stages of tissue processing. Steps 3-12 performed in an automated tissue- 
processor (no microwave processors). 
 
 
Step/Process 
Solution 
Time 
1. Fixation 
10% buffered formalin 
12-24 hours 
2.Dehydration 
70% Ethanol 
30 minutes or up to 
72 hours 
3.Dehydration 
95% Ethanol 
30 minutes 
4.Dehydration 
95% Ethanol 
30 minutes 
5.Dehydration 
100% Ethanol 
30 minutes 
6.Dehydration 
100% Ethanol 
30 minutes 
7.Dehydration 
100% Ethanol 
30 minutes 
8.Clearing 
Xylene 
30 minutes 
9.Clearing 
Xylene 
30 minutes 
10.Infiltration 
Paraffin Wax 
30 minutes 
11.Infiltration 
Paraffin Wax 
30 minutes 
12.Infiltration 
Paraffin Wax 
30 minutes 
13.Blocking Out 
Paraffin Wax 
n/a 

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APPENDIX F 
SKIN PUNCH BIOPSY VERIFICATION FORM 
 
 
Please have the Clinician* responsible for signing out this patient’s case complete the following: 
 
ETCTN Universal Patient ID:  
 
 
ETCTN Patient Study ID:   
 
 
Date of Procedure (mm/dd/yyyy):   
 
 
Tissue Type (circle one): 
Normal Skin 
Other:___________ 
 
Time point (circle one):  Baseline 
Other:_______________ 
 
Site Tissue Taken From:   
 
 
 
 
 
I agree that this tissue may be released for research purposes only and that the release of this tissue 
will not have any impact on the patient’s care. 
 
 
 
Clinician Signature 
Date 
 
 
 
Clinician Printed Name 
 
*Note: For the purposes of this form, Clinician could include the Nurse Practitioner, Registered Nurse, Pathologist, 
Radiologist, Interventional Radiologist, Surgeon, Oncologist, Internist, or other medical professional responsible for the 
patient’s care.  
 
 
 
 
 
Version: 1 
Effective Date: 9/2022