BLockade of PD-1 Added to Standard Therapy to Target Measurable Residual Disease in Acute Myeloid Leukemia 2 (BLAST MRD AML-2): A Randomized Phase 2 Study of the Venetoclax, Azacitadine, and Pembrolizumab (VAP) Versus Venetoclax and Azacitadine as First Line Therapy in Older Patients With Acute Myeloid Leukemia (AML) Who Are Ineligible or Who Refuse Intensive Chemotherapy
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 # 10334 Version 
Date: November 27, 2024   
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` 
 
       SUMMARY OF CHANGES – Protocol  
 
For Protocol Revision 16 to: BLockade of PD-1 Added to Standard Therapy to target Measurable Residual       
Disease in Acute Myeloid Leukemia 2 (BLAST MRD AML-2): A randomized Phase 2 Study of the 
Venetoclax, Azacitidine, and Pembrolizumab (VAP) Versus Venetoclax and Azacitidine as First Line Therapy 
in Older Patients with Acute Myeloid Leukemia (AML) who are Ineligible or who Refuse Intensive 
Chemotherapy 
 
NCI Protocol #: 10334 
Local Protocol #: 2000031641 
 
NCI Version Date:  11/27/2024 
Protocol Date: 11/27/2024 
 
I. Recommendations per Review of Amendment #10 by Brian Ko, MD dated 11/04/2024.  
 
# 
Section 
Comments 
1. 
All 
Updated Version Date in Header 
 
2. 
Title 
Page 
Revised Protocol Type / Version# / Version Date  
3. 
Table of 
Contents 
Updated Table of Contents  
4. 
5.7 
5.9.6.2 
Please specify “skin” punch biopsies.  
 
PI Response: This has been updated.  
5. 
5.9.9.1 
Please make sure that the address for shipment of specimens for methylation 
assay is correct. Beatriz Sanchez-Espiridion’s name was removed from other 
assays. 
 
PI Response: Thank you for the comment. We are awaiting confirmation 
of the address but needed to update the study personnel right away. The 
address change will be provided once MD Anderson has confirmed.  
 
6. 
13.5-
13.6 
Please delete both sections. 
PI Response: These sections have been deleted.  
 
 
 
 

NCI Protocol # 10334 Version 
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II. Additional Changes by Principal Investigator  
 
# 
Section 
Comments 
7. 
Title 
Page  
Added Jan Bewersdorf as Co-Principal Investigator 
8. 
All 
Formatting, typos corrected throughout. 
 

NCI Protocol # 10334  
Version Date: November 27, 2024  
1 
 
 
 
 
  
 
NCI Protocol #: 
10334 
 
Local Protocol #: 
2000031641 
 
ClinicalTrials.gov Identifier: NCT04284787 
 
TITLE: BLockade of PD-1 Added to Standard Therapy to target Measurable Residual Disease 
in Acute Myeloid Leukemia 2 (BLAST MRD AML-2): A randomized Phase 2 Study of the 
Venetoclax, Azacitidine, and Pembrolizumab (VAP) Versus Venetoclax and Azacitidine as First 
Line Therapy in Older Patients with Acute Myeloid Leukemia (AML) who are Ineligible or who 
Refuse Intensive Chemotherapy 
 
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: 203-785-7232 
amer.zeidan@yale.edu 
 
Co-Principal Investigator/Study Chair: 
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: 
 

NCI Protocol # 10334  
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Jerald Radich, MD 
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 
EDDOP / Early Drug Development Opportunity Program  
 
 
 
 
Statistician: 
Study Coordinator: 
Ondrej Blaha                                                         Anne Caldwell, RN, BSN, OCN 
300 George Street, Suite 511 
                            Yale University 
 
New Haven, CT 06511                                           300 George Street, Suite 120 
 
 
Telephone: 203-737-5906                                     Telephone: 475-434-7321 
Fax: 203-737-8880                                                Fax: 203-737-3401 
 
Responsible Research Nurse: 
Responsible Data Manager: 
Anne Caldwell, R.N, BSN, OCN 
Anne Caldwell R.N, BSN, OCN 
300 George Street, Suite 120 
300 George Street, Suite 120 
New Haven, CT 06511 
New Haven CT 06511 
 
anne.caldwell@yale.edu 
 
 
 
NCI-Supplied Agent: MK-3475 (pembrolizumab) (NSC # 776864) 
Commercial Agents: Azacitidine (NSC # 102816), Venetoclax (NSC # 766270) 
 
IND #: 
IND Sponsor: DCTD, NCI 
 
Protocol Type / Version # / Version Date: 
Original / May 30, 2019 
Revision 1 / December 3, 2019 

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Revision 2 / January 9, 2020 
Revision 3 / January 31, 2020 
Revision 4a / March 11, 2020 
Revision 5 / April 7, 2020 
Revision 6 / May 14, 2020Revision 7 / June 9, 2020 
Revision 8 / September 5, 2020 - Disapproved 
Revision 9 / October 24, 2020 
Revision 10 / October 11, 2021 
Revision 11 / January 10, 2022 
Revision 12 / April 11, 2022 
Revision 13 / October 25, 2022 
Revision 14 / February 10, 2023 
Revision 15 / October 11, 2024 
Revision 16 / November 27, 2024  
 

4
NCI Protocol # 10334 
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2024
SCHEMA
a.
Complete Step 0: Initial Registration at time of sample submission.
b. Complete Step 1: Treatment Registration at time of completion of eligibility.
c.
Patient may come off trial at any point after Induction to proceed to allogeneic stem cell transplant as per investigator 
discretion.

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TABLE OF CONTENTS 
SCHEMA ......................................................................................................................................... 4 
1. 
OBJECTIVES ...................................................................................................................... 8 
1.1 
Primary Objectives ................................................................................................... 8 
1.2 
Secondary Objectives ............................................................................................... 8 
1.3 
Exploratory Studies .................................................................................................. 8 
2. 
BACKGROUND .................................................................................................................. 9 
2.1 
Study Disease(s) ....................................................................................................... 9 
2.2 
CTEP IND Agent ................................................................................................... 16 
2.3 
Other Agent(s) ........................................................................................................ 17 
2.4 
Rationale ................................................................................................................ 20 
2.5 
Standard of care assessments: ................................................................................ 22 
2.6 
Correlative Studies Background ............................................................................. 23 
3. 
PATIENT SELECTION .................................................................................................... 31 
3.1 
Eligibility Criteria .................................................................................................. 31 
3.2. 
Exclusion Criteria ................................................................................................... 34 
3.3. 
Inclusion of Women and Minorities ....................................................................... 36 
4. 
REGISTRATION PROCEDURES .................................................................................... 36 
4.1. 
Investigator and Research Associate Registration with CTEP ............................... 36 
4.2. 
Site Registration ..................................................................................................... 38 
4.3. 
Patient Registration ................................................................................................ 40 
4.4. 
General Guidelines ................................................................................................. 42 
5. 
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES ......................................... 42 
5.1 
Summary Table for Specimen Collection .............................................................. 42 
5.2. Specimen Procurement Kits and Scheduling .............................................................. 45 
5.3 
Specimen Tracking System Instructions ................................................................ 47 
5.4 
Specimen Collection and Processing ..................................................................... 51 
5.5 
Shipping Specimens from Clinical Site to the EET Biobank. ................................ 54 
5.6 
Shipping of Specimens from Clinical Site to Brent Wood’s Lab, 
Department of Pathology and Laboratory Medicine, Children’s Hospital, 
Los Angeles ............................................................................................................ 57 
5.7. 
Biomarker Plan ....................................................................................................... 59 
5.8. 
Integral Laboratory Studies .................................................................................... 64 
5.9 
Exploratory Correlative Studies ............................................................................. 64 
6. TREATMENT PLAN ................................................................................................................ 70 
6.1 Agent Administration ................................................................................................... 70 
6.2. General Concomitant Medication and Supportive Care Guidelines ........................... 75 
6.3. Duration of Therapy .................................................................................................... 77 
6.4. Duration of Follow-Up ................................................................................................ 78 

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6.5. Criteria to Resume Treatment .................................................................................. 78 
6.6. Contraception and Pregnancy ..................................................................................... 79 
6.7 Treatment Up to 2 Years .............................................................................................. 81 
7. DOSING DELAYS/DOSE MODIFICATIONS ........................................................................ 81 
7.1 
Pembrolizumab (MK-3475) ................................................................................... 81 
7.2 
Venetoclax .............................................................................................................. 92 
7.3 
Azacitidine ............................................................................................................. 94 
8 
PHARMACEUTICAL AGENT INFORMATION ............................................................ 95 
8.1 
CTEP IND Agent ................................................................................................... 95 
8.2 
Commercial Agent(s) ............................................................................................. 98 
9 
STATISTICAL CONSIDERATIONS ............................................................................... 101 
9.1 
Study Design/Endpoints ......................................................................................... 101 
9.2 
Sample Size/Accrual Rate ...................................................................................... 102 
9.3 
Stratification Factors .............................................................................................. 103 
9.4 
Analysis of Secondary Endpoints .......................................................................... 103 
9.5 
For phase 2 protocols only: Reporting and Exclusions ........................................  109 
9.6 
Data Safety Monitoring Board .............................................................................  110 
10 
ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS ...........................  111 
10.1 
Comprehensive Adverse Events and Potential Risks List(s) (CAEPRs) .............  111 
10.2 
Adverse Event Characteristics ..............................................................................  123 
10.3 
Expedited Adverse Event Reporting ....................................................................  124 
10.4 
Routine Adverse Event Reporting ........................................................................  127 
10.5 
Pregnancy .............................................................................................................  127 
10.6 
Secondary Malignancy .........................................................................................  127 
10.7 
Second Malignancy ..............................................................................................  128 
11 
STUDY CALENDAR ......................................................................................................  129 
12 
MEASUREMENT OF EFFECT ......................................................................................  134 
12.1 
Definitions ............................................................................................................  134 
12.2 
Hematologic Response .........................................................................................  134 
12.3 
Other Response Parameters ..................................................................................  135 
13 
STUDY OVERSIGHT AND DATA REPORTING / REGULATORY 
REQUIREMENTS ........................................................................................................... 136 
13.1. Study Oversight .................................................................................................... 136 
13.2. Data Reporting ..................................................................................................... 136 
13.3. Data Quality Portal ............................................................................................... 138 
13.4. Collaborative Agreements Language ................................................................... 139 
 
14. 
REFERENCES ................................................................................................................. 141 
APPENDIX A 
PERFORMANCE STATUS CRITERIA ................................................. 152 

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APPENDIX B 
FORMULA TO ESTIMATE RENAL FUNCTION USING 
SERUM CREATININE ................................................................................................... 153 
APPENDIX C 
PATIENT CLINICAL TRIAL WALLET CARD ................................... 154 
APPENDIX D 
RESPONSE  CRITERIA  (per  ELN  2017 
RECOMMENDATIONS) ................................................................................................ 155 
APPENDIX E 
PATIENT DIARY ................................................................................... 157 
APPENDIX F 
CUTTING AND EMBEDDING SKIN .............................................. 161 
APPENDIX G 
BONE MARROW CORE AND SKIN PUNCH BIOPSY 
SAMPLES 
165 

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1. OBJECTIVES 
 
1.1 Primary Objectives 
 
1.1.1 Assess the percentage of patients with minimal residual disease (MRD) negative CR 
(MRD-CR) or complete remission with incomplete count recovery (MRD-CRi) with 
AZA + VEN with pembrolizumab during the first 6 cycles and compare to control arm. 
 
1.2 Secondary Objectives 
 
1.2.1 Assess the investigator-assessed rates of CR/CRi/partial remission (PR)/morphological 
leukemia free state (MLFS) as defined per the modified International Working Group 
(IWG) 2003 response criteria with AZA + VEN with pembrolizumab (Cheson et al., 
2003), as well as rates of MRD negative MLFS. 
 
1.2.2  Rates of complete remission with partial count recovery (CRh) and Hematologic 
improvement (HI) to red blood cells and platelets 
 
1.2.3 Assess time to MRD negativity and duration of MRD negative state, event free survival 
(EFS), relapse free survival (RFS), calculated as the time from initial treatment to either 
disease relapse or death, 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). 
 
1.2.4 Assess the proportion of patients who develop severe toxicity. Severe toxicity is defined 
as the occurrence of any of the adverse events (AEs) (section 9.5.1) during the first 35 
days from start of Pembrolizumab (MK-3475) therapy 
 
1.3 Exploratory Studies 
 
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 
 
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 
efficacy using O-link cytokine panel and RNA-seq, respectively 

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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. Profiling of DNA methylation patterns before and after treatment to assess for 
correlation to response to treatment. 
1.3.7 Correlate gut microbiome at baseline and changes in the microbiome with clinical 
response, both in standard chemotherapy and immunotherapy/chemotherapy therapy 
settings. 
 
1.3.8 MRD assessment using duplex sequencing strategy for circulating cell-free tumor 
DNA and correlation with long-term outcomes 
 
1.3.9 Exploring different thresholds of MRD negativity using flow cytometry aside from the 
0.1% level that will be used for the primary endpoint purposes (e.g. 0.01%). 
 
 
 
2. BACKGROUND 
 
2.1 Study Disease(s) 
 
2.1.1 Acute myeloid leukemia (AML) 
 
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). 

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AML in elderly adults (>65 years of age) represent major therapeutic challenges, including both 
a more aggressive disease biology as well as a population that is more susceptible to the toxic 
effects of chemotherapy. Survival outcomes of AML in older patients are historically dismal, 
with a 3-year survival of < 10% (Menzin et al., 2002; Alibhai et al., 2009). Elderly patients are 
often not candidates for conventional cytotoxicity induction due to associated comorbidities and 
poor performance status (Pettit and Odenike, 2008). The de facto standard of care lower- 
intensity regimens for patients unfit for induction chemotherapy consist predominately of 
hypomethylating agents (HMAs) such as 5-azacitidine (azacitidine) and decitabine or low-dose 
cytarabine. Both the HMAs are currently approved by the Food and Drug Administration (FDA) 
for myelodysplastic syndrome (MDS) and commonly are used off-label for front-line or 
refractory disease in patients with AML. HMAs are especially used for older patients with high 
risk cytogenetics (Ravandi et al., 2009), however, median overall survival (OS) estimates with 
HMA monotherapy are poor at <1 year (Al-Ali et al., 2012; Dombret et al., 2015; Cashen et al., 
2010; Kantarjian et al., 2012). Furthermore, HMA monotherapy is associated with low response 
rates (10-50%), require several months to achieve best response, and are not curative (Dombret 
et al., 2015; Cashen et al., 2010; Kantarjian et al., 2012; Al-Ali et al., 2012). To this end, efforts 
are underway at developing rationally selected HMA combination approaches that can yield 
rapid clinical responses, which are more deep and durable (DiNardo et al., 2019). 
 
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 
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 

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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). 
 
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 

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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 
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). Kronig 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 

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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. 
 
2.1.2.2.3 
Early clinical 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. 

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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 azacitidine 
(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 Hypomethylating agents modulate the immune system 
 
The hypomethylating agents (HMAs), azacitidine and decitabine are valuable therapeutic options 
in older patients with AML deemed unfit for induction chemotherapy. Both of these agents, 
which initially were developed as antimetabolites, were later shown to target leukemogenic 
epigenetic changes (Fenaux et al., 2010). Mechanisms underlying response to HMAs include 
direct cytotoxic effects, terminal differentiation, and apoptosis (Juttermann et al., 1994). 
Mechanistic studies suggest that higher doses of therapy are primarily cytotoxic while 
hypomethylation activity is more prominent at lower levels of exposure (Christman, 2002). 
Apart from their cytotoxic and hypomethylating effects, these agents actively modulate the 
immune system, exerting a dual effect on anti-tumor immunity. In this context, an important 
mechanism of tumor immune response evasion by cancer cells lies in their ability to alter the 
expression of tumor-expressing antigens, resulting in deficient antigen presentation (Heninger et 
al., 2015). HMAs can have a favorable effect on anti-tumor immune response by several 
mechanisms including upregulating a range of IFN-gamma pathway viral defense and 
immunomodulatory pathway-related genes such as cancer testis antigens, increasing class I HLA 
expression on tumor cells, and upregulating co-stimulatory molecules (CD28, CD40L) (Li et al., 
2014; Srivastava et al., 2016; Coral et al., 1999; Coral et al., 2002). In addition to the favorable 
effects on anti-tumor immunity, HMA treatment can also dampen immune response by 
upregulating the expression of inhibitory check point receptor PD-1 on T cells and inhibitory 
ligands PD-L1 and PD-L2 on tumor cells. The increased expression of PD-1 on T cells is 
brought about by azacitidine induced hypomethylation of the PD-1 promotor region 

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(Youngblood et al, 2011). The increased PD-L1 expression on tumor specific T cells, if left 
unchecked, promotes their exhaustion. 
 
In a study by Ørskov et al, treatment with azacitidine was accompanied by DNA demethylation 
in 44% of patients with AML/MDS. Hypo/demethylation of the PD-1 promoter status was 
correlated with significantly worse response rates and a trend toward a shorter OS (Ørskov et al, 
2015). In another study by Yang et al, azacitidine upregulated PD-1 and PDL1 mRNA 
expression on the PB mononuclear cells in approximately 50% of patients with AML or MDS. 
PD-1 and its ligands, PD-L1 and PD-L2, and to a lesser extent CTLA-4, were also aberrantly 
upregulated (≥2-fold) on BM CD34 + cells by mRNA expression, in approximately 30–40% of 
patients with AML or MDS (Yang et al, 2014). Patients resistant to azacitidine-based therapy 
had relative higher increments in gene expression on the PB mononuclear cells and BM CD34 + 
cells as compared with patients who achieved response, suggesting this could be a mechanism of 
resistance (Yang et al, 2014). Azacitidine also enhances anti-tumoral activity of the anti-CTLA- 
4 antibody by inducing hypomethylation of human endogenous retroviral elements eventually 
leading to upregulation of immune effector pathway signaling genes, thereby leading to 
sensitization to anti-CTLA4 antibody therapy (Chiappinelli et al., 2017; Roulius et al., 2015). 
Apart from their effects on tumor antigen and immune check point receptor-ligand expression, 
HMAs can also impact T-cell numbers and phenotypes such as increasing peripheral blood T-reg 
cells, decreasing Th1 and Th2 cells, and shifting CD8 lymphocytes towards CD8+/IFN-gamma+ 
T subtype (which expresses a higher level of PD-1) (Catakovic et al., 2017; Costantini et al., 
2013). 
 
Given that the immune check point molecular upregulation represents an important mechanism 
of HMA resistance, a number of clinical trials combining HMAs with PD-1/PD-L1- based 
therapies have recently started enrollment for AML and MDS, including azacitidine with the 
anti-PD-1 antibody nivolumab (NCT02397720). This phase II study enrolled 73 patients with 
R/R AML; the median age was 70-years (range,22-90) and the median number of prior therapies 
was 2 (range,1-7) (Daver et al., 2018b). The ORR was 58% and 22%, in HMA-naive (n=25) and 
HMA pre-treated (n=45) patients, respectively. Notably, grade 3-4 immune-related adverse 
events (irAE) occurred in eight (11%) patients. The irAE profile differed slightly from that seen 
in solid tumors with the more common irAEs being pneumonitis, nephritis, colitis, and dermatitis 
compared to endocrinopathies, skin rash, and transaminitis described more frequently in solid 
tumors. Pre-therapy bone marrow and peripheral blood CD3 and CD8 were significantly 
predictive for response on flow-cytometry (Daver et al., 2018b). CTLA-4 was significantly up- 
regulated on CD4+ T effector cells in non-responders after 2, 4, and 6 doses of nivolumab 
suggesting that combination blockade of both these major co-inhibitory pathways may improve 
response rates and durability of responses. 
 
The combination of azacitidine with nivolumab was also evaluated in frontline MDS setting and 
demonstrated to be effective, with a response rate of 80% (NCT02530463) (Garcia-Manero et 
al., 2016). This phase 2 study also evaluated nivolumab alone and ipilimumab alone in patients 
with MDS who had failed prior therapy with a HMA. Interestingly, while ipilimumab 
demonstrated response in 33% of patients with high risk relapsed/refractory MDS, nivolumab 
showed no single agent activity suggesting that there may be a differential efficacy profile for 
PD-1 and CTLA-4 inhibition in myeloid diseases. Overall, clinical trial data from HMA-ICPi 

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combination approaches have shown encouraging response rates and durable responses without 
resorting to stem cell transplant. 
 
2.2 CTEP IND Agent 
 
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 
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 

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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). 
Pembrolizumab (MK-3475) 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 Venetoclax 
 
B-cell lymphoma 2 (BCL-2) is a mitochondrial pathway anti-apoptosis protein that plays a key 
role in survival and persistence of AML blasts (Vo et al., 2012; Konopleva et al., 2006). BCL-2 
overexpression has been implicated in chemotherapy resistance and inferior overall survival in 
AML (Konopleva et al., 2006). Venetoclax (ABT-199/GDC-0199) is a potent and highly 
selective small molecule BCL-2 inhibitor that has shown preclinical and clinical activity across a 
range of hematologic malignancies, including AML. Targeted BCL-2 inhibition with venetoclax 
has shown to induce cell death in AML cell lines and leukemic blasts and stem cells while 
sparing normal hematopoietic stem cells. (Vo et al., 2012; Konopleva et al., 2006; Lagadinou et 
al., 2013). In the relapsed-refractory (R/R) AML setting, a phase 2 study examined the single 
agent activity of venetoclax. The CR rate was 19% with another 19% having some form of 
myeloblast reduction. The drug was well tolerated with AEs consistent with expectations in the 
R/R AML population. Following its modest clinical activity and acceptable safety profile as 
monotherapy, venetoclax was investigated in phase 1 studies in combination with HMAs 
(decitabine or azacitidine) (NCT02203773) (Konopleva et al., 2016). This drug combination 
strategy is based on ex vivo pre-clinical evidence that combining venetoclax with azacitidine was 
potently synergistic in inducing AML cell death (Bogenberger et al., 2015). Studies suggest that 
azacitidine downregulates anti-apoptotic Mcl-1 protein concentrations, which is a known 
mediator of resistance to venetoclax (Tsao et al., 2012). Furthermore, the combination was 
shown to induce apoptosis in a p53 independent manner which has potential therapeutic 
implications in TP53 AML (Tsao et al., 2012). 

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A combination of venetoclax with hypomethylating agents in treatment naïve elderly (65 years 
or above) AML was shown to lead to high response rates, which were comparable to standard 
induction therapy, with acceptable toxicity (DiNardo et al., 2018a). In the final report of this 
phase 1b AML study 145 patients were enrolled and received oral venetoclax in combination 
with either decitabine (20 mg/m2, Days 1-5) or azacitidine (75 mg/m2, Days 1-7) (DiNardo et al., 
2019). In the expansion phase, venetoclax 400 mg or 800 mg with either HMA was given. With 
a median duration of follow up of 15.1 months (range, 9.8-31.7), 67% and 83% of the total study 
population had achieved a CR/CRi (97 of 145) and overall leukemia response rate 
(CR+CRi+partial remission+median leukemia free state), respectively. Response rates were 
similar between the venetoclax 400 mg + azacitidine and venetoclax 400 mg + decitabine 
cohorts. Notably, responses were noted even in high risk patient groups, such as poor-risk 
cytogenetics, ≥75 years, and secondary AML. Median times to best response (CR/CRi), duration 
of CR/CRi, and OS in the overall population were 2.1, 11.3, and 17.5 months, respectively; mOS 
has not been reached for the 400 mg venetoclax cohort. The CR/CRi rate, median duration of 
CR/CRi, and mOS for venetoclax 400 mg + azacitidine cohort were 76%, not reached (NR) 
(95% CI, 5.6-NR), and NR (95% CI, 9.0-NR), respectively. Data obtained from multiparameter 
flow cytometry (<10-3) demonstrated MRD negativity, at least once during study treatment, in 
29% of patients with CR/CRi. In summary, the CR+CRi rate of 67% and the mOS of 17.5 
months achieved with the HMA-venetoclax combination is notable compared with CR+CRi rate 
and mOS of 10-50% and <12 months, after HMA monotherapy (Dombret et al., 2015; Cashen et 
al., 2010; Kantarjian et al., 2012; Al-Ali et al., 2012). Based on these data, the FDA granted 
approval in November 2018 of the combination of HMAs with venetoclax in management of 
untreated older patients who are unfit for intensive chemotherapy. 
 
2.3.1.1 
Venetoclax’s effects on the immune system 
 
The BCL-2 family of proteins play a crucial role in the maintenance and survival of immune 
cells, as well as in regulating anti-tumor responses of the immune system (Bougras et al., 2004; 
Hawkins and Vaux, 1997). Given the critical role of BCL-2 in immune system regulation, 
venetoclax may affect the anti-tumor activity of immune checkpoint inhibitors. Two recent 
reports investigated the effects of venetoclax mediated BCL-2 inhibition on the viability and 
function of immune-cell subsets (Mathew et al., 2018; Lasater et al., 2018). Data from these 
studies suggest that venetoclax treatment results in loss of B-cell and naïve T-cell subsets but not 
memory CD8+ and CD4+ T effector cells. The lack of impairment in survival or activation of 
resting CD8+ T-cell memory subsets is possibly due to increased levels of BCL-XL and MCL-1 
(Lasater et al., 2018). In mixed lymphocyte reaction and cytomegalovirus recall assays, 
venetoclax did not affect the viability, the induction or frequency of memory T cells (Mathew et 
al., 2018). Furthermore, venetoclax did not antagonize the therapeutic effect of anti-PD-1 in in 
vivo syngeneic murine models (Mathew et al., 2018; Lasater et al., 2018). On the contrary, 
venetoclax-exposed memory T-cells were able to rapidly acquire effector and cytotoxic function 
to eliminate cancer cells, thus supporting the rationale for combinability of venetoclax with 
immune check point inhibitors (Mathew et al., 2018). The enhanced efficacy of the venetoclax- 
anti-PD-1 combination appears to be immune-mediated in nature (Mathew et al., 2018). To best 
of our knowledge, the venetoclax-ICPi combination strategy is yet to be explored in clinical 
trials. 

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2.3.2 Azacitidine 
 
Azacitidine (Vidaza®, NSC 102816) is a pyrimidine nucleoside analogue of cytidine that is 
incorporated into both RNA and DNA, leading to loss of DNA methylation and cytotoxicity 
(Stresemann and Lyko, 2008). Azacitidine is metabolized to 5-aza-2’-deoxycytidine 
triphosphate after cellular uptake, incorporated into DNA in place of cytosine, and forms 
covalent bonds to DNA methyltransferases (DNMT), leading to loss of DNA methylation by 
impairing DNMT function and triggering of DNA damage signaling and DNMT degradation. 
For more detailed information, please consult the Vidaza® package insert (2016). 
 
2.3.2.1 
Azacitidine activity in treatment-naïve, relapsed or refractory AML, and AML with 
MRD 
Epigenetic changes, including DNA methylation, play an important role in myeloid 
leukemogenesis, and the DNA hypomethylating agent azacitidine targets aberrant DNA 
methylation in AML (Döhner et al., 2015). A subset analysis of the AZA-001 study, which 
compared azacitidine to conventional care for patients with MDS, showed a CR rate of 17% for 
azacitidine-treated patients (Fenaux et al., 2009). The AZA-001-AML study compared 
azacitidine to conventional care for older patients with AML with >30% blasts and showed a 
CR/complete response with incomplete hematologic recovery (CRi) rate of 27.8% and a trend 
towards improved OS (Dombret et al., 2015). In both studies, azacitidine was well tolerated 
with primarily hematologic toxicity. Several studies have also demonstrated clinical activity of 
azacitidine in R/R AML (Craddock et al., 2016; Itzykson et al., 2015; Ivanoff et al., 2013; Roboz 
et al., 2014; Tawfik et al., 2014). The results are summarized in Table 1. In a phase 3 study 
comparing elacytarabine to investigator choice, the azacitidine arm showed a median OS of 
approximately 6-7 months and 12-month survival of 25%. CR/CRi rate was not reported. Other 
studies evaluating the activity of azacitidine in R/R AML showed CR/CRi rates ranging from 4- 
21%, median OS 8.2-9 months, and 12-month survival of 25-30%. These studies included a 
single center experience, a trial in patients after prior intense chemotherapy, a trial in older 
patients, and a trial after allogeneic HCT. 
 
Table 1: Azacitidine efficacy in R/R AML. 
Study 
CR/CRi 
Median OS 
1-year Survival 
Phase 3 
NR 
7 months 
25% 
Single-Center 
4% 
8.2 months 
25% 
After prior intense chemotherapy 
21% 
9 months 
30% 
Elderly patients 
17% 
8.4 months 
25% 
After allogeneic HCT 
15% 
NR 
12% (2-year) 
NR = Not reported; CR/CRi = complete response plus complete response with incomplete count 
recovery; OS = overall survival; HCT = hematopoietic cell transplant. 
 
Measurable/minimal residual disease (MRD) refers to low levels of disease that can be detected 
in some patients without frank hematologic relapse (<5% blasts). Methods used to measure 
MRD include multiparameter flow cytometry (MFC), real-time quantitative polymerase chain 
reaction (RT-qPCR) and targeted next-generation sequencing (NGS). Several studies have 
shown that detection of MRD after CR is highly prognostic for survival. For example, in one 

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study examining a large cohort of patients ages <60 years, presence of MRD after induction was 
associated with a 43% 5-year OS versus 63% in those without MRD (Freeman et al. 2017). 
Another study showed that MRD detected by both MFC and targeted NGS were independently 
prognostic and together had additive prognostic value (Jongen-Lavrencic et al. 2018). 
Importantly, several studies are congruent in showing that pre-transplant MRD predicts for 
survival similar to that of going to transplant with active AML. For example, in one study of 359 
patients (median age at transplant = 50 years) in MRD negative CR, MRD positive CR, or with 
active AML before allogeneic transplant, the 3-year OS was 73% for MRD negative patients 
compared to 26% for MRD positive patients and 23% for patients with active AML (Araki et al., 
2016). Thus, improving outcomes for patients with detectable MRD has the potential to prevent 
or delay relapse and improve survival. To this end, the RELAZA2 trial showed that azacitidine 
can prevent or delay hematologic relapse in patients (median age 59, range 52-69) with AML in 
CR with very early relapse as evidenced by newly detectable MRD. Treatment with standard- 
dose azacitidine led to a 58% response rate after six cycles and prevented relapse in 51% of 
patients. The 12-month relapsed free survival (RFS) and OS was 46% and 75%, respectively 
(Platzbecker et al., 2018). Overall, these findings support the further evaluation of azacitidine- 
based therapy as an approach to delay or prevent relapse and improve outcomes after transplant 
in patients with very early relapse (<5% blasts). 
 
2.4 Rationale 
 
2.4.1 Rationale for combining azacitidine + venetoclax combination therapy with 
immunotherapy (anti-PD-L1 antibody pembrolizumab) to stimulate immune attack 
against AML blasts 
 
The recent trial data from the azacitidine + venetoclax (AZA + VEN) combination in front-line 
AML suggests a high clinical activity with complete response rates approaching 70% and 
durable responses, with a median duration of complete response of 11.3 months (DiNardo et al., 
2019). Furthermore, 30% of the complete responders were MRD negative. Combining AZA + 
VEN combination chemotherapy with an immune check point inhibitor (ICPi) could lead to 
synergy by combining the immunomodulatory abilities of checkpoint inhibition and the AZA + 
VEN. Chemotherapy with AZA + VEN 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 (Kroemer et al., 2013; Zitvogel et al; 2013). Interferon-gamma 
(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 chemotherapy. 
Furthermore, induced expression of immune checkpoint pathway-related genes (PD-1, PD-L1, 
PD-L2) by HMAs leads to exhaustion of T cells and is a frequently described immune evasion 
mechanism of HMA resistance. The functionality of these ‘exhausted’ T cells may be restored 
by concomitant PD-L1/PD-L1 blockade. Furthermore, venetoclax-exposed CD8+ memory T- 
cells are able to rapidly acquire effector and cytotoxic function to eliminate cancer cells thus 
supporting the rationale for combinability of venetoclax with ICPis. Adding the anti PD-1 
antibody pembrolizumab to AZA+ VEN chemotherapy may lead to more effective CTL 
mediated destruction of leukemic blasts. The goal with combining pembrolizumab to AZA+ 

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VEN chemotherapy is to facilitate a more effective CTL mediated destruction of leukemic blasts 
resulting in improved rates of CR without MRD, which would hopefully increase duration of 
response and lower relapse rates. 
 
Figure 1: Potential synergistic effect of azacitidine + venetoclax combination and 
checkpoint inhibition with anti PD-1 antibody pembrolizumab in inducing an immune 
response against AML blasts. (Kroemer et al., 2013; Daver et al., 2018a) 
 
2.4.2 Safety of the combination of epigenetic and therapy and chemotherapy with checkpoint 
inhibition 
 
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 (high dose cytarabine followed by 
pembrolizumab) (Zeidner et al., 2017) and epigenetic therapy (azacitidine and nivolumab) 

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(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 (azacitidine 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 (Zeidner et al., 2017; Daver 
et al., 2017). Importantly, preliminary results from AML trials using anti-PD1 therapy in 
combination with intensive chemotherapy in the relapsed/refractory setting (Zeidner, 2017) and 
high doses of cytarabine/idarubicin in frontline setting (Ravandi et al., 2017) and in combination 
with HMAs (Ørskov, et al; 2015) among the patients who proceeded to HSCT after checkpoint 
inhibitor therapy found NO signal for increased risk of graft-versus-host disease or transplant- 
related toxicity (Ravandi et al., 2017a; Zeidner et al., 2017; Daver et al., 2017). Therefore, it is 
highly unlikely that there would be an increase in post-transplant toxicities using this 
combination among the very few patients who might proceed to HSCT. 
 
2.5 Standard of care assessments: 
 
2.5.1.1 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 for patients who undergo intensive chemotherapy. 
However, as the use of FLT3 inhibitors in older unfit patients who are not receiving intensive 
chemotherapy is not a standard of care or defined yet, those patients will be allowed on the trial. 
Given FLT3 assessment will not be required for eligibility or stratification, the results of FLT3 
testing are not required to be available at time of screening or randomization as they patients will 
not be getting intensive chemotherapy. 
 
2.5.2 Cytogenetics 
 
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 

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(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). While categorization into intermediate-risk AML is 
primarily based on cytogenetics, the definition of this broad and heterogenous category continues 
to evolve, as reflected by the incorporation of molecular markers of prognostic importance such 
as FLT3, NPM1, and CEBPA. Unlike the case with the favorable and intermediate risk AML, 
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 usecytogenetics as a stratification factor to account for heterogeneity in outcomes 
based on AML risk status. NPM1, FLT3, CEBPA and other genetic alternations are required for 
the risk group assignment in ELN and NCCN risk stratifications. Since the results of tests for 
these molecular markers are often not available at time of therapy initiation, we will use a risk 
stratification system that relies only on cytogenetic abnormalities for sake of stratification at 
randomization and not one that incorporates molecular markers. Patients with core binding factor 
(CBF) AML, rare among older patients, will be excluded. Patients with no-CBF AML will be 
stratified into two groups based on cytogenetic results available at time of randomization: 
Adverse cytogenetics vs. intermediate/unknown risk karyotype based on the NCCN/ELN 
karyotype risk stratification. 
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. Adverse karyotype can be 
determined based on FISH results (e.g. loss of chromosome 7 or 5 or 3 or more abnormalities) 
based on the specific probes used in the FISH. If results of full metaphase karyotype are not 
available and the available FISH results do not suggest an adverse karyotype, and there is a need 
to initiate therapy before those full results are available, then the patient can be stratified into the 
unknown/intermediate group for randomization purposes. In any case, results from FISH or 
karyotype should show that CBF abnormalities are NOT present by at time of randomization as 
the presence of CBF abnormalities is an exclusion factor. 
 
 
Refer to Section 9.3 for further details on stratification factors. 
 
 
2.6 Correlative Studies Background 
 
2.6.1 Multiplex Immunofluorescence (mIF) 

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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 
Multiplex Immunofluorescence (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 (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. 
 
 
2.6.2 Mass Cytometry (CyTOF) 
 
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). For this 
assay, the staining will be performed using MaxPar Human T-Cell Phenotyping Panel Kit 
(CD11a, CD4, CD8a, CD16, CD25, CD45, CCR7, CD69, CD45RO, CD44, CD27, CD45RA, 
CD3, CD57, HLA-DR, and CD127; along with CD28, CD117, and CD95), modified MaxPar 
human AML phenotyping panel (added CD10,CD13, CD16 on top of CD19, CD117, CD11b, 
CD64, CD7, CD123, CD45, CD33, CD15, CD34, CD3, CD44, CD38, HLA-DR, and CXCR4) 
for surface staining as per manufacturer’s protocol. These markers may change as the 
technology evolves and develops. 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) 
 
2.6.3 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, 

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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 peripheral blood in collaboration 
with Dr. Sacha Gnajtic at Mount Sinai CIMAC. 
 
2.6.4 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. 
 
2.6.5 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. 
 
2.6.6 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- 
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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. 
 
2.6.7 Leukemia-specific T-cell response 
 
Although AML cells express antigens recognizable to host T-cells, established leukemia is rarely 
eradicated by the host T-cells (Teague and Kline, 2013). AML cells employ a number of 
immune evasion mechanisms, which inhibit the generation or functional execution of anti-tumor 
immune responses including negative co-stimulatory ligands such as PD-L1 and the expansion 
and/or induction of suppressive cell types (Tregs), myeloid-derived suppressor cells (MDSC). 
Animal models have shown that blockade of the PD-1 pathway can boost leukemia antigen- 
specific T-cell responses (Zhang et al., 2009). Splenocytes, isolated from WT and PD-1-/- mice 
12 days after they were challenged with C1498. GFP AML cells, were exposed to irradiated 
tumor cells as an antigenic source for T-cell re-stimulation. In IFN-γ ELISpot assays, 
significantly greater numbers of IFN-γ spot-forming cells were detected from PD-1-/- compared 
with WT spleens (319209 spots vs. 3019 spots, respectively; P=0.001) demonstrating improved 
anti-leukemia directed T-cell response with interruption of the PD-1 pathway. Similarly, higher 
numbers of IFN-γ spot-forming cells were detected in spleens of mice treated with the PD-L1 
blocking antibody compared with the isotype control antibody (50.1 ±21.1 spots vs. 25.9 ±14.9 
spots, respectively; P=0.001). Furthermore, survival following AML induction was significantly 
superior in PD-1-deficient mice as well as WT mice following administration of anti-PD-L1 
antibodies compared to WT mice without anti-PD-L1 antibody treatment. These studies have led 
us to test the question whether the combination of chemotherapy with the anti-PD-1 antibody 
pembrolizumab (MK-3475) can lead to an improvement in leukemia antigen-specific T-cell 
response. 
 
Patient monocytes or CD34+ progenitors will be cultured in vitro with granulocyte macrophage- 
colony stimulating factor (GM-CSF) plus interleukin-4 (IL-4) in order to differentiate these cells 
into DC as described in detail elsewhere (Sallusto and Lanzavecchia, 1994). These DC will then 
be loaded with and primed by the patients’ leukemia cell lysate (Rainone et al., 2016). The 
stimulatory capacity of antigen loaded DCs will be tested by flow cytometric IFN-γ cytokine 
analysis of the patient’s own CD3+ naïve responder cells cultured with the patient’s own 
leukemia lysate pulsed DCs. Brefeldin A will be added to the cultures during the last 6 hours of 
stimulation to block protein secretion. Cells will be washed in PBS split in different flow 
cytometry tubes. Following blocking with anti-FCγR to reduce a-specific signals for 15 mins, 
cells will be then stained with anti-CD3, anti-CD4, anti-CD8 antibodies. Cells will then be 
fixed, permeabilized, and then stained with mAbs specific for IFN-γ. After a 20-minute 
incubation at 4°C in the dark, cells will be washed and fixed in 1% paraformaldehyde in PBS. 
 
2.6.8 DNA methylation assays 
 
DNA methylation by HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) 
Genomic DNA will be extracted from the mononuclear cell fraction of bone marrow specimens 
collected from enrolled patients at baseline, after Cycles 1, 2, 4, 6, at 1 year of treatment, and end 
of treatment/disease progression using the Puregene kit from Qiagen (Valencia, CA). HELP 

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representations will be prepared as previously described (Figueroa, 2009) and hybridized onto a 
custom long-oligonucleotide microarray designed to cover 25,626 HpaII amplifiable fragments 
(~50,000 unique CpG sites annotated to ~22,000 unique RefSeq genes) (Roche-NimbleGen, 
design ID: 2006-10- 26_HG17_HELP_Promoter) at the Weill Cornell Medical College 
Epigenomics Core Facility. Labeling, hybridization, and scanning will be performed as 
previously described (Figueroa, 2008). Quality control and array normalization were performed 
following our previously described methods (Figueroa, 2010; Thompson, 2008) and any arrays 
that did not pass our quality control were excluded from the analysis. In order to correct for the 
introduction of hybridization batches caused by a technical change in the microarray printing 
process introduced by NimbleGen while the trial was still ongoing, we subjected each individual 
channel to batch correction using the ComBat algorithm (Johnson, 2007) and the adjusted 
channels were then used to calculate the final HpaII/MspI ratio. 
 
 
2.6.9 Assessment of MRD 
 
2.6.9.1 
MRD assessment by multicolor flow cytometry (MFC) as an integral biomarker 
 
Achievement of MRD negativity at complete remission will be the primary endpoint of the trial, 
however, no therapeutic decisions will be made using the data generated. This application 
proposes to use multiparameter flow cytometry (MPF) to detect MRD in adults with AML. The 
results of these determinations will be compared with similar residual disease data generated by 
dual sequencing, which will be obtained an exploratory biomarker. Patients with AML will have 
MRD measured in the bone marrow (BM) and peripheral blood (PB) at the time of study entry to 
determine leukemia-associated immunophenotypic abnormalities and then BM will be evaluated 
for residual disease and after the fourth cycle of therapy 
 
The central MRD studies will be performed solely in the flow cytometry laboratory located in 
the Hematopathology Laboratory at 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 

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AML M1 
HLA-DR 
CD15 
CD33 
CD19 
CD117 
CD13 
CD38 
CD34 
CD71 
CD45 
AML M2 
HLA-DR 
CD64 
CD123 
CD4 
CD14 
CD13 
CD38 
CD34 
CD16 
CD45 
AML M4 
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. 
 
2.6.9.2 
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 
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 
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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 3, below). 
 
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. Dr. 
Radich’s lab, after review of all published AML genomes, 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. 
 
 
 
 
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 
(Unpublished Data). 
 
2.6.9.3 
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 

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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 (alloHCT) 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 researchers will 
be blinded to MFC results. 
 
2.6.9.4. 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 8-10mL 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. 
 
 
2.6.10 
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 

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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. Microbial metabolites are also 
important regulators of immune-responses. 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 and its metabolites modulate 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. 
 
 
 
3. PATIENT SELECTION 
 
3.1 Eligibility Criteria 
 
3.1.1 Newly diagnosed and pathologically-confirmed, previously untreated AML as defined by 
World Health Organization (WHO) criteria. 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 arising from 
prior myelodysplastic syndrome (MDS), as long as they have not received more than full 
cycle of hypomethylating agent therapy for MDS,] and t-AML) are also allowed. AML 
arising from antecedent hematologic disorders defined as prior MDS, MPN, or aplastic 
anemia are 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. Note 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 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 exclude presence of CBF abnormalities by time of 
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3.1.2 Age ≥60 years. 
 
3.1.3 Patients who are ineligible for intensive chemotherapy according to treating physician’s 
assessment or who refuse intensive chemotherapy. 
 
3.1.4 ECOG performance status of 0-3 (See Appendix A). 
 
3.1.5 Prior use of lenalidomide, erythropoiesis-stimulating agents (ESAs), and growth factors 
is allowed if used to treat prior MDS. AML must be previously untreated except for 
hydroxyurea, or ATRA for suspicion of APL but both should be discontinued prior to 
initiation of study therapy. Hypomethylating agents are not allowed to have been used for 
AML therapy. If hypomethylating agent therapy was used for prior MDS or MPN therapy 
then it should not have exceeded one full cycle. Note: One dose of prophylactic 
intrathecal therapy is allowed during or before screening if a lumbar puncture is 
performed to rule out CNS involvement. 
 
3.1.6 Hydroxyurea, or leukopheresis are allowed for management of hyperleukocytosis, as well 
as ATRA, before initiation of study therapy. White blood cell (WBC) count must be < 
25 ×109/L to start on study therapy per venetoclax label. Hydroxyurea and ATRA may 
be administered up to one day prior to start of study treatment. 
 
3.1.7 Intermediate-risk or poor risk AML as well as favorable risk by NCCN/ELN with the 
exception of “good-risk” cytogenic profile (i.e. for eligibility patient should lack the presence of 
t(8;21), (inv[16] or t[16;16]), or t(15;17) by full cytogenetics or FISH). Clarification: 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. Adverse karyotype can be determined based on FISH results (e.g. loss of 
chromosome 7 or 5 or 3 or more abnormalities) based on the specific probes used in the FISH. If 
results of full metaphase karyotype are not available and the available FISH results do not 
suggest an adverse karyotype, and there is a need to initiate therapy before those full results are 
available, then the patient can be stratified into the unknown/intermediate NCCN cytogenetic 
group for randomization purposes. In any case, results from FISH or karyotype should show that 
CBF abnormalities are NOT present by at time of randomization as the presence of CBF 
abnormalities is an exclusion factor. 
 
 
3.1.8 Patients must have adequate organ function: 
 
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 

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33 
 
 
 
System 
Laboratory Value 
 
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 
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. 
 
3.1.9. Patients who are 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. 
 
3.1.10 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. 
 
3.1.11 Patients who have undergone major surgery must have recovered adequately from the 
toxicity and/or complications from the intervention prior to starting therapy. 
 
3.1.12 Female patients of childbearing potential must have a negative urine or serum pregnancy 
test within 72 hours prior to receiving the first dose of study medication. 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 24 consecutive months (i.e., has had menses at any time in 
the preceding 24 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 who have a female partner 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. 

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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 CBF-AML and acute promyelocytic leukemia (APL). 
 
3.2.2. Received a prior anti-cancer mAb within 4 weeks 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. 
 
3.2.3. Prior therapy with an anti-PD-1, anti-PD-L1, or anti-PD-L2 agent. 
 
3.2.4. Patients who have had chemotherapy, targeted small molecule therapy (aside from 
imatinib, dasatinib, or nilotinib, hydroxyurea, or ATRA), or radiotherapy within 4 weeks 
(6 weeks for nitrosoureas or mitomycin C) prior to entering the study. 
 
3.2.5. Left ventricular ejection fraction <50% as determined by either echocardiogram or 
MUGA. 
 
3.2.6. Patients who have not recovered from AEs due to prior anti-cancer therapy (i.e., 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). 
 
3.2.7. 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. 
 
3.2.8. History of hypersensitivity to pembrolizumab (MK-3475) or any of its excipients, or 
other agents used in this study. 
 
3.2.9. Current use of systemic corticosteroids or immunosuppressive agents. 
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), inhaled corticosteroids, or topical steroids are permitted. 
 
3.2.10. 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). 

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3.2.11. 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. 
 
3.2.12. Patients who received prior allogenic transplant. 
 
3.2.13. 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. 
 
3.2.14. Patient with a diagnosis of immunodeficiency or receiving high dose systemic steroid 
therapy or any other form of immunosuppressive therapy within 7 days prior to the first 
dose of treatment. 
 
3.2.15. Patient 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. 
 
3.2.16. Patient with a known history of non-infectious pneumonitis that required the use of 
steroids or current non-infectious pneumonitis 
 
3.2.17. Patient with active uncontrolled infection. 
 
3.2.18. Patient with a known history of active TB (Bacillus Tuberculosis). 
 
3.2.19. Patients with uncontrolled intercurrent illness. 
 
3.2.20. Patients with psychiatric illness/social situations that would limit compliance with study 
requirements 
 
3.2.21. 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, breastfeeding should be discontinued if the 

NCI Protocol # 10334  
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36 
 
 
mother is treated with pembrolizumab. These potential risks may also apply to other 
agents used in this study 
 
 
3.2.22. Patients with no bone marrow involvement (i.e., those with only extramedullary disease). 
 
3.2.23. Patients who received prior hypomethylating agent (HMA) therapy for more than one full 
cycle in treatment for prior MDS. Patient must not have received HMA therapy for 
treatment of AML. 
 
3.2.24. Patients that 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. 
 
3.2.25. Patients with 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. 
 
 
3.3. 
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 and National Cancer Institute (NCI) policy 
require all individuals contributing to NCI-sponsored trials to register 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 

NCI Protocol # 10334  
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37 
 
 
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. 
 
RCR utilizes five person registration types. 
x IVR: MD, DO, or international equivalent, 
x NPIVR: advanced practice providers (e.g., NP or PA) or graduate level researchers (e.g., 
PhD), 
x 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,), 
x Associate (A): other clinical site staff involved in the conduct of NCI-sponsored trials, 
and 
x 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: 
 
x Addition to a site roster, 
x Assign the treating, credit, consenting, or drug shipment (IVR only) tasks in OPEN, 
x Act as the site-protocol Principal Investigator (PI) on the IRB approval, and 
x 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. 

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38 
 
 
 
 
4.2. 
Site Registration 
 
This study is supported by the NCI Cancer Trials Support Unit (CTSU). 
 
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 IRB Manager 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). 
 
 
Sites using their local IRB or REB must submit their approval to the CTSU Regulatory Office 
using the Regulatory Submission Portal located in the Regulatory section of the CTSU website. 
Acceptable documentation of local IRB/REB approval includes: 
x Local IRB documentation, 
x IRB-signed CTSU IRB Certification Form, and/or 
x Protocol of Human Subjects Assurance Identification/IRB Certification/Declaration of 
Exemption Form. 
 
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: 
x Holds an Active CTEP status, 
x Rostered at the site on the IRB/REB approval (applies to US and Canadian sites only) and 
on at least one participating roster, 
x If using NCI CIRB, rostered on the NCI CIRB Signatory record, 
x Includes the IRB number of the IRB providing approval in the Form FDA 1572 in the 
RCR profile, and 
x Holds the appropriate CTEP registration type for the protocol. 
 
Additional Requirements 
 
Additional requirements to obtain an approved site registration status include: 
x An active Federalwide Assurance (FWA) number, 
x An active roster affiliation with the Lead Protocol Organization (LPO) or a Participating 
Organization, and 
x Compliance with all protocol-specific requirements (PSRs). 

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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. 
 
x Log on to the CTSU members’ website (https://www.ctsu.org) using your CTEP-IAM 
username and password, 
x 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 / 
Yale University Cancer Center LAO, and protocol number 10334 
x 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 10334 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. 
x 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. 

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40 
 
 
 
To access the Regulatory Submission Portal, log on to the CTSU members’ website Î
Regulatory Î 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 
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. 
 
x Log on to the CTSU members’ website 
x Click on Regulatory at the top of your screen 
x Click on Site Registration 
x 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 

NCI Protocol # 10334  
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41 
 
 
will populate the patient enrollment data in NCI’s clinical data management system, Medidata 
Rave. 
 
Requirements for OPEN access: 
 
x A valid CTEP-IAM account. 
x 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. 
x If a DTL is required for the study, the registrar(s) must hold the OPEN Registrar task on 
the DTL for the site. 
x 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. 
 
Prior to accessing OPEN, site staff should verify the following: 
 
x Patient has met all eligibility criteria within the protocol stated timeframes, and 
x 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. OPEN/IWRS User Requirements 
 
OPEN/IWRS users must meet the following requirements: 
x Have a valid CTEP-IAM account (i.e., CTEP username and password). 
x To enroll patients or request slot reservations: Be on an ETCTN Corresponding or 
Participating Organization roster with the role of Registrar. Registrars must hold a 
minimum of an AP registration type. 
x To approve slot reservations or access cohort management: Be identified to Theradex as 
the “Client Admin” for the study. 
x Have regulatory approval for the conduct of the study at their site. 

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42 
 
 
Prior to accessing OPEN/IWRS, site staff should verify the following: 
x All eligibility criteria have been met within the protocol stated timeframes. 
x If applicable, all patients have signed an appropriate consent form and Health Insurance 
Portability and Accountability Act (HIPAA) authorization form. 
 
4.3.3. Special Instructions for Patient Enrollment 
 
This Study will use the ETCTN Specimen Tracking System (STS). 
 
x All biospecimens collected for this trial must be submitted using the ETCTN Specimen 
Tracking System (STS) unless otherwise noted. 
x 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. 
x Please refer to the Medidata Account Activation and Study Invitation Acceptance link on the 
CTSU website under the Rave/DQP tab. 
x 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 
 
4.3.4. OPEN/IWRS Questions? 
 
Further instructional information on OPEN is provided on the OPEN tab 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. 
 
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 SPECIAL STUDIES 
 
5.1 Summary Table for Specimen Collection 

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43 
 
 
 
Time Point 
Specimen 
Send Specimens To: 
Baseline 
 
x 5mL bone marrow aspirate (mandatory) 
Local CLIA labs 
 
x 10 mL blood in EDTA lavender top vacutainer 
tube (mandatory) 
x 10 mL bone marrow aspirate (mandatory) 
Brent Wood’s lab, Children’s 
Hospital Los Angeles 
 
x 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred Hutchinson 
Cancer Research Center 
 
x 10mL blood in EDTA lavender top vacutainer 
tube (mandatory) 
x 1 Bone marrow core biopsy, processed to 
FFPE block (optional) 
x 30mL blood in sodium Heparin green top 
vacutainer tubes (mandatory) 
x 16 mL bone marrow aspirate in EDTA 
lavender top vacutainer tube (mandatory) 
x Skin punch biopsy, processed to FFPE block 
(mandatory) 
x Stool (OMNIgene-GUT kit) (mandatory) 
EET Biobank 
Post-Cycle 1 
 
x 10 mL blood in EDTA lavender top vacutainer 
tube (mandatory) 
x 10 mL bone marrow aspirate (mandatory) 
Brent Wood’s lab, Children’s 
Hospital Los Angeles 
 
x 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred Hutchinson 
Cancer Research Center 
 
x Stool (OMNIgene-GUT kit) (mandatory) 
EET Biobank 
Post-Cycle 2 
 
x 10 mL blood in EDTA lavender top vacutainer 
tube (mandatory) 
x 10 mL bone marrow aspirate (mandatory) 
Brent Wood’s lab, Children’s 
Hospital Los Angeles 
 
x 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred Hutchinson 
Cancer Research Center 
Post-Cycle 4 
 
x 10 mL blood in EDTA lavender top vacutainer 
tube (mandatory) 
x 10 mL bone marrow aspirate (mandatory) 
Brent Wood’s lab, Children’s 
Hospital Los Angeles 
 
x 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred Hutchinson 
Cancer Research Center 

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44 
 
 
 
 
x 10mL blood in EDTA lavender top vacutainer 
tube (mandatory) 
x 30 mL blood in sodium Heparin green top 
vacutainer tubes (mandatory) 
x 16 mL bone marrow aspirate in EDTA 
lavender top vacutainer tube (mandatory) 
x 1 Bone marrow core biopsy, processed to 
FFPE block (optional) 
EET Biobank 
Post-Cycle 6 
 
x 10 mL blood in EDTA lavender top vacutainer 
tube (mandatory) 
x 10 mL bone marrow aspirate (mandatory) 
Brent Wood’s lab, Children’s 
Hospital Los Angeles 
 
x 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred Hutchinson 
Cancer Research Center 
 
x 10mL blood in EDTA lavender top vacutainer 
tube (mandatory) 
x 30 mL blood in sodium Heparin green top 
vacutainer tubes (mandatory) 
x 16mL bone marrow aspirate in EDTA lavender 
top vacutainer tube (mandatory) 
x 1 Bone marrow core biopsy, processed to 
FFPE block (optional) 
EET Biobank 
Every 3 months during maintenance 
 
x 10 mL blood in EDTA lavender top vacutainer 
tube (optional) 
x 10 mL bone marrow aspirate (optional) 
Brent Wood’s lab, Children’s 
Hospital Los Angeles 
 
x 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred Hutchinson 
Cancer Research Center 
After 1 year of maintenance 
 
x 10 mL blood in EDTA lavender top vacutainer 
tube (mandatory) 
x 10 mL bone marrow aspirate (mandatory) 
Brent Wood’s lab, Children’s 
Hospital Los Angeles 
 
x 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred Hutchinson 
Cancer Research Center 

NCI Protocol # 10334  
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45 
 
 
 
 
x 10mL blood in EDTA lavender top vacutainer 
tube (mandatory) 
x 30 mL blood in sodium Heparin green top 
vacutainer tubes (mandatory) 
x 16 mL bone marrow aspirate in EDTA 
lavender top vacutainer tube (mandatory) 
x 1 Bone marrow core biopsy, processed to 
FFPE block (optional) 
EET Biobank 
Relapse 
 
x 1 Bone marrow core biopsy, processed to 
FFPE block (optional) 
x 10mL blood in EDTA lavender top vacutainer 
tube (mandatory) 
x 30 mL blood in sodium Heparin green top 
vacutainer tubes (mandatory) 
x 16 mL bone marrow aspirate in EDTA 
lavender top vacutainer tube (mandatory) 
EET Biobank 
Progression/End of Treatment 
 
x 10 mL blood in EDTA lavender top vacutainer 
tube (mandatory) 
x 10 mL bone marrow aspirate (mandatory) 
Brent Wood’s lab, Children’s 
Hospital Los Angeles 
 
x 8-10 mL blood in Streck tube (mandatory) 
Radich laboratory, Fred Hutchinson 
Cancer Research Center 
 
x 30 mL blood in Heparin green top vacutainer 
tubes (mandatory) 
x 10mL blood in EDTA lavender top vacutainer 
tube (mandatory) 
x 16 mL bone marrow aspirate in EDTA 
lavender top vacutainer tube (mandatory) 
x 1 Bone marrow core biopsy, processed to 
FFPE block (optional) 
EET Biobank 
Note: 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. 
 
5.2. Specimen Procurement Kits and Scheduling 
 
5.2.1 Specimen Procurement Kits 
 
Kits for the 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 

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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 or jradich@fredhutch.org. 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: 
x 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. 
x Fresh blood and bone marrow specimens may be collected and shipped Monday through 
Friday. 
x Stool samples may be collected any day and may be shipped on Monday through 
Thursday. 
 
 
5.2.3 Scheduling of Specimen Collections for the Brent Wood’s Lab at 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. In any case, please send notification of shipment to lbeppu@fredhutch.org; 

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jradich@fredhutch.org; anne.caldwell@yale.edu, or call the lab at Fred Hutch at 206-667- 
6966. 
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: 
x Protocol Number 
x Investigator Identification 
o Institution and affiliate name 
o Investigator’s name 
x Eligibility Verification: Patients must meet all the eligibility requirements listed in 
Section 3. 
x 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 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. 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. 

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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 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 or bone marrow specimens (including whole blood and frozen, 
processed blood products – like serum and plasma): 
x Patient Study ID
x Universal Patient ID (UPID)
x Specimen ID (automatically generated by Rave)
x Time point
x Specimen type (e.g., blood, serum)
x Collection date and time (to be added by hand)
x 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., cassette): 
x Patient Study ID
x Universal Patient ID (UPID)
x Specimen ID (automatically generated by Rave)
x Time point
x Specimen type (e.g., Bone Marrow Aspirate, etc.)
x BM Core Biopsy Laterality (e.g., Right (R) or Left (L) Side if both submitted) – to be 
added by hand
x Surgical pathology ID (SPID) number (if applicable)
x Collection date
 
5.3.2.3 
Stool Specimen Labels 
Include the following on all tissue specimens or containers (e.g., cassette): 
x Patient Study ID
x Universal Patient ID (UPID)
x Specimen ID (automatically generated by Rave)
x Time point
x Specimen type (e.g., stool etc.)
x 
Collection date

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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, time and laterality.
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
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

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assignments. If specimen analysis is required to determine eligibility, the protocol will be setup 
with multi-step registration. 
 
Registration without eligibility specimen analysis: 
 
1. Site enters registration data into OPEN during one or more steps. 
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 all applicable registration data directly to Rave at the end of the final 
registration step. 
 
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: 
x Specimen Tracking Enrollment CRF: Enter Time Point, Specimen Category, 
Specimen Type, Block number, Tissue type, Surgical Path ID, and 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. 
x 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.
x After collection, store labeled specimens as described in Section 5.3.2.1.
x Apply an extra specimen label to each report before scanning. Return to the Histology 
and Disease form to upload any initial Pathology, Bone Marrow, Radiology, Molecular 
Reports (up to 4), and Surgical (or Operative) reports. Return to Specimen Tracking 
Enrollment CRF to upload any molecular report (one per specimen) and/or specimen 
specific pathology or related report (one per specimen) and/or the Tissue 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, 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. 
x Specimen Transmittal Form: Enter collection date and time and other required 
specimen details.

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Step 4: When ready to ship, enter shipment information. 
x 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.
x 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. 
x Shipping List report is available at the site level.
x Print two copies of the shipping list, one to provide in the box, the other for your own 
records.
x 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. 
x 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 
 
Proper tissue embedding and orientation are necessary in order to support the sample and prevent 
tissue damage or loss during sectioning (as well as preserve diagnostic histological features). 
Improperly embedded tissue (e.g., needle cores) can provide incomplete information if 
diagnostic material is not properly sectioned in a timely manner. Improper orientation of certain 
samples (e.g., skin tumors) can prevent the evaluation of histological features that may affect 
survival or recurrence, like depth of tumor invasion and involvement of surgical margins of 
resection. In order to prevent tissue embedding and orientation errors, refer to the guidelines in 
Appendix F. 
 
 
Pre-Analytical 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. 
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. 

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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 capture the 
chronological order in which the biopsy cores were obtained. 
 
5.4.1 Formalin-fixed paraffin-embedded (FFPE) Bone Marrow Core Biopsy Collection and 
Processing 
 
5.4.1.1 
Collection of Bone Marrow Core Biopsy 
 
Bone marrow core biopsies should be collected as per institutional guidelines and standard 
operating procedures. 
 
5.4.1.2 
Processing of Bone Marrow Core Biopsy 
 
Bone marrow core biopsies should be processed to separate FFPE blocks 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 G) 
4. Sites should follow embedding protocols where the total processing time from 
70% ethanol to block embedding exceeds 4 hours. 
5. Label the FFPE bone marrow biopsy according to instructions in section 5.3.2.2. 
 
 
5.4.2 Bone Marrow Aspirate Collection 
 
5.4.2.1 
Collection of Bone Marrow Aspirate 
1. Label EDTA tubes according to the instructions in section 5.3.2.1. 
2. First Syringe: (mandatory) obtain 10 mL of bone marrow aspirate and aliquot into an 
EDTA tube. 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. 

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3. Second Syringe (optional): obtain 16 mL of bone marrow aspirate and aliquot 8 mL into 
each EDTA tube. Mix the EDTA tubes, by inversion, 8-10 times. This tube will be sent 
to the EET Biobank for immune correlative studies. 
4. Ship on the day of collection. FedEx Priority Overnight preferred for shipments to the 
EET Biobank. 
 
**For baseline bone marrow, obtain an additional 5 mL of bone marrow aspirate and aliquot in 
EDTA tube (to be sent to local CLIA lab) 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 Blood Collection 
 
5.4.3.1 Collection of Blood in EDTA Tubes for Shipment to Brent Wood’s Lab at Children’s 
Hospital in Los Angeles 
 
1. Label EDTA tubes according to the instructions in section 5.3.1. 
2. Collect 10 mL of blood in 2 EDTA tubes, 1 each 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 at Children’s Hospital in Los 
Angeles and the biorepository, 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. 
 
5.4.3.2 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 5.2.4. 
. 
 
5.4.3.3. Collection of Blood in Green Top Sodium Heparin Tubes for Shipment to the EET 
Biobank 
 
1. Label Green Top tubes according to the instructions in section 5.3.2.1 
2. Collect a total of 30 mL of blood in sodium Heparin tubes and gently invert tube 8-10 
times to mix. 

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3. After collection, gently invert tube(s) 8-10 times to ensure adequate mixing of sodium 
heparin. Maintain specimens at ambient temperature (room temperature) during 
collection and transport. 
4. Blood should be shipped ambient FedEx Priority Overnight to the EET Biobank the 
day of collection. 
 
 
5.4.4. 
FFPE Skin Punch Biopsies 
 
1. Label formalin-filled containers according to instructions in Section 5.3.2.2. 
2. Obtain one ≥5mm punch biopsy, and place in cassette. Refer to guidelines in Appendix F 
to prevent tissue embedding and orientation errors. 
3. Snap the cassette lid closed and place cassette into pre-labeled container(s). filled with 
10% neutral buffered formalin as soon as possible after collection to prevent air drying. 
. Samples should be fixed for 24-48 hours. 
4. Process and embed following instructions in Appendix G. 
.Label the FFPE tissue block according to instructions in section 5.3.2.2. 
5. Keep FFPE tissue at room temperature until shipment to the EET Biobank. 
 
 
5.4.5 
Collection of Stool in OMNIgene-GUT Tubes for Shipment to the EET Biobank 
 
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 
5.3.2.3 prior to being provided to the patient. 
2. Baseline and post Cycle 1 timepoints will use OMNIgene GUT kits (OMR-200.100—shipped 
ambient) which include a DNA stabilizing solution. 
3. Collection kits will include directions for specimen collection and shipment as well as 
collection and shipping supplies. 
4. The patient will collect the stool sample and return the sample to 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. 
5. The stool sample and a shipping manifest should be shipped on day of collection (whenever 
possible) according to instructions provided in the kit and in section 5.6.2. 
 
 
5.5 Shipping Specimens from Clinical Site to the EET Biobank. 
 
General Shipping Information 
 
Fresh bone marrow aspirate and fresh blood should be shipped ambient. Formalin-fixed 
paraffin-embedded (FFPE) tissue, including bone marrow biopsy and skin punch specimens are 
also shipped at ambient temperature. In winter months, please include extra insulation, such as 
bubble wrap, around the specimen(s) inside the shipping container. 

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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. 
Tissue 
Required Forms 
Bone Marrow Aspirate or 
Biopsy 
1. Shipping List 
2. Corresponding Bone Marrow Report 
Other (blood, blood product, 
stool, etc.) 
1. Shipping List 
 
 
Specimen Shipping Instructions 
 
FFPE tissue may be shipped on Monday through Thursday. 
 
Fresh blood or bone marrow aspirate may be shipped on Monday through Friday. Please select 
“Saturday Delivery” when shipping fresh blood on a Friday. 
 
Shipping FFPE Blocks in Your Own Container 
 
1. Before packaging blocks, verify that each specimen is labeled according to Section 
5.3.2.2. 
2. Blocks should be placed in a hard-sided container, preferably a special block holder, to 
protect the specimen. 
3. Place the blocks in a reinforced cardboard shipping box with appropriate packaging filler 
to minimize movement of specimens within the shipping box. 
4. Include a copy of the forms listed above and a shipping manifest from the Specimen 
Tracking System with each shipment. 
5. Please include a cold pack when shipping on hot days and extra insulation on cold days. 
6. Ship specimens to the address listed below. FedEx Priority Overnight is strongly 
recommended to prevent delays in package receipt. 
 
Shipping Blood and Bone Marrow in Your Own Container 
 
1 Before packaging specimens, verify that the collection tube is labeled according to 
instructions in section 5.3.2. 
2 Place the blood collection tube into a zip-lock bag. 
3 Place zip-lock bag 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 into a sturdy shipping 
container. In winter months, please use an insulated container and include extra 

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insulation, such as bubble wrap, inside the shipping container to prevent specimens from freezing. 
6 Close the container 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. 
 
Shipping Stool in an Ambient Shipper 
 
The clinical site will ship each specimen to the 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 samples are provided in the kit. 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. 
 
Contact Information for Assistance 
 

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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 Medicine, Children’s Hospital, Los Angeles 
 
5.6.1. Shipping of Specimens to Brent Wood’s Lab, Department of Pathology and Laboratory 
Medicine, 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. 
2. Specimen must be placed in a leak proof vacutainer. 
3. Multiple fragile vacutainers must be individually wrapped or separated to prevent contact. 
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 CHLA Department of Pathology and Laboratory Medicine is open Monday-Saturday 8:00- 4:30. As the hospital 
loading dock is closed on Saturdays, for Saturday delivery to be successful, please: 
1. Add on shipping label: "Friday shipping for Saturday delivery. Deliver to Front Desk, 
Children’s Hospital Los Angeles." 
2. Notify the CHLA laboratory at plmclinicalimmunologylaboratory@chla.usc; bwood@chla.usc.edu 
and cc amer.zeidan@yale.edu; anne.caldwell@yale.edu. 
3. Provide CHLA the FedEx tracking number and subject number so the lab can track the sample shipment. 

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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 2-290Los Angeles, CA 90027 Phone: (877) 543-9522 
 
An STS Shipping Manifest Form must be generated and shipped with all specimen submissions. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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5.7. 
Contact Information for AssistanceBiomarker 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 
Immunofluorescence 
Multiplex IF 
CLIA: N 
Exploratory 
Correlate with 
clinical 
response 
FFPE Bone 
Marrow Core 
Biopsy 
Baseline 
Post cycle 4 
Post cycle 6 
After 1 year 
Relapse 
End of 
treatment/progression 
O 
CIMAC 
Ignacio Wistuba 
iiwistuba@mdanderson.org 
Bone Marrow Aspirate Biomarkers 
1 
FLT3 mutations 
FLT3 PCR 
or NGS 
Panel 
CLIA: Y 
Integral 
 
Stratification 
factor 
Bone marrow 
aspirate 
Baseline 
M 
Local CLIA labs 
2 
Karyotype 
Conventional 
cytogenetics 
and AML 
FISH 
translocation 
panel 
CLIA: Y 
Integral 
Stratification 
factor 
Bone marrow 
aspirate 
Baseline 
M 
Local CLIA labs 

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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 
CLIA: Y 
Integral 
Primary 
endpoint 
Bone marrow 
aspirate 
Baseline 
Post-cycle 1 
Post-cycle 2 
Post-cycle 4 
Post-cycle 6 
Every 3 months during 
maintenance 
After 1 year 
End of 
treatment/progression 
M (O during 
maintenance) 
Brent Wood’s Lab, 
Children’s Hospital, Los 
Angeles 
4 
MRD Status 
Duplex 
sequencing 
CLIA: N 
Exploratory 
Exploratory 
endpoint 
Bone marrow 
aspirate 
(same as #3) 
Baseline 
Post-cycle 1 
Post-cycle 2 
Post-cycle 4 
Post-cycle 6 
Every 3 months during 
maintenance 
After 1 year 
End of 
treatment/progression 
M (O during 
maintenance 
phase) 
Fred Hutchinson Cancer 
Research Center 
(shipped from Brent 
Wood’s lab) 
Jerald Radich 
jradich@fhcrc.org 
5 
Immune cell subset 
analysis 
CyTOF 
CLIA: N 
Exploratory 
 
Correlate with 
clinical 
response 
Bone marrow 
aspirate in 
EDTA 
(mononuclear 
cells) 
Baseline 
Post-cycle 4 
Post-cycle 6 
After 1 year 
Relapse 
End of 
treatment/progression 
O 
CIMAC 
Ignacio Wistuba 
iiwistuba@mdanderson.org 
6 
Olink (cytokine 
panel) 
Olink 
CLIA: N 
Exploratory 
 
Correlate with 
clinical 
response 
Bone marrow 
aspirate in 
EDTA 
(plasma) 
Baseline 
Post-cycle 4 
Post-cycle 6 
After 1 year 
Relapse 
End of 
treatment/progression 
O 
CIMAC 
Sacha Gnjatic 
Sacha.gnjatic@mssm.edu 

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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 
7 
Whole exome 
sequencing 
WES 
(tumor) 
CLIA: N 
Exploratory 
Correlate with 
clinical 
response 
DNA from 
marrow 
aspirate 
(EDTA) 
Baseline 
Relapse 
End of 
treatment/progression 
O 
CIMAC 
Ignacio Wistuba 
iiwistuba@mdanderson.org 
WES 
(germline) 
CLIA:N 
Exploratory 
 
Correlate with 
clinical 
response 
DNA from 
FFPE skin 
punch 
biopsies 
Baseline 
O 
CIMAC 
Ignacio Wistuba 
iiwistuba@mdanderson.org 
8 
RNA-Seq 
RNA-Seq 
CLIA: N 
Exploratory 
 
Correlate with 
clinical 
response 
RNA from 
bone marrow 
aspirate in 
EDTA 
Baseline 
Relapse 
End of 
treatment/progression 
O 
CIMAC 
Ignacio Wistuba 
iiwistuba@mdanderson.org 
9 
TCR-seq 
TCR-Seq 
CLIA: N 
Exploratory 
 
Correlate with 
clinical 
response 
DNA from 
Bone marrow 
aspirate in 
EDTA 
Baseline 
Post-cycle 4 
Post-cycle 6 
After 1 year 
Relapse 
End of 
treatment/progression 
O 
CIMAC working with 
Adaptive 
Ignacio Wistuba 
iiwistuba@mdanderson.org 
10 
Leukemia-specific 
T cell response 
Flow 
cytometry 
 
CLIA: N 
Exploratory 
 
Correlate with 
clinical 
response 
Mononuclear 
cells from 
bone marrow 
aspirate in 
EDTA tubes 
Baseline 
Post-cycle 4 
Post-cycle 6 
After 1 year 
Relapse 
End of 
treatment/progression 
O 
MD Anderson Cancer 
Center (Non-CIMAC) 
Gheath Al-Atrash 
galatras@mdanderson.org 
11 
DNA methylation 
assay 
Microarray 
CLIA: N 
Exploratory 
 
Correlate with 
clinical 
response 
Mononuclear 
cells from 
Bone marrow 
aspirate in 
EDTA tubes 
Baseline 
Post-cycle 4 
Post-cycle 6 
After 1 year 
Relapse 
End of 
treatment/progression 
O 
MD Anderson Cancer 
Center (Non-CIMAC) 
Gheath Al-Atrash 
galatras@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 
Blood-based Biomarkers 
1 
MRD Status 
Duplex 
sequencing 
CLIA: N 
Exploratory 
Exploratory 
endpoint 
Peripheral 
blood in 
EDTA tubes 
Baseline 
Post-cycle 1 
Post-cycle 2 
Post-cycle 4 
Post-cycle 6 
Every 3 months during 
maintenance 
After 1 year 
End of 
treatment/progression 
M (O during 
Maintenance) 
Fred Hutchinson Cancer 
Research Center 
(shipped from Brent 
Wood’s lab) 
Jerald Radich 
jradich@fhcrc.org 
2 
Immune cell subset 
analysis 
CyTOF 
CLIA: N 
Exploratory 
 
Correlate with 
clinical 
response 
PBMCs from 
blood 
collected in 
sodium 
heparin tubes 
Baseline 
Post-cycle 4 
Post-cycle 6 
After 1 year 
Relapse 
End of 
treatment/progression 
M 
CIMAC 
Ignacio Wistuba 
iiwistuba@mdanderson.org 
3 
Olink (cytokine 
panel) 
Olink 
CLIA: N 
Exploratory 
 
Correlate with 
clinical 
response 
Plasma (from 
blood 
collected in 
sodium 
heparin 
tubes) 
Baseline 
Post-cycle 4 
Post-cycle 6 
After 1 year 
Relapse 
End of 
treatment/progression 
M 
CIMAC 
Sacha Gnjatic 
Sacha.gnjatic@mssm.edu 
4 
TCR-seq 
TCR-Seq 
 
CLIA: N 
Exploratory 
 
Correlate with 
clinical response 
DNA from 
PBMCs from 
blood 
collected in 
EDTA or 
sodium 
heparin tubes 
Baseline 
Post-cycle 4 
Post-cycle 6 
After 1 year 
Relapse 
End of 
treatment/progression 
M 
CIMAC working with 
Adaptive 
Ignacio Wistuba 
iiwistuba@mdanderson.org 

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4 
Leukemia-specific 
T cell response 
Flow 
cytometry 
CLIA: N 
Exploratory 
 
Correlate with 
clinical 
response 
Mononuclear 
cells from 
Peripheral 
blood in 
EDTA tubes 
Baseline 
Post-cycle 4 
Post-cycle 6 
After 1 year 
Relapse 
End of 
treatment/progression 
M 
MD Anderson Cancer 
Center (Non-CIMAC) 
Gheath Al-Atrash 
galatras@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 
5 
DNA methylation 
assay 
Microarray 
CLIA: N 
Exploratory 
Correlate with 
clinical 
response 
Mononuclear 
cells from 
Peripheral 
blood in 
EDTA tubes 
Baseline 
Post-cycle 4 
Post-cycle 6 
After 1 year 
Relapse 
End of 
treatment/progression 
M 
MD Anderson Cancer 
Center (Non-CIMAC) 
Gheath Al-Atrash 
galatras@mdanderson.org 
6. 
cfDNA 
Duplex 
Sequencing 
(DS) 
CLIA: N 
Exploratory 
Exploratory 
Endpoint 
Peripheral 
blood 
(Streck) 
Baseline 
Post-cycle 1 
Post-cycle 2 
Post-cycle 4 
Post-cycle 6 
Every 3 months during 
maintenance 
After 1 year 
End of 
treatment/progression 
M 
To be shipped to Radich 
laboratory, Fred 
Hutchinson Cancer 
Research Center. 
Molecular Oncology 
Laboratory at Fred 
Hutchinson Cancer 
Research Center 
Jerald Radich 
jradich@fhcrc.org 
Stool-based biomarkers 
1 
Gut microbiome 
characterization 
16sRNA V4 
region 
sequencing of 
bacterial 
genomic DNA 
x Exploratory 
Correlate with 
clinical response 
Stool from 
OMNIgene 
GUT sample 
collection kit 
x Baseline 
x Post cycle 1 
M 
MDACC 
CIMAC 
Gheath Al-Atrash 
galatras@mdanderson.org 
* CyTOF, WES, RNAseq, TCR and Olink analyses in bone marrow will be performed using cells and plasma from a single BM 
aspirate. 

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5.8. 
Integral Laboratory Studies 
 
5.8.1 FLT3 mutations 
 
5.8.1.1 
Specimen Receipt and Processing at Local Labs 
 
Samples should be received and processed as per standard operating procedures. 
 
5.8.1.2 
Site Performing Correlative Study 
This study will be conducted by local labs. 
5.8.2 Karyotype/FISH 
 
5.8.2.1 
Specimen Receipt and Processing at Local Labs 
 
Samples should be received and processed as per standard operating procedures. 
 
5.8.2.2 
Site Performing Correlative Study 
This study will be conducted by local labs. 
5.8.3 MRD Status by Multicolor Flow Cytometry 
 
5.8.3.1 Specimen Receipt and Processing at Brent Wood’s Lab, Children’s Hospital, Los 
Angeles 
Bone marrow aspirate will be processed as per laboratory standard operating procedures. 
 
5.8.3.2 
Site(s) Performing Correlative Study 
 
The study will be performed in the laboratory of Dr. Brent Wood, Children’s Hospital, Los 
Angeles 
 
5.9 
Exploratory Correlative Studies 
 
5.9.1 Multiplex Immunofloresence: 
 
5.9.1.1 Specimen(s) Receipt and Processing at the EET Biobank 
 
The EET Biobank will receive and barcode FFPE bone marrow biopsy blocks, and will store 
them at room temperature until distribution. The EET Biobank will ship the FFPE bone biopsy 
core tissue from all time points to MDACC CIMAC. 
 
UT MD Anderson Cancer Center  
Life Science Plaza Building  
2130 W. Holcombe Blvd, LSP9.3003  
Houston, TX 77030 

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c/o Mei Jiang 
Mjiang1@mdanderson.org 
Phone: 713-563-0373 
 
5.9.1.2 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). 
 
5.9.2 RNA-Seq 
 
5.9.2.1 Specimen(s) Receipt and Processing at the EET Biobank 
 
Upon receiving the bone marrow aspirate in EDTA tubes, 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 slow- 
frozen in freezing media and stored in a liquid nitrogen vapor phase freezer.  
 
DNA and RNA will be co-extracted from bone marrow mononuclear cell. An aliquot of RNA 
from the bone marrow mononuclear cell will be used for this assay. 
The EET Biobank will ship this sample to MDACC CIMAC. 
 
UT MD Anderson Cancer Center 
Room 4SCR5.2085 
1881 East Rd. 
Houston, TX 77054 
c/o Habibul Islam 
MHIslam@mdanderson.org 
Phone: 713-794-1068 
 
 
5.9.2.2 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) (shipping address above). 
 
 
 
5.9.3 MRD Status by Duplex Sequencing 

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5.9.3.1 Specimen Receipt and Processing at Brent Wood’s Lab, Department of Pathology and 
Laboratory Medicine Children’s Hospital Los Angeles 
 
Bone marrow aspirate and blood 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. Jerald Radich at the Fred Hutchinson 
Cancer Research Center. 
 
5.9.4 Immune cell subset analysis by CyTOF 
 
5.9.4.1 Specimen(s) Receipt and Processing at the EET Biobank 
 
The Biorepository 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 slow- 
frozen in freezing media and stored in a liquid nitrogen vapor phase freezer. 
 
The EET Biobank will ship this sample to MDACC CIMAC.  
 
UT MD Anderson Cancer Center  
Room 2SCR2.3219 
7435 Fannin St 
Houston, TX 77054 
713-745-6999 
c/o Karen Millerchip 
kamillerchip@mdanderson.org 
 
 
 
 
5.9.4.2 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). 
 
5.9.5 O-link Cytokine panel 
 
5.9.5.1 Specimen(s) Receipt and Processing at the EET Biobank 
 
The Biorepository 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 

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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 slow- 
frozen in freezing media and stored in a liquid nitrogen vapor phase freezer. 
The EET Biobank will ship this frozen plasma to the Mt. Sinai CIMAC. 
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 
 
 
5.9.5.2 
Site(s) Performing Correlative Study 
 
This study will be performed at the Mount Sinai CIMAC by Sacha Gnajtic. Email: Sacha. 
gnjatic@mssm.edu 
 
5.9.6 Whole exome sequencing 
 
5.9.6.1 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 slow-frozen in 
freezing media and stored in a liquid nitrogen vapor phase freezer. 
 
DNA and RNA will be co-extracted from bone marrow mononuclear cells. An aliquot of DNA 
will be used for this assay.  
 
Germline DNA will be extracted from skin punch tissue. 
 
The EET Biobank will ship this sample to MDACC CIMAC.  
 
UT MD Anderson Cancer Center 
Room 4SCR5.2085 
1881 East Rd. 
Houston, TX 77054 
c/o Habibul Islam 
MHIslam@mdanderson.org 
Phone: 713-794-1068 
 
5.9.6.2 Specimen(s) Receipt and Processing at the EET Biobank for FFPE skin punch biopsies 

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Upon receiving of the skin punch biopsies from the collection site, the EET Biobank will process 
according to section 5.4.4 Germline DNA will be extracted from FFPE skin punch biopsies. 
 
 
The EET Biobank will ship this sample to MDACC CIMAC. 
 
 
UT MD Anderson Cancer Center 
Room 4SCR5.2085 
1881 East Rd. 
Houston, TX 77054 
c/o Habibul Islam 
MHIslam@mdanderson.org 
Phone: 713-794-1068 
 
5.9.6.3 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). 
 
5.9.7 TCR-Seq 
 
5.9.7.1 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.  
 
Additionally,  PBMCs from peripheral blood samples collected in EDTA or heparin tubes will 
also be used for TCRseq. 
 
DNA and RNA 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). 
. 
The EET Biobank will ship the samples to Adaptive Biotechnologies, who will work with MDACC 
CIMAC.  
 
 
 
 
 
5.9.7.2 Site(s) Performing Correlative Study 
 
This assay will be performed at Adaptive Biotechnologies, which will work with MDACC 
CIMAC (PI: Dr. Ignacio I Wistuba, Email: iiwistuba@mdanderson.org). 

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5.9.8 Leukemia-Specific T cell response 
 
5.9.8.1 Specimen(s) Receipt and Processing at the EET Biobank 
Bone marrow aspirate and blood will be processed as per laboratory standard operating 
procedures. The Biorepository will receive fresh BM aspirate collected in EDTA tubes and blood 
samples collected in EDTA 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 slow-frozen in freezing media and stored in a liquid nitrogen vapor phase freezer. 
The EET Biobank will ship this sample to MD Anderson Cancer Center.  
 
Attn: Lisa St. John, PhD 
MD Anderson Cancer Center 
Hematopoietic Biology and Malignancy 
2SCR4.2215 
7435 Fannin St. 
Houston, TX 77054 
 
5.9.8.2 Site(s) Performing Correlative Study 
 
This assay will be performed at MD Anderson Cancer Center in the laboratory of Gheath Al- 
Atrash (Email: galatras@mdanderson.org) 
 
5.9.9 DNA methylation assays 
 
5.9.9.1 Specimen(s) Receipt and Processing at the EET Biobank 
 
Bone marrow aspirate and blood will be processed as per laboratory standard operating 
procedures. The Biorepository will receive fresh BM aspirate collected in EDTA tubes and blood 
samples collected in the EDTA 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 
slow-frozen in freezing media and be stored in a liquid nitrogen vapor phase freezer. 
The EET Biobank will ship this sample to MD Anderson Cancer Center. 
Attn: Beatriz Sanchez-Espiridion 
Institutional Tissue Bank (ITB) 
1515 Holcombe Blvd, Rm G1.3586 
Houston, TX 7703 
Phone: 713-745-7047 
Email: bsanchez2@mdanderson.org 

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5.9.9.2 Site(s) Performing Correlative Study 
 
This assay will be performed at MD Anderson Cancer Center in the laboratory of Gheath Al- 
Atrash (Email: galatras@mdanderson.org) 
 
5.9.10 Gut microbiome 
 
5.9.10.1 Specimen(s) receipt and processing at MDACC 
 
Upon receipt of the sample in the OMNIgene-Gut kits, the stool sample will be processed as per 
laboratory standard operating procedures. Stool aliquots will be stored in a -80°C freezer until 
distribution. 
 
The EET Biobank will ship this sample to the MDACC CIMAC lab.  
 
UT 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 
CChang2@mdanderson.org   
 
 
 
 
5.9.10.2. Site(s) Performing Correlative Study 
 
The microbiome assay will be performed at MDACC CIMAC in the laboratory of Gheath Al- 
Atrash (Email: galatras@mdanderson.org. 
 
 
6. TREATMENT PLAN 
 
 
6.1 Agent Administration 
 
Treatment will be administered on an inpatient or outpatient basis. Reported adverse events 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. 
 
6.1.1 Induction therapy phase 
Eligible patients will be randomized before proceeding to AZA + VEN or within one week of 
starting AZA + VEN in 1:1 ratio to AZA + VEN alone or AZA + VEN + pembrolizumab. As 
shipment of pembrolizumab to site can take up to 4 business days and can only be ordered after 
randomization, every effort should be made to randomize the patient as soon as possible. If patient is 
randomized after initiation of Aza+Ven therapy, the patient should continue to meet all eligibility 

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criteria up to randomization date. Planned stratification factors include 1) cytogenetics 
(intermediate/unknown vs. adverse karyotype) 2) antecedent hematologic disorder defined as 
prior MDS, MPN, or aplastic anemia (present vs. absent); and 3) reason to not receive intensive 
chemotherapy (ineligibility vs refusal). All patients will receive azacitidine at 75 mg/m2 IV over 
10-40 minutes or SQ x 7 days on Days 1-7 or Days 5-2-2 (to avoid weekend administration) and, 
including a ramp-up phase to 400 mg /day venetoclax on Days 1-4. Venetoclax will be 
administered at 400 mg/day (or as dose adjusted for concomitant therapy) on the remaining days 
of the cycle for the first cycle and on Days 1-21 or 1-28 for subsequent cycles (depending on 
count recovery). Half of the patients will receive pembrolizumab intravenously starting on Day 
8 of the first cycle and then q 3 weeks through cycles 2-6 (intervention arm). The cycles of 
pembrolizumab run independently from aza+ve therapy. The other half will not receive 
pembrolizumab (control arm). At time of count recovery (approximately 28-42 days following 
administration of AZA+VEN), patients will undergo a bone marrow aspirate/biopsy in order to 
assess for response and MRD. In addition, following count recovery after Cycles 2, 4, and 6, a 
bone marrow biopsy will be performed to assess for response and MRD status. Patients will be 
allowed to continue on study as long as the investigator believes they are deriving clinical benefit 
and not experiencing prohibitive toxicity. 
 
Induction Therapy Regimen Description 
Agent 
Premedications 
; Precautions 
Dose* 
Route# 
Schedule 
Cycle 
Length 
Pembrolizumab (for 
intervention arm 
only) 
Premedication 
with antipyretic 
and 
antihistamine 
may be 
considered. No 
premedication 
with steroids 
200 mg 
See 
Section 
8.1.1. 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. 
Day 8 
(Q3W ±3 
days) 
continuous 
from Day 8 
of 
induction 
28 days 

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Induction Therapy Regimen Description 
Agent 
Premedications 
; Precautions 
Dose* 
Route# 
Schedule 
Cycle 
Length 
Azacitidine 
IV formulation 
is incompatible 
with 5% 
dextrose, 
Hespan, or 
bicarbonate 
solutions. 
Patients should 
be pre- 
medicated for 
nausea and 
vomiting. 
75 mg/m2 
IV over 10-40 
minutes or SQ 
Refer to Section 
8.2.1 for 
additional 
information. 
Days 1-7 or 
days 5-2-2 
Venetoclax 
None. 
400 mg 
(refer to 
ramp up 
dosing 
scheme in 
Table 
below) 
Refer to 
Section 
8.2.2 for 
additional 
informatio 
n. 
P.O. 
Days 1-28 
for Cycle 1, 
Days 1-21 
or 1-28 for 
subsequent 
Cycles 
*Dose rounding per institutional guidelines is allowed. 
#Order should be pembrolizumab, followed by azacitidine, then venetoclax on days when 
multiple medications are given. 
 
The patient will be requested to maintain a medication diary of each dose of venetoclax. The 
medication diary will be returned to clinic staff at the end of each course (see Appendix E). 
 
The following ramp-up scheme will be utilized for venetoclax: 
 
Administer the venetoclax dose according to a daily ramp-up schedule over 4 days to the 
recommended daily dose of 400 mg as shown in Dosing Scheme for the Ramp-up Phase below. 
Instruct patients to take venetoclax tablets with a meal and water at approximately the same time 
each day. Venetoclax tablets should be swallowed whole and not chewed, crushed, or broken 
prior to swallowing. 

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Dosing Scheme for Ramp-Up Phase 
Day 
Venetoclax 
Daily Dose 
1 
100 mg 
2 
200 mg 
3 
400 mg 
4 and beyond 
400 mg 
 
The Starting Pack provides the first 4 days of Venetoclax according to the ramp-up schedule. 
 
Patients treated with Venetoclax may develop tumor lysis syndrome. Refer to the appropriate section 
below for specific details on management. Assess patient-specific factors for level of risk of tumor 
lysis syndrome (TLS) and provide prophylactic hydration and anti-hyperuricemics to patients prior to 
first dose of Venetoclax to reduce risk of TLS as clinically indicated. 
 
Acute Myeloid Leukemia 
x 
All patients should have white blood cell count less than 25 × 10
9/L prior to initiation of 
Venetoclax. Cytoreduction prior to treatment may be required. 
x 
Prior to first Venetoclax dose, provide all patients with prophylactic measures including 
adequate hydration and anti-hyperuricemic agents and continue during ramp-up phase as 
clinically indicated. 
x 
Assess blood chemistry (potassium, uric acid, phosphorus, calcium, and creatinine) and 
correct pre-existing abnormalities prior to initiation of treatment with Venetoclax. 
x 
Monitor blood chemistries for TLS at pre-dose, daily with each new dose during ramp-up and 
24 hours after reaching final dose. 
x 
For patients with risk factors for TLS (e.g., circulating blasts, high burden of leukemia 
involvement in bone marrow, elevated pretreatment lactate dehydrogenase (LDH) levels, or 
reduced renal function) additional measures should be considered, including hospitalization, 
hydration, rasburicase, more frequent monitoring of TLS labs, and other measures as deemed 
necessary by treating physician. 
 
6.1.2. Maintenance therapy phase 
 
The first 6 cycles of azacitidine and venetoclax constitute induction phase for purposes of this 
protocol. After induction phase is completed, it is planned that an additional 2 years, up to a 
maximum of 36 doses of pembrolizumab will be administered for patients in response from this 
regimen during the maintenance phase. For patients on the control arm, treatment with 
azacitidine and venetoclax may continue for 2 years after the 6 cycles (up to additional 24 
cycles) during the maintenance phase. No dose ramp-up is required during the maintenance 
therapy phase. Patients whose best response to therapy after 6 cycles is stable disease can only 
remain on study therapy if they derive clinical benefit from therapy that is defined by transfusion 
independence (defined as at least 8 consecutive weeks without transfusions of either red blood 
cells or platelets for patients who were receiving transfusions prior to or during therapy) or by 
hematologic improvement criteria listed in section 12.2.5. of the protocol. 

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Continuation of azacitidine and venetoclax for patients completing 24 cycles of therapy may be 
continued for patients in response or with stable disease at discretion of treating investigator. 
Patients who do not remain in clinical response will be taken off treatment/study. Patients in 
response will proceed with AZA + VEN with pembrolizumab (intervention arm) vs. AZA + 
VEN (control arm) every Q3W dosing schedule for up to 2 years. Bone marrow samples will be 
obtained every 3 months while patients are receiving maintenance to assess for MRD and 
perform correlative studies (optional during maintenance). Patients, who are deemed to require 
HSCT, will forgo any remaining protocol-defined 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 graft versus host disease (GVHD). 
 
Maintenance Regimen Description 
Agent 
Premedications 
; Precautions 
Dose* 
Route# 
Schedule 
Cycle 
Length 
Pembrolizumab (for 
intervention arm 
only) 
Premedication 
with antipyretic 
and 
antihistamine 
may be 
considered. No 
premedication 
with steroids 
200 mg 
See 
Section 
8.1.1 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 
28 days 
Azacitidine 
IV formulation 
is incompatible 
with 5% 
dextrose, 
Hespan, or 
bicarbonate 
solutions. 
Patients should 
be pre- 
medicated for 
nausea and 
vomiting. 
75 mg/m2 
Refer to 
Section 
8.2.1 for 
additional 
informatio 
n. 
IV over 10-40 
minutes or SQ 
Days 1-7 or 
days 5-2-2 
(to avoid 
weekend 
administrat 
ion) 

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Maintenance Regimen Description 
Agent 
Premedications 
; Precautions 
Dose* 
Route# 
Schedule 
Cycle 
Length 
Venetoclax 
None. 
400 mg 
(refer to 
ramp up 
dosing 
scheme) 
Refer to 
Section 
8.2.2 for 
additional 
informatio 
n. 
P.O. 
Days 1-21 
or 1-28 
dosing 
(depending 
count 
recovery) 
for all 
cycles. 
*Dose rounding per institutional guidelines is allowed. 
#Order should be pembrolizumab first, followed by azacitidine at least an hour later on days 
when both medications are given. 
 
6.1.3. CTEP IND Agent 
 
Trial treatment of Pembrolizumab will be administered Day 8 of the induction phase 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 
 
6.2. 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. 
 
6.2.1. Pembrolizumab 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 

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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 the patient. 
 
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: 
 
 
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. 
 
Anti-leukemia investigational 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 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 

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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. 
 
6.2.2. 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. 
 
6.2.3. Venetoclax Concomitant Medications 
 
Venetoclax is predominantly metabolized by CYP3A4 in vitro, thus CYP3A4 inhibitors or 
inducers are expected to cause changes in venetoclax exposures (Venetoclax Investigator's 
Brochure, 2018). Clinical studies have supported the in vitro observations for venetoclax as a 
sensitive substrate of CYP3A4: >5-fold increase in AUC when co-dosed with ketoconazole, and 
71% decrease in AUC when co-dosed with rifampin. Venetoclax is a substrate for the efflux 
transporters P-gp and BCRP, and inhibitors or inducers of these transporters are expected to 
cause change in the exposure of venetoclax. Venetoclax is a P-gp and BCRP inhibitor and may 
interact with substrates for these transporters. Venetoclax may inhibit OATP1B1 and cause 
weak interaction with drugs that are substrates of this transporter. Refer to the venetoclax label 
for dose adjustment concerning concurrent medications. 
 
6.2.3.1. Excluded Concomitant Medications and Procedures 
CYP3A inhibitors and inducers are allowed on study as clinically indicated and per standard 
institutional uses but their use including dose adjustment of venetoclax and other drugs should 
follow the FDA drug label for venetoclax 
 
 
6.3. Duration of Therapy 

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In the absence of treatment delays due to adverse event(s), treatment may continue for up to 24 
cycles or 2 years of maintenance therapy or until one of the following criteria applies: 
 
x 
Disease progression 
x 
Once patient proceeds to HSCT 
x 
Intercurrent illness that prevents further administration of treatment 
x 
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 Principal Investigator must be 
consulted. 
o Tumor assessments should continue as per protocol even if dosing is interrupted. 
x 
Unacceptable AE(s) 
x 
Patient decides to withdraw from the study 
x 
General or specific changes in the patient's condition render the patient unacceptable 
for further treatment in the judgment of the investigator 
x 
Clinical progression 
x 
Severe, significant and persistent Patient non-compliance 
x 
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. 
x 
Termination of the study by sponsor 
x 
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 
 
6.4. Duration of Follow-Up 
 
Patients will be followed in clinic or phone call for at least every 6 months for up to 3 years after 
treatment or until death, whichever occurs first. Patients removed from study for unacceptable 
AE(s) will be followed until resolution or stabilization of the AE. 
 
6.5. 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: 

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x Patients may resume treatment in the presence of grade 2 fatigue. 
x 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. 
x Patients with combined grade 2 AST/ALT and total bilirubin values meeting study 
parameters outlined in Section 7.1 which are attributed to study therapy should have 
treatment permanently discontinued. 
x Non-drug-related toxicity including hepatic, pulmonary toxicity, diarrhea, or colitis, must 
have resolved to baseline before treatment is resumed. 
x 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 may be allowed to continue on study on 
case-by-case basis based on consultation with study chair and CTEP 
 
 
6.6. Contraception and Pregnancy 
 
6.6.1 Contraception 
 
Pembrolizumab may have adverse effects on a fetus in utero. Furthermore, it is not known if 
Pembrolizumab has transient adverse effects on the composition of sperm. 
 
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: 
 
x 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 

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x Have had a hysterectomy and/or bilateral oophorectomy, bilateral salpingectomy or 
bilateral tubal ligation/occlusion, at least 6 weeks prior to screening; 
OR 
x 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: 
 
x Practice abstinence† from heterosexual activity; 
OR 
x Use (or have their partner use) acceptable contraception during heterosexual activity. 
 
Acceptable methods of contraception are‡: 
 
Single method (1 of the following is acceptable): 
x intrauterine device (IUD) 
x vasectomy of a female patient’s male partner 
x contraceptive rod implanted into the skin 
Combination method (requires use of 2 of the following): 
x diaphragm with spermicide (cannot be used in conjunction with cervical 
cap/spermicide) 
x cervical cap with spermicide (nulliparous women only) 
x contraceptive sponge (nulliparous women only) 
x male condom or female condom (cannot be used together) 
x 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 
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 

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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. 
 
6.6.2 Use in Pregnancy 
 
If a patient inadvertently becomes pregnant while on treatment with Pembrolizumab, 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. 
 
6.6.3 Use in Nursing Women 
 
It is unknown whether Pembrolizumab 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. 
 
6.7 Treatment Up to 2 Years 
 
Maintenance treatment will continue for up to 2 years (up to 24 cycles from start of maintenance, 
and for those randomized to intervention arm, maximum of 36 doses of Pembrolizumab (MK- 
3475), 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. 
 
7. DOSING DELAYS/DOSE MODIFICATIONS 
 
Dosing delays and dose modifications for therapy should follow institutional guidelines and/or 
treating physicians opinion. 
 
7.1 Pembrolizumab (MK-3475) 
 
7.1.1 Immune-related AEs 
 
Immune-related AEs (irAEs), defined as AEs of unknown etiology, associated with drug 
exposure and consistent with an immune phenomenon, may be predicted based on the nature of 
the pembrolizumab compound, its mechanism of action, and reported experience with 
immunotherapies that have a similar mechanism of action. Special attention should be paid to 
AEs that may be suggestive of potential irAEs. An irAE can occur shortly after the first dose or 
several months after the last dose of treatment. All AEs of unknown etiology associated with 
drug exposure should be evaluated to determine if they are possibly immune-related. If an irAE 

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is suspected, efforts should be made to rule out neoplastic, infectious, metabolic, toxin or other 
etiologic causes prior to labeling an AE as an irAE. 
 
The table below includes guidelines for managing irAEs. 
 
General Dose Modification Guidelines for Drug-Related Immune-Related Adverse Events 
 
irAE 
Withhold/Discontinue 
pembrolizumab (MK-3475)? 
Supportive Care 
Grade 1 
No action. 
Provide symptomatic treatment. 
Grade 2 
May withhold pembrolizumab 
Consider systemic corticosteroids in addition to 
appropriate symptomatic treatment. 
Grade 3 and 
Grade 4 
Withhold pembrolizumab 
Discontinue if unable to reduce 
corticosteroid dose to <10 mg per 
day prednisone equivalent within 
12 weeks of toxicity. 
Systemic corticosteroids are indicated in addition to 
appropriate symptomatic treatment. May utilize 1 to 2 
mg/kg prednisone or equivalent per day. 
Steroid taper should be considered once symptoms 
improve to grade 1 or less and tapered over at least 4 
weeks. 
 
7.1.2 Non-immune-related Adverse Events 
 
Additionally, pembrolizumab (MK-3475) will be withheld for other drug- 
related grade 4 hematologic toxicities, drug-related non-hematological toxicity 
≥ grade 3 including laboratory abnormalities, and severe or life-threatening 
AEs. The table below includes dose modification guidelines for other 
toxicities that do not appear to be irAEs. Note: For hematological AEs, these 
guidelines are intended to be applied when the investigator determines the events 
to be Pembrolizumab-related. Dose modifications of MK- 3475 
(pembrolizumab) for hematological AEs do not apply during the induction 
phase of treatment. Note: if after the evaluation the event is determined not to be 
related, the investigator does not need to follow the treatment guidance. 
Therefore, these recommendations should be seen as guidelines and the treating 
physician should exercise individual clinical judgment based on the patient. 

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Dose Modification Guidelines for Non-IRAE-Related Adverse Events 
 
 
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 
x Grade 2 
alopecia 
x 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 
4 
Yes 
N/A 
N/A 
Subject must be discontinued 
*Only if deemed related to pembrolizumab (MK-3475) treatment 
 
In case toxicity does not resolve to grade 0-1 within 12 weeks after last infusion, protocol 
treatment should be discontinued. With Study Chair 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. 
 
7.1.3 Pembrolizumab Dose Modifications and Supportive Care Guidelines for Drug-
Related Adverse Events  

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7.1.4 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 *** 
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. 
 
7.1.5. 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. 
 
7.1.6  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.  
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. 

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Table 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 
Patients who do not respond 
to corticosteroids should 
be seen by a 
gastroenterologist for 
confirmation of the 
diagnosis and 
consideration of 
secondary immune 
suppression 
 
 
Monitor participants for signs 
and symptoms of 
enterocolitis (i.e., 
diarrhea, abdominal pain, 
blood or mucus in stool 
with 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 
Recurrent Grade 3 or 
Grade 4 
Permanently 
discontinue 

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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 
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 E-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 
x 
Stop Infusion. 
x 
Additional appropriate medical therapy 
may include but is not limited to: 
x 
IV fluids 
x 
Antihistamines 
x 
NSAIDs 
x 
Acetaminophen 
x 
Narcotics 
x 
Increase monitoring of vital signs as 
medically indicated until the participant is 
deemed medically stable in the opinion of 
the investigator. 
x 
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 
x 
Stop Infusion. 
x 
Additional appropriate medical therapy 
may include but is not limited to: 
x 
Epinephrine** 
x 
IV fluids 
x 
Antihistamines 
x 
NSAIDs 
x 
Acetaminophen 
x 
Narcotics 
x 
Oxygen 
x 
Pressors 
x 
Corticosteroids (e.g. methylprednisolone 2 
mg/kg/day or dexamethasone 10 mg every 
6 hours) 
x 
Increase monitoring of vital signs as 
medically indicated until the participant is 
deemed medically stable in the opinion of 
the investigator. 
x 
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 
x 
Continue pembrolizumab. 
x 
Investigate etiology. 
x 
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 
x 
Withhold pembrolizumab for up to 12 weeks after event onset. a 
x 
Investigate etiology and refer patient to neurologist. 
x 
Initiate treatment as per institutional guidelines. 
x 
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 
x 
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 
x 
Permanently discontinue pembrolizumab. c  
x 
Refer patient to neurologist. 
x 
Initiate treatment as per institutional guidelines. 
Myasthenia gravis and 
Guillain-Barré syndrome 
(any grade) 
x 
Permanently discontinue pembrolizumab. c 
x 
Refer patient to neurologist. 
x 
Initiate treatment as per institutional guidelines. 
x 
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 

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corticosteroids (if initiated) to be reduced to the equivalent of d10 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 t1 month to the equivalent of d10 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). 
 
7.2 Venetoclax 
 
Monitor blood counts frequently through resolution of cytopenias. Management of some 
adverse reactions may require dose interruptions or permanent discontinuation of 
VENCLEXTA. shows the dose modification guidelines for hematologic toxicities. Below are 
guidelines and ultimate decision on dose modifications of venetoclax is per the treating 
physician’s judgement. 
 
Event 
Occurrence 
Action 
Hematological Toxicity 
Grade 4 neutropenia with or without 
fever or infection; or Grade 4 
thrombocytopenia 
Occurrence prior to 
achieving remission 
Transfuse blood products, 
administer prophylactic and 
treatment anti-infectives as 
clinically indicated. In most 
instances, VENCLEXTA and 
azacitidine cycles should not 
be interrupted due to 
cytopenias prior to achieving 
remission 
First occurrence 
achieving remission 
and lasting at least 7 
days 
Delay subsequent treatment 
cycle of VENCLEXTA and 
azacitidine and monitor blood 
counts. 
Administer granulocyte-
colony stimulating factor (G-
CSF) if clinically indicated for 
neutropenia. Once the toxicity 
has resolved to Grade 1 or 2, 
resume VENCLEXTA 
therapy at the same dose in 
combination with azacitidine. 

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Subsequent 
occurrences in cycles 
after achieving 
remission and lasting 7 
days or longer 
Delay subsequent treatment 
cycle of VENCLEXTA and 
azacitidine, and monitor blood 
counts. Administer G-CSF if 
clinically indicated for 
neutropenia. Once the toxicity 
has resolved to Grade 1 or 2, 
resume VENCLEXTA therapy 
at the same dose and the 
duration reduced. 
Adverse reactions were graded using NCI CTCAE version 4.0. 

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7.3 
Azacitidine 
 
Below are guidelines for azacitidine dose modification that are per the drug label. However, the 
ultimate decision on dose modifications of azacitidine is per the treating physician’s judgement. 
Generally, dose reductions of azacitidine for hematological values are not recommended before 
achievement of morphologic remission. 
 
For patients with baseline (start of treatment) white blood count (WBC) ≥3 x109/L, 
ANC ≥1.5 x109/L, and platelets ≥75 x109/L, adjust the dose as follows, based on nadir counts for 
any given cycle: 
 
Nadir counts 
Azacitidine dose in the next 
course (mg/m2) 
ANC (x109/L) 
Platelets (x109/L) 
<0.5 
<25 
37.5 
0.5-1.5 
25-50 
50 
>1.5 
>50 
75 
ANC=absolute neutrophil count 
 
For patients whose baseline counts are WBC <3 x109/L, ANC <1.5 x109/L, or platelets <75 
x109/L, dose adjustments should be based on nadir counts and bone marrow biopsy cellularity at 
the time of the nadir as noted below, unless there is clear improvement in differentiation 
(percentage of mature granulocytes is higher and ANC is higher than at onset of that course) at 
the time of the next cycle, in which case the dose of the current treatment should be continued. 
 
WBC or platelet 
nadir % decrease in 
counts from baseline 
Bone marrow biopsy cellularity % 
At time of nadir 
30-60 
15-30 
<15 
Azacitidine dose in the Next Course (mg/m2) 
50-75 
75 
37.5 
25 
>75 
56.5 
37.5 
25 
WBC=white blood count 
 
If a nadir as defined in the table above has occurred, the next course of treatment should be given 
28 days after the start of the preceding course, provided that both the WBC and the platelet 
counts are >25% above the nadir and rising. If a >25% increase above the nadir is not seen by 
Day 28, counts should be reassessed every 7 days. If a 25% increase is not seen by Day 42, then 
the patient should be treated with 50% of the scheduled dose (37.5 mg/m2). 
 
If unexplained reductions in serum bicarbonate levels to less than 20 mEq/L occur, the dosage 
should be reduced by 50% (to 37.5 mg/m2) on the next course. Similarly, if unexplained 
elevations of blood urea nitrogen (BUN) or serum creatinine occur, the next cycle should be 
delayed until values return to normal or baseline and the dose should be reduced by 50% (to 37.5 
mg/m2) on the next treatment course. (See Vidaza® Package Insert) 
 
Azacitidine and its metabolites are known to be substantially excreted by the kidney, and the risk 
of toxic reactions to this drug may be greater in patients with impaired renal function. Because 

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elderly patients are more likely to have decreased renal function, care should be taken in dose 
selection, and it may be useful to monitor renal function. (See Vidaza® Package Insert) 
 
8 
PHARMACEUTICAL AGENT 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. 
 
8.1 
CTEP IND Agent 
 
8.1.1 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. 

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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 
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). 
 
8.1.2 Agent Ordering and Agent Accountability 
 
8.1.2.1 
NCI-supplied agents may be requested by eligible participating Investigators (or their 
authorized designee) at each participating institution. The CTEP-assigned protocol 

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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 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 PMB Online Agent Order Processing (OAOP) 
application. Access to OAOP 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 website for specific policies and guidelines related to agent 
management. 
 
8.1.2.2 
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. 
 
8.1.3 Investigator Brochure Availability 
 
The current versions of the IBs for the agents will be accessible to site investigators and research 
staff through the PMB OAOP application. Access to OAOP 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. 
 
8.1.4 Useful Links and Contacts 
 
x CTEP Forms, Templates, Documents: http://ctep.cancer.gov/forms/
x NCI CTEP Investigator Registration: RCRHelpDesk@nih.gov
x PMB policies and guidelines: http://ctep.cancer.gov/branches/pmb/agent_management.htm
x 
PMB Online Agent Order Processing (OAOP) application: 
https://ctepcore.nci.nih.gov/OAOP
x CTEP Identity and Access Management (IAM) account: https://ctepcore.nci.nih.gov/iam/
x CTEP IAM account help: ctepreghelp@ctep.nci.nih.gov
x IB Coordinator: IBCoordinator@mail.nih.gov
x PMB email: PMBAfterHours@mail.nih.gov

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x PMB phone and hours of service: (240) 276-6575 Monday through Friday between 8:30 am 
and 4:30 pm (ET)
 
8.2 
Commercial Agent(s) 
 
8.2.1 Azacitidine NSC# 102816 
Molecular Formula: C8H12N4O5 
M.W.: 244 
Description: White to off-white solid. 
 
How Supplied: The finished product is supplied in a sterile form for reconstitution as a 
suspension for subcutaneous injection or reconstitution as a solution with further dilution for 
intravenous infusion. Azacitidine is supplied as a lyophilized powder in 100 mg single-use vials. 
Store unreconstituted vials at 25° C (77° F); excursions permitted to 15°-30° C (59°-86° F). 
Preparation for SubQ: Azacitidine should be reconstituted aseptically with 4 mL sterile water 
for injection. The diluent should be injected slowly into the vial. Vigorously shake or roll the vial 
until a uniform suspension is achieved. The suspension will be cloudy. The resulting suspension 
will contain azacitidine 25 mg/mL. Do not inject the slurry intravenously. Doses greater than 4 
mL should be divided equally into 2 syringes, or per institutional standard. The product may be 
held at room temperature for up to 1 hour, but must be administered within 1 hour after 
reconstitution. 
Storage and Stability for SubQ: The reconstituted product may be kept in the vial or drawn 
into a syringe. Doses greater than 4 mL should be divided equally into 2 syringes. The product 
must be refrigerated immediately, and may be held under refrigerated conditions (2°C - 8°C, 
36°F - 46°F) for up to 8 hours. After removal from refrigerated conditions, the suspension may 
be allowed to equilibrate to room temperature for up to 30 minutes prior to administration. 
SubQ Administration: To provide a homogeneous suspension, the contents of the dosing 
syringe must be re-suspended immediately prior to administration. To re-suspend, vigorously roll 
the syringe between the palms until a uniform, cloudy suspension is achieved. Azacitidine 
suspension is administered subcutaneously. Doses greater than 4 mL should be divided equally 
into 2 syringes (or per institutional standard), and injected into 2 separate sites. Rotate sites for 
each injection (thigh, abdomen, or upper arm). New injections should be given at least one inch 
from an old site and never into areas where the site is tender, bruised, red, or hard. 
 
Preparation for IV: Reconstitute the appropriate number of vials to achieve the desired dose. 
Reconstitute each vial with 10 mL sterile water for injection. Vigorously shake or roll the vial 
until all solids are dissolved. The resulting solution will contain azacitidine 10 mg/mL. 
Withdraw the required amount of azacitidine to deliver the desired dose and inject into a 50 -100 
mL infusion bag of either 0.9% Sodium Chloride Injection or Lactated Ringer’s Injection. 

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Storage and Stability for IV: Azacitidine reconstituted for intravenous administration may be 
stored at 25°C (77°F), but administration must be completed within 1 hour of reconstitution. 
 
IV Administration: IV infusion over a period of 10-40 minutes. 
Agent Ordering: Azacitidine is available from commercial sources. 
For more detailed information, please consult the package insert. 
8.2.2 Venetoclax NSC#766270 
 
Chemical Name: 4-(4-{[2-(4-chlorophenyl)-4,4- dimethylcyclohex-1-en-1- 
yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4- 
ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide) 
 
Other Names: ABT-199, A-1195425.0, GDC-0199, RO5537382 
 
CAS registry number: 1257044-40-8 
Molecular Formula: C45H50ClN7O7S 
M.W.: 868.44 g/mol 
Approximate Solubility: very low aqueous solubility 
 
Mode of Action: B-cell lymphoma-2 (BCL-2) inhibitor 
 
Description: Venetoclax tablets for oral administration are supplied as pale yellow or beige 
tablets that contain 100 mg venetoclax as the active ingredient. Each tablet contains the 
following inactive ingredient: copovidone, colloidal silicone dioxide, polysorbate 80, sodium 
stearyl fumarate, and calcium phosphate dibasic. In addition, the 100 mg coated tablets include 
the following: iron oxide yellow, iron oxide red, iron oxide black, polyvinyl alcohol, talc, 
polyethylene glycol, and titanium dioxide. Each tablet is embossed with “V” on one side and 
“100” corresponding to the tablet strength on the other side. 
 
How Supplied: 100 mg tablets for each cycle 
Dosing Scheme for Ramp-Up Phase 
Day 
Venetoclax 
Daily Dose 
1 
100 mg 
2 
200 mg 
3 
400 mg 
4 and beyond 
400 mg 

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Storage: Store at or below 86°F (30°C) 
Route(s) of Administration: PO 
 
Method of Administration: Tablets should be taken orally once daily, at approximately the 
same time, with a meal and water. Do not chew, crush, or break tablets. 
 
If a dose is missed by less than 8 hours, take the missed dose right away and take the next dose 
as usual. If a dose is missed by more than 8 hours, the patient should wait and take the next dose 
at the usual time. 
 
Patients should not take an additional dose if they vomit after taking venetoclax. They should 
take the next dose at the usual time the following day. 
 
Potential Drug Interactions: Venetoclax is predominantly metabolized by CYP3A4/5. 
Concomitant use of venetoclax with strong inhibitors of CYP3A at initiation and during ramp-up 
phase is discouraged. If used during ramp-up, appropriate dose adjustments of venetoclax ramp- 
up should be followed. For patients who have completed the ramp-up phase and are on a steady 
daily dose, reduce the dose of venetoclax by at least 75% when used concomitantly with strong 
CYP3A inhibitors. Resume the venetoclax dose that was used prior to initiating the CYP3A 
inhibitor or the full venetoclax dose 2 to 3 days after discontinuation of the inhibitor. 
 
Azoles are standard prophylactic and therapeutic anti-fungal agents used in leukemia patients 
and their use is allowed on trial. When used in patients with AML who are receiving venetoclax, 
careful monitoring and dose adjustments as per standard clinical care is recommended. 
 
When possible. avoid concomitant use of moderate CYP3A inhibitors or P-gp inhibitors. If a 
moderate CYP3A inhibitor or a P-gp inhibitor must be used, reduce the dose by at least 50%. 
Resume the dose that was used prior to initiating the CYP3A inhibitor or P-gp inhibitor 2 to 3 
days after discontinuation of the inhibitor. If a narrow therapeutic index P-gp substrate must be 
used, it should be taken at least 6 hours before venetoclax. 
 
When possible., avoid concomitant use with strong CYP3A inducers or moderate CYP3A 
inducers. 
 
Venetoclax may cause a significant increase in Cmax and AUC of warfarin. International 
normalized ratio (INR) should be monitored closely in patients receiving warfarin. 
 
Venetoclax had no large effect on QTc interval (i.e., >20 ms) and there was no relationship 
between venetoclax exposure and change in QTc interval. 
 
Gastric acid reducing agents (e.g., proton pump inhibitors, H2-receptor antagonists, antacids) do 
not affect venetoclax bioavailability. 
 
Avoid grapefruit products, Seville oranges, and starfruit during treatment 

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Patient Care Implications: Tumor Lysis Syndrome (TLS) prophylaxis - Patients should be 
instructed to drink 6 to 8 glasses of water each day, starting 2 days before the first dose, on the 
day of the first dose of venetoclax, and each time the dose is increased. Consider IV hydration 
and/or uric acid reducing agents in patients at risk for TLS. 
 
Immunization: Do not administer live attenuated vaccines prior to, during, or after venetoclax 
treatment until B-cell recovery has occurred. 
 
Females of reproductive potential should use effective contraception during treatment and for at 
least 30 days after the last dose. 
 
Nursing women should discontinue breastfeeding during treatment with venetoclax. 
 
Availability 
 
Venetoclax is available from commercial sources. 
 
For more detailed information, please consult the package insert. 
 
 
9 
STATISTICAL CONSIDERATIONS 
 
9.1 
Study Design/Endpoints 
 
This is an open label randomized phase 2 study. The primary objective of the phase 2 study is to 
determine the efficacy of MK3475 (pembrolizumab) in combination with azacitidine and 
venetoclax in elderly (≥60-year-old) patients with newly diagnosed AML not eligible for 
intensive chemotherapy. A total of 76 patients will be included (38 patients in the intervention 
arm and 38 patients in the control arm, see below for sample calculation). 
 
Early Safety assessment: After the first 6 patients have been enrolled on the experimental VAP 
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 
toxicity data to identify any concerning drug-related safety signals before enrolling further 
patients on the experimental arm. To be considered evaluable for purposes of the early safety rule, 
the patient must have received one pembrolizumab dose at least. If severe Pembrolizumab -related 
toxicity was identified in 2 or more of these first 6 patients, the safety review committee will 
make a recommendation to adjust dosing/schedule or any other changes before enrollment 
resumes. The evaluability period includes the first 35 days after pembrolizumab first dose, till 
hematologic recovery, or till starting cycle 2 of therapy, whichever occurs first. 
 
Futility analysis: After 15 patients in each of the two study arms have their primary endpoint 
results available (per Section 9.5.2), a formal futility analysis will be conducted. Enrollment to 
study will not be held while the futility analysis is being conducted. If the rate of MRD negative 
CR (primary efficacy end point) is lower in the experimental arm than the control arm by any 
amount, the trial would stop for futility. 

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Toxicity analysis: After 15 patients in each of the two study arms have their primary endpoint 
results available (per Section 9.5.2), a formal toxicity analysis will be conducted along with 
futility analysis. If the schedule results in unacceptable toxicity (per Sections 9.4 and 9.5.1), we 
will discuss alternative approaches with CTEP 
 
For patients who neither relapse nor die, the duration of response (DOR) will be censored on the 
date of their last evaluable disease assessment. Median relapse-free survival (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 two years from first dose. An analysis of the survival and RFS data 
will be conducted based on the two-year follow up. 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. 
 
 
9.1.1 Primary endpoint 
 
x Assess the percentage of patients with MRD-CR or MRD-Cri with AZA + VEN with 
pembrolizumab (MK-3475) during the first 6 cycles of therapy. 
9.1.2 MRD assessment by multicolor flow cytometry (MFC) as an integral biomarker 
We will use a cutoff of 0.1% as the threshold to distinguish MRD-positive from MRD- 
“negative” patients as this threshold was found relevant in most published studies to date and 
was included in the official consensus document on minimal residual disease by the European 
LeukemiaNet MRD Working Party. However, it should be noted that MRD tests with MRD 
quantified below <0.1% may still be consistent with residual leukemia, and several studies have 
shown prognostic significance of MRD levels below 0.1%. Thus, cutoff levels below 0.1% (e.g., 
<0.01%) may define patients with particularly good outcome. 
 
9.2 
Sample Size/Accrual Rate 
 
In a recent study of azacitidine and venetoclax therapy in newly diagnosed elderly AML, a 
complete response was achieved in 73%, of all patients, of whom 30% attained MRD negativity, 
thereby yielding approximately 20% MRD- CR rate.(DiNardo et al, 2019). We hypothesize that 
the addition of pembrolizumab (MK-3475) to AZA+VEN therapy will lead to an improvement 
of MRD negativity at/after attainment of CR when compared to AZA+VEN therapy alone. 
Assuming MRD-negative CR/CRi rates of 0.2 and 0.45 for Aza + Ven and Aza + Ven + 
MK3475 (pembrolizumab), respectively, a total sample of 60 evaluable patients would provide 
80% power to detect a between-arm differencing based on a two-sample test of proportions 
(normal approximation) with one-sided type-I error of 10%. Allowing for up to 20% dropout, 
the total target accrual would be 76 patients (38 per arm). 

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PLANNED ENROLLMENT REPORT 
 
 
Racial Categories 
Ethnic Categories 
Total 
Not Hispanic or Latino 
Hispanic or Latino 
 
Female 
Male 
Female 
Male 
American Indian/ Alaska 
Native 
 
1 
1 
1 
1 
4 
 
Asian 
 
1 
1 
1 
1 
4 
Native Hawaiian or 
Other Pacific Islander 
 
1 
1 
1 
1 
4 
Black or African 
American 
 
2 
3 
1 
1 
7 
 
White 
 
20 
25 
4 
4 
53 
 
More Than One Race 
 
1 
1 
1 
1 
4 
 
Total 
 
26 
32 
9 
9 
76 
PHS 398 / PHS 2590 (Rev. 08/12 Approved Through 8/31/2015) 
OMB No. 0925-0001/0002 
 
 
9.3 
Stratification Factors 
Planned stratification factors include cytogenetics by FISH or karyotype (intermediate/unknown 
vs. adverse karyotype), antecedent hematologic disorder defined as prior MDS, MPN or aplastic 
anemia (present vs. absent), and reason to not receive therapy (ineligibility vs refusal). Dose 
escalation and MTD determination are not parts of this study. 
 
Refer to Section 2.5.1 for further details on assessment of karyotyping. 
 
9.4 Analysis of Secondary Endpoints 
 
Safety Endpoints 
 
Assessment of the proportion of patients who develop severe toxicity, defined as the occurrence 
of any of the below AE during the first 35 days from start of pembrolizumab (MK-3475) 
therapy. Severe pembrolizumab-related toxicity is defined as the occurrence of any of the below 
AEs that are attributed to pembrolizumab (MK-3475). The evaluability period includes the first 35 
days after pembrolizumab first dose, till hematologic recovery, or till starting second AZA+Ven 
cycle therapy, whichever occurs first: 

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x Any grade 3 or 4 non-hematologic toxicity, that is deemed not related to the underlying 
disease or chemotherapy, with the following exceptions: a) Transient laboratory 
abnormalities that can be treated or resolve to grade 2 or less within 7 days, or 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. 
x Grade 4 thrombocytopenia or neutropenia associated with 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 especially with use of venetoclax, they will not be used to define unacceptable 
toxicity except if associated with prolonged treatment-associated aplasia AND not 
associated with active disease that is attributed to pembrolizumab. 
 
x Patients with grade 3 and 4 immune related adverse events (with exception of events that 
resolve with steroids to grade 1 or less and do not require continued steroid therapy 
beyond 4 weeks). 
 
9.4.1 Exploratory Endpoints 
 
Descriptive statistics will be provided for each biomarker of interest in overall sample and/or by 
treatment arms across different time points of the measurements (e.g., baseline, end of induction, 
post-cycle1, 2, 4, and 6, every 3 month during maintenance, one year, and end of treatment when 
applicable). Continuous biomarkers (e.g., count of biomarkers from tumor infiltrating 
lymphocytes, gene expression from RNASeq, Nanostring, and WES, inflammation biomarkers 
from Olink, DNA methylation, and immune cell expression from CyTOF) will be summarized 
using mean, standard deviation, median, inter-quartile range, and range. Categorical biomarkers 
(e. g, mutation, PD-1 status, and tumor mutation burden, microbiome data) will be summarized 
using frequency counts and percentages. Boxplots, bar plots or variation of boxplots (e.g., 
swarmplot, violinplot) will be generated for visualization. Distribution of normality of markers 
will be tested, and transformation of data will be applied if needed. Classification variables may 
be considered using median, tertile cut-off points, or recursive partitioning methods. A false 
discovery rate of 5% will be used to account for multiple comparisons and the associated 
inflation of false detection rates. 
 
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, and RNA seq/TCRseq, and 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-Hochberg1 correction to 
control for false discovery rates. 

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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-cycle1, 2, 4, and 6, every 3 months during maintenance, one year, and end of 
treatment when applicable. Landmark analysis1, 2 or joint modeling3 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, VZA+VEN vs. VAP 
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 sun / 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. 
 
 
9.4.1.1 
MRD assessment by DS as an exploratory biomarker 
 
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 

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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: ~1μg 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, 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. 
 
9.4.1.1.1 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. 
 
9.4.1.1.2 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. 

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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 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 

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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. 
 
 
9.4.1.2 
Assessment of cytokine panel 
 
For data analysis, IFN-γ+/CD3+/ CD4+ or IFN-γ+/CD3+/CD8+ events will be gated and 
percentages of the total CD4+ and CD8+ T cells will be determined, although these may change 
as the technology evolves and develops. Percentages of IFN-γ+ events in corresponding gates 
from un-stimulated control samples will be subtracted. This analysis will be done both at day 30 
(after administration of pembrolizumab and count recovery) and after cycles 2,4, and 6 of 
therapy to compare levels of leukemia specific T-cell prior to and after anti-PD-L1 therapy. 
 
9.4.1.3 
Evaluate whether the expression of PD-1, PD-L1 in AML BM correlates with the 
response to anti-PD-1 therapy and characterize dynamic change of immune cell 
subsets in AML patients pre- and post- anti-PD-1 therapy: 
 
Multiplex immunofluorescent assay (IF) will be performed in samples collected at the beginning 
of trial, post cycle 4, post cycle 6, after 1 year, at relapse, and at end of treatment/progression 
using PD-L1 (clone: E1L3N, Cell Signaling Technology) antibody and PD-1 (clone: EH33, 
Cell Signaling Technology) antibody. We will assess PD-L1 expression and PD-1 expression in 
CD34+ or CD33+ population (depending on initial phenotype of AML blasts). 
 
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 2x2 table of frequencies. The dependent variable will 
be defined as response (yes versus 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. 
 
9.4.1.4 
Dynamic change of immune subsets 
 
We will assess these immune cell markers at different time points of therapy: at baseline (prior 
to application of AZA+VEN therapy), at Day 30 (at time of count recovery), as well as after 
cycles 2, 4, and 6 of therapy. We also plan to measure CD47 levels on leukemic blasts prior to 
and after AZA+VEN therapy and pembrolizumab (MK-3475) 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 

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9.4.1.5 .. Correlation between microbial changes with immune-checkpoint expression and 
kinetics of immune cell subset recovery and programming 
 
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 six cycles will also be explored 
 
9.5 
For phase 2 protocols only: Reporting and Exclusions 
 
9.5.1 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 (MK-3475). The evaluability period includes the first 35 days after 
pembrolizumab first dose, till hematologic recovery, or till starting second AZA+Ven cycle 
therapy, whichever occurs first: 
 
x Any grade 3 or 4 non-hematologic toxicity, that is deemed not related to the underlying 
disease or chemotherapy with the following exceptions: a) transient laboratory 

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abnormalities that can be treated or resolve to grade 2 or less within 7 days; or 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. 
 
x Grade 4 thrombocytopenia or neutropenia, associated with 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 especially with use of venetoclax, they will not be used to define unacceptable 
toxicity except if associated with prolonged treatment-associated aplasia AND not 
associated with active disease that is attributed to pembrolizumab. 
 
x Patients with grade 3 and 4 irAEs (with exception of events that resolve with steroids to 
grade 1 or less and do not require continued steroid therapy beyond 4 weeks). 
 
9.5.2 Evaluation of Response 
 
All patients included in the study 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 following categories: 1) complete response, 2) partial response, 3) stable disease, 4) 
progressive disease, 5) early death from malignant disease, 6) early death from toxicity, 7) early 
death because of other cause, or 9) unknown (not assessable, insufficient data). [Note: By 
arbitrary convention, category 9 usually designates the “unknown” status of any type of data in a 
clinical database.] 
 
All of the patients who met the eligibility criteria (with the possible exception of those who 
received no study medication) should be included in the main analysis of the response rate. 
Patients in response categories 4-9 should be considered to have a treatment failure (disease 
progression). Thus, an incorrect treatment schedule or drug administration does not result in 
exclusion from the analysis of the response rate. Precise definitions for categories 4-9 will be 
protocol specific. 
 
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 subanalyses 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. 
 
9.6 
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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10 ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS 
 
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. 
 
10.1 
Comprehensive Adverse Events and Potential Risks List(s) (CAEPRs) 
 
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. 
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. 
 
10.1.1 CAEPRs for CTEP IND Agent 
 
10.1.1.1 
CAEPR for pembrolizumab (MK-3475) 
 
Comprehensive Adverse Events and Potential Risks list (CAEPR) 
for  
Pembrolizumab (MK-3475, 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. 
 
 
 
 
 
 
 
 
 

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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  
 (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 
 
  

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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)  
 
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 
 
  
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 
  

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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)  
 
 
 
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 - 
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 
  

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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)  
 
 
 
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 
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 

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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. 
 
 
 
10.1.1.2 
CAEPR for azacitidine 
 
Comprehensive Adverse Events and Potential Risks list (CAEPR) 
for 
Azacitidine (NSC 102816) 
 
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 1800 patients. Below is the CAEPR for 
Azacitidine. 
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 2.7, July 30, 20191 
 
Adverse Events with Possible 
Relationship to Azacitidine 
(CTCAE 5.0 Term) 
[n= 1800] 
Specific Protocol Exceptions 
to Expedited Reporting 
(SPEER) 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
BLOOD AND LYMPHATIC SYSTEM DISORDERS 
Anemia 
 
Anemia (Gr 3) 
 
Febrile neutropenia 
Febrile neutropenia (Gr 3) 
CARDIAC DISORDERS 
 
Heart failure 
Heart failure (Gr 2) 
 
Pericardial effusion 
Pericardial effusion (Gr 2) 
 
Sinus tachycardia 
Sinus tachycardia (Gr 2) 
 
Supraventricular tachycardia 
Supraventricular tachycardia (Gr 
2) 
GASTROINTESTINAL DISORDERS 
 
Abdominal pain 
Abdominal pain (Gr 3) 
 
Colitis 
Colitis (Gr 2) 
Constipation 
 
Constipation (Gr 2) 
Diarrhea 
 
Diarrhea (Gr 3) 
 
Esophagitis 
Esophagitis (Gr 2) 
 
Gastrointestinal hemorrhage2 
 
Mucositis oral 
Mucositis oral (Gr 2) 
Nausea 
 
Nausea (Gr 3) 
Vomiting 
 
Vomiting (Gr 3) 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS 
 
Chills 
Chills (Gr 2) 
 
Edema limbs 
Edema limbs (Gr 2) 
Fatigue 
 
Fatigue (Gr 3) 

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Fever 
 
Fever (Gr 3) 
Injection site reaction 
 
Injection site reaction (Gr 2) 
 
 
 
Adverse Events with Possible 
Relationship to Azacitidine 
(CTCAE 5.0 Term) 
[n= 1800] 
Specific Protocol Exceptions 
to Expedited Reporting 
(SPEER) 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
IMMUNE SYSTEM DISORDERS 
 
 
Allergic reaction 
Allergic reaction (Gr 2) 
 
 
Anaphylaxis 
INFECTIONS AND INFESTATIONS 
Infection3 
 
Infection3 (Gr 4) 
INJURY, POISONING AND PROCEDURAL COMPLICATIONS 
 
Bruising 
Bruising (Gr 2) 
INVESTIGATIONS 
 
Alanine aminotransferase 
increased 
Alanine aminotransferase 
increased (Gr 4) 
 
Alkaline phosphatase increased 
Alkaline phosphatase increased 
(Gr 2) 
 
Aspartate aminotransferase 
increased 
Aspartate aminotransferase 
increased (Gr 4) 
 
Blood bilirubin increased 
Blood bilirubin increased (Gr 2) 
 
GGT increased 
GGT increased (Gr 2) 
 
Lymphocyte count decreased 
Lymphocyte count decreased (Gr 
4) 
Neutrophil count decreased 
 
Neutrophil count decreased (Gr 4) 
Platelet count decreased 
 
Platelet count decreased (Gr 4) 
 
Weight loss 
Weight loss (Gr 2) 
 
White blood cell decreased 
White blood cell decreased (Gr 4) 
METABOLISM AND NUTRITION DISORDERS 
 
Acidosis 
Acidosis (Gr 2) 
 
Anorexia 
Anorexia (Gr 3) 
 
Hypokalemia 
 
 
Tumor lysis syndrome 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS 
 
Arthralgia 
Arthralgia (Gr 2) 
 
Back pain 
Back pain (Gr 2) 
 
Generalized muscle weakness 
Generalized muscle weakness (Gr 
2) 
 
Myalgia 
Myalgia (Gr 2) 
 
Pain in extremity 
Pain in extremity (Gr 2) 
NERVOUS SYSTEM DISORDERS 
 
Dizziness 
Dizziness (Gr 2) 
 
Headache 
Headache (Gr 2) 
 
Peripheral motor neuropathy 
Peripheral motor neuropathy (Gr 
2) 
 
Somnolence 
Somnolence (Gr 2) 
PSYCHIATRIC DISORDERS 
 
Anxiety 
 
Confusion 
Confusion (Gr 2) 
 
Insomnia 
RENAL AND URINARY DISORDERS 
 
 
Acute kidney injury 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS 

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Adverse Events with Possible 
Relationship to Azacitidine 
(CTCAE 5.0 Term) 
[n= 1800] 
Specific Protocol Exceptions 
to Expedited Reporting 
(SPEER) 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
 
 
Bronchopulmonary 
hemorrhage 
 
Cough 
Cough (Gr 2) 
 
Dyspnea 
Dyspnea (Gr 4) 
 
Epistaxis 
Epistaxis (Gr 2) 
 
Pharyngolaryngeal pain 
 
Postnasal drip 
Postnasal drip (Gr 2) 
 
Respiratory, thoracic and 
mediastinal disorders - Other 
(abnormal breath sound)4 
Respiratory, thoracic and 
mediastinal disorders - Other 
(abnormal breath sound)4 (Gr 2) 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS 
 
Alopecia 
Alopecia (Gr 2) 
 
Pruritus 
Pruritus (Gr 2) 
 
Purpura 
Purpura (Gr 2) 
 
Rash maculo-papular 
Rash maculo-papular (Gr 3) 
VASCULAR DISORDERS 
 
Hematoma 
Hematoma (Gr 2) 
 
Hypotension 
Hypotension (Gr 3) 
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. 
 
2Gastrointestinal hemorrhage includes Anal hemorrhage, Cecal hemorrhage, Colonic hemorrhage, 
Duodenal hemorrhage, Esophageal hemorrhage, Esophageal varices hemorrhage, Gastric hemorrhage, 
Hemorrhoidal hemorrhage, Ileal hemorrhage, Intra-abdominal hemorrhage, Jejunal hemorrhage, Lower 
gastrointestinal hemorrhage, Oral hemorrhage, Pancreatic hemorrhage, Rectal hemorrhage, 
Retroperitoneal hemorrhage, and Upper gastrointestinal hemorrhage under the GASTROINTESTINAL 
DISORDERS SOC. 
 
3Infection may include any of the 75 infection sites under the INFECTIONS AND INFESTATIONS SOC. 
 
4Abnormal breath sounds include rales and rhonchi. 
 
 
Adverse events reported on azacitidine trials, but for which there is insufficient evidence to 
suggest that there was a reasonable possibility that azacitidine caused the adverse event: 
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS - Blood and lymphatic system disorders - Other 
(agranulocytosis); Blood and lymphatic system disorders - Other (lymphadenopathy); Blood and lymphatic 
system disorders - Other (pancytopenia); Blood and lymphatic system disorders - Other (splenomegaly); 
Blood and lymphatic system disorders - Other (transfusion: platelets); Bone marrow hypocellular; 
Hemolysis; Leukocytosis 
CARDIAC DISORDERS - Atrial fibrillation; Atrial flutter; Atrioventricular block complete; Cardiac arrest; 
Cardiac disorders - Other (cardiac valve vegetation); Cardiac disorders - Other (Wolff-Parkinson-White 
syndrome); Chest pain - cardiac; Myocardial infarction; Palpitations; Pericarditis; Restrictive 
cardiomyopathy; Sinus bradycardia; Ventricular fibrillation 
EAR AND LABYRINTH DISORDERS - Hearing impaired; Tinnitus 

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EYE DISORDERS - Eye disorders - Other (eye/conjunctival hemorrhage); Eye disorders - Other (retina 
hemorrhage); Papilledema; Uveitis 
GASTROINTESTINAL DISORDERS - Abdominal distension; Ascites; Duodenal ulcer; Dyspepsia; 
Dysphagia; Enterocolitis; Esophageal pain; Esophageal ulcer; Flatulence; Gastritis; Gastrointestinal 
disorders - Other (enteritis); Gastrointestinal disorders - Other (inguinal hernia, obstructive); 
Gastrointestinal disorders - Other (intestinal ischemia); Gastrointestinal disorders - Other 
(intussusception); Gastrointestinal pain; Hemorrhoids; Pancreatitis; Periodontal disease; Small intestinal 
obstruction; Visceral arterial ischemia 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS - Death NOS; Edema face; Flu 
like symptoms; Gait disturbance; General disorders and administration site conditions - Other (systemic 
inflammatory response syndrome); Generalized edema; Malaise; Multi-organ failure; Non-cardiac chest 
pain; Pain; Sudden death NOS 
HEPATOBILIARY DISORDERS - Cholecystitis; Hepatic failure; Hepatobiliary disorders - Other (bile duct 
stone); Hepatobiliary disorders - Other (hepatic cirrhosis) 
IMMUNE SYSTEM DISORDERS - Autoimmune disorder; Immune system disorders - Other (GVHD) 
INJURY, POISONING AND PROCEDURAL COMPLICATIONS - Burn; Fall; Fracture; Hip fracture; Injury, 
poisoning and procedural complications - Other (excoriation); Injury, poisoning and procedural 
complications - Other (transfusion reaction); Postoperative hemorrhage; Wound dehiscence 
INVESTIGATIONS - Activated partial thromboplastin time prolonged; Blood lactate dehydrogenase 
increased; Creatinine increased; Electrocardiogram QT corrected interval prolonged; INR increased; 
Investigations - Other (blood urea increased); Investigations - Other (cardiac murmur); Investigations - 
Other (coagulopathy); Investigations - Other (protein total decreased); Investigations - Other 
(thrombocytosis); Lipase increased; Lymphocyte count increased; Serum amylase increased 
METABOLISM AND NUTRITION DISORDERS - Dehydration; Hyperglycemia; Hyperkalemia; 
Hyperphosphatemia; Hyperuricemia; Hypoalbuminemia; Hypocalcemia; Hypomagnesemia; 
Hyponatremia; Hypophosphatemia; Metabolism and nutrition disorders - Other (gout exacerbation); 
Metabolism and nutrition disorders - Other (hypovolemia); Metabolism and nutrition disorders - Other (low 
carbon dioxide) 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS - Arthritis; Bone pain; Chest wall pain; 
Flank pain; Muscle cramp; Muscle weakness lower limb; Musculoskeletal and connective tissue disorder - 
Other (chondritis); Musculoskeletal and connective tissue disorder - Other (intervertebral disc protrusion); 
Musculoskeletal and connective tissue disorder - Other (musculoskeletal stiffness); Neck pain 
NEOPLASMS BENIGN, MALIGNANT AND UNSPECIFIED (INCL CYSTS AND POLYPS) - 
Myelodysplastic syndrome; Neoplasms benign, malignant and unspecified (incl cysts and polyps) - Other 
(colonic polyp, vaginal polyp); Neoplasms benign, malignant and unspecified (incl cysts and polyps) - 
Other (metastases to central nervous system); Treatment related secondary malignancy 
NERVOUS SYSTEM DISORDERS - Dysesthesia; Dysgeusia; Hydrocephalus; Intracranial hemorrhage; 
Lethargy; Memory impairment; Nervous system disorders - Other (head injury); Paresthesia; Peripheral 
sensory neuropathy; Seizure; Stroke; Syncope 
PSYCHIATRIC DISORDERS - Delirium; Depression; Hallucinations; Psychiatric disorders - Other (mental 
status changes) 
RENAL AND URINARY DISORDERS - Chronic kidney disease; Dysuria; Hematuria; Proteinuria; Renal 
and urinary disorders - Other (bladder distention); Renal and urinary disorders - Other (calculus urinary); 
Renal calculi; Urinary frequency; Urinary retention 
REPRODUCTIVE SYSTEM AND BREAST DISORDERS - Erectile dysfunction; Reproductive system and 
breast disorders - Other (benign prostatic hyperplasia); Uterine hemorrhage; Vaginal hemorrhage 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS - Atelectasis; Hypoxia; Laryngeal 
hemorrhage; Nasal congestion; Oropharyngeal pain; Pleural effusion; Pleuritic pain; Pneumonitis; 
Pneumothorax; Productive cough; Pulmonary edema; Respiratory failure; Respiratory, thoracic and 
mediastinal disorders - Other (chronic obstructive pulmonary disease); Respiratory, thoracic and 
mediastinal disorders - Other (pharyngeal erythema); Rhinorrhea; Sinus pain; Wheezing 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS - Dry skin; Hyperhidrosis; Palmar-plantar 
erythrodysesthesia syndrome; Skin and subcutaneous tissue disorders - Other (skin laceration); Skin and 
subcutaneous tissue disorders - Other (skin lesion); Skin and subcutaneous tissue disorders - Other (skin 
nodule); Skin and subcutaneous tissue disorders - Other (Sweet's Syndrome); Skin induration; Urticaria 

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VASCULAR DISORDERS - Flushing; Hypertension; Thromboembolic event; Vascular disorders - Other 
(pallor); Vascular disorders - Other (poor venous access); Vasculitis 
 
 
Note: Azacitidine 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. 
 
 
 
 
10.1.1.3 
CAEPR for venetoclax 
 
. Frequency is provided based on 1298 patients. Below is the CAEPR for Venetoclax (ABT-199). 
 
 
Version 2.1, May 8, 2019 
 
Adverse Events with Possible 
Relationship to Venetoclax (ABT-199) 
(CTCAE 5.0 Term) 
[n= 1298] 
Specific Protocol 
Exceptions to Expedited 
Reporting (SPEER) 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
BLOOD AND LYMPHATIC SYSTEM DISORDERS 
Anemia 
 
Anemia (Gr 2) 
 
Febrile neutropenia 
GASTROINTESTINAL DISORDERS 
 
Constipation 
Constipation (Gr 2) 
Diarrhea 
 
Diarrhea (Gr 2) 
Nausea 
 
Nausea (Gr 2) 
 
Vomiting 
Vomiting (Gr 2) 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS 
Fatigue 
 
Fatigue (Gr 2) 
 
Fever 
Fever (Gr 2) 
INFECTIONS AND INFESTATIONS 
Infection2 
 
Infection2 (Gr 2) 
INVESTIGATIONS 
 
Lymphocyte count decreased 
Neutrophil count decreased 
 
Neutrophil count decreased 
(Gr 2) 
 
Platelet count decreased 
Platelet count decreased 
(Gr 2) 
 
White blood cell decreased 
METABOLISM AND NUTRITION DISORDERS 
 
Hypocalcemia 
 
Hypokalemia 
Hypokalemia (Gr 2) 

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Adverse Events with Possible 
Relationship to Venetoclax (ABT-199) 
(CTCAE 5.0 Term) 
[n= 1298] 
Specific Protocol 
Exceptions to Expedited 
Reporting (SPEER) 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
 
Hypophosphatemia 
 
 
Tumor lysis syndrome 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS 
 
Arthralgia 
NERVOUS SYSTEM DISORDERS 
 
Headache 
Headache (Gr 2) 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS 
 
Cough 
Cough (Gr 2) 
VASCULAR DISORDERS 
 
Hypertension 
 
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. 
 
2Infection includes all 75 sites of infection under the INFECTIONS AND INFESTATIONS SOC. 
 
 
Adverse events reported on venetoclax (ABT-199) trials, but for which there is insufficient 
evidence to suggest that there was a reasonable possibility that venetoclax (ABT-199) caused the 
adverse event: 
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS - Hemolysis; Thrombotic thrombocytopenic purpura 
CARDIAC DISORDERS - Atrial fibrillation; Cardiac disorders - Other (coronary artery disease); Heart 
failure; Myocardial infarction; Sinus tachycardia; Ventricular arrhythmia 
EAR AND LABYRINTH DISORDERS - Tinnitus; Vertigo 
GASTROINTESTINAL DISORDERS - Abdominal pain; Belching; Dry mouth; Dyspepsia; Dysphagia; 
Gastrointestinal disorders - Other (Crohn's disease); Small intestinal obstruction 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS - Death NOS; Edema limbs; Flu 
like symptoms; General disorders and administration site conditions - Other (general physical heath 
deterioration); General disorders and administration site conditions - Other (multiple organ dysfunction 
syndrome); Injection site reaction; Non-cardiac chest pain; Pain; Sudden death NOS 
HEPATOBILIARY DISORDERS - Hepatobiliary disorders - Other (hepatic function abnormal) 
IMMUNE SYSTEM DISORDERS - Cytokine release syndrome 
INJURY, POISONING AND PROCEDURAL COMPLICATIONS - Infusion related reaction; Injury, 
poisoning and procedural complications - Other (laceration) 
INVESTIGATIONS - Aspartate aminotransferase increased; Blood bilirubin increased; Weight loss 
METABOLISM AND NUTRITION DISORDERS - Anorexia; Dehydration; Hyperglycemia; Hyperkalemia; 
Hyperphosphatemia; Hyperuricemia; Hypoalbuminemia; Hypomagnesemia; Hyponatremia; Metabolism 
and nutrition disorders - Other (failure to thrive) 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS - Back pain; Bone pain 
NEOPLASMS BENIGN, MALIGNANT AND UNSPECIFIED (INCL CYSTS AND POLYPS) - 
Myelodysplastic syndrome; Neoplasms benign, malignant and unspecified (incl cysts and polyps) - Other 
(acute myeloid leukemia); Treatment related secondary malignancy 

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NERVOUS SYSTEM DISORDERS - Dizziness; Dysesthesia; Intracranial hemorrhage; Ischemia 
cerebrovascular; Nervous system disorders - Other (neuropathy peripheral); Peripheral sensory 
neuropathy; Syncope 
PSYCHIATRIC DISORDERS - Confusion; Insomnia 
RENAL AND URINARY DISORDERS - Acute kidney injury; Cystitis noninfective; Dysuria 
REPRODUCTIVE SYSTEM AND BREAST DISORDERS - Ovarian rupture 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS - Allergic rhinitis; Aspiration; Dyspnea; 
Epistaxis; Hypoxia; Nasal congestion; Oropharyngeal pain; Pleural effusion; Respiratory failure; 
Respiratory, thoracic and mediastinal disorders - Other (asphyxia) 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS - Eczema; Pruritus; Rash acneiform; Rash maculo- 
papular 
VASCULAR DISORDERS - Hypotension; Thromboembolic event 
 
 
Note: Venetoclax (ABT-199) 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. 
 
 
10.1.2 Adverse Effects of Dexamethasone 
Routes: Intraocular; Ophthalmic; Oral; Topical 
Common Adverse Effects 
x Cardiovascular: Hypertension (Diabetic macular edema, 13%) 
x Dermatologic: Atrophic condition of skin, Finding of skin healing, Impaired 
x Endocrine metabolic: Cushing's syndrome, Decreased body growth 
x Immunologic: At risk for infection 
x Ophthalmic: Abnormal vision (9%), Cataract (Diabetic macular edema, 68%; retinal 
vein occlusion and uveitis, 5%), Conjunctival edema (5%), Conjunctivitis (6%), 
Discomfort, Eye (10%), Dry eye syndrome (5%), Raised intraocular pressure (Diabetic 
macular edema, 35%; retinal vein occlusion and uveitis, 25%), Vitreous floaters (5%) 
x Psychiatric: Depression, Euphoria 
x Respiratory: Pulmonary tuberculosis 
 
For complete information on potential AEs for dexamethasone, please refer to Dexamethasone 
Prescribing Information (Roxane Laboratories, Inc., 2015). 
 
10.1.3 Unknown or Potential Risks Associated with the Use of Pembrolizumab (MK-3475) in 
AML Therapy 
 
Immune check point blockade studies have demonstrated the feasibility and safety of combining 
immunotherapy with intensive chemoinduction (Ravandi et al., 2019) and azacitidine (Daver et 
al., 2018) in patients with AML. 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 1.5g/m2, 24-hour 
infusion of cytarabine daily on Days 1–4 (three days only for patients >60 years), alongside 

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12mg/m2 daily on days 1–3 of idarubicin. Nivolumab was given on Day 24 at a dose of 3mg/kg 
which was repeated every two weeks for a year in responders. Adverse events are detailed in the 
table below. 
 
Adverse events 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 
 
There were no nivolumab treatment-associated deaths and post-transplant severe GVHD was not 
significantly increased and manageable. 
 
Similarly, Daver et al reported on the safety outcomes of combining nivolumab with azacitidine 
in patients with R/R AML. In the phase 2 study enrolling 70 patients, grade 3-4 and grade 2 
immune related adverse events (irAEs) were observed in 8 (11%) and 8 (11%) patients, 
respectively. Of the 16 (23%) patients with grade 2-4 immune toxicities, 9 episodes were 
pneumonitis, 6 were nephritis, 3 were immune related skin rash, and 2 were transaminitis (some 
patients had more than 1 irAE). Fourteen of the 16 (88%) toxicities responded to steroids, and 
these 14 patients were safely rechallenged with nivolumab. The non-immune toxicities with this 
combination were similar to other HMA-based salvage therapies. 
 
 
10.2 
Adverse Event Characteristics 
 
x 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. 
 
x For expedited reporting purposes only: 

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- 
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. 
- 
Other AEs for the protocol that do not require expedited reporting are outlined in 
Section 10.3.4. 
 
x 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. 
 
10.3 
Expedited Adverse Event Reporting 
 
10.3.1 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: 
 
x The reporting period (course/cycle) is correct, and 
x 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 

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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: 
 
x Study specific documents: Protocols > Documents > Education and Promotion, 
and 
x Expedited Safety Reporting Rules Evaluation user guide: Resources > CTSU 
Operations Information > User Guides. 
 
NCI requirements for SAE reporting are available on the CTEP website: 
 
x NCI Guidelines for Investigators: Adverse Event Reporting Requirements is 
available at 
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/aeguid 
elines.pdf. 
 
 
 
10.3.2 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. 
 
10.3.3 Expedited Reporting Guidelines 
 
Use the NCI protocol number and the protocol-specific patient ID assigned during trial 
registration on all reports. 
 
Note: A death on study requires both routine and expedited reporting, regardless of 
causality. 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. 

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126 
 
 
 
Phase 1 and Early Phase 2 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/Intervention 1, 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 1-2 Timeframes 
Grade 3-5 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. 
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 
x 
Within 5 calendar days for Grade 3-5 SAEs 
x 
Within 10 calendar days for Grade 1-2 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 
 
10.3.4 Additional Protocol-Specific Expedited Adverse Event Reporting Exclusions 

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127 
 
 
For this protocol only, the AEs/grades listed below do not require expedited reporting via CTEP- 
AERS. However, they still must be reported through the routine reporting mechanism (Section 
10.4): 
 
CTCAE SOC 
Adverse Event 
Grade 
≥24h 
Hospitalizationa 
Blood and lymphatic system 
disorders 
Febrile neutropenia 
≥ 3 
Regardless 
a Indicates that an adverse event required hospitalization for ≥24 hours or prolongation of 
hospitalization by ≥24 hours of a patient. 
 
 
10.4 
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 
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. 
 
10.5 
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. 
 
10.6 
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: 

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128 
 
 
x Leukemia secondary to oncology chemotherapy (e.g., acute myelocytic leukemia 
[AML]) 
x Myelodysplastic syndrome (MDS) 
x 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. 
 
 
10.7 
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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129 
11 STUDY CALENDAR 
Pre- 
Study 
(21 
days) 
Cycle 1 
 
 
 
Cycle 2- 6 
Maintenance 
Therapy 
Cycles 7-30 
After 
1 
year 
Relaps 
e 
End 
of 
Study 
/ 
Progr 
essio 
n 
Post 
- 
Trea 
tme 
nt 
Foll 
ow- 
Upb 
Baseline 
D1 
(+/- 
3 
days) 
D 
2 
D 
3 
D 
4 
D 
5 
D 
6 
D 
7 
D8 
D14 
 
D 
28 
D1 
(+/- 3 
days) 
 
D 
2 
 
D 
3 
 
D 
4 
 
D 
5 
 
D 
6 
 
D 
7 
 
D 
8 
 
 
D14 
 
 
D21 
D28 
Days 1-28 
(+/- 3 days ) 
AzacitidineC 
X 
X 
X 
X 
X 
X 
X 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
Xs 
Venetoclaxd 
X -------------------------------------------------------- X 
X --------------------------------------------------- X 
X 
Pembrolizum 
ab (MK- 
3475)e 
X 
Q3 
wks 
 
 
Q3 
wks 
from 
C1D8 
 
 
 
 
 
 
 
 
 
Q3 weeks 
Informed 
consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical 
history 
X 
 
 
 
 
 
 
 
 
 
 
 
EKG 
Xf 
 
 
 
 
 
 
 
 
 
 
 
ECHO or 
MUGA 
X 
 
 
 
 
 
 
 
 
 
 
 
Vital signs 
and physical 
exam 
X 
X 
 
Xx 
 
 
 
 
 
 
 
 
 
 
X 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 

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Pre- 
Study 
(21 
days) 
Cycle 1 
 
 
 
Cycle 2- 6 
Maintenance 
Therapy 
Cycles 7-30 
After 
1 
year 
Relaps 
e 
End 
of 
Study 
/ 
Progr 
essio 
n 
Post 
- 
Trea 
tme 
nt 
Foll 
ow- 
Upb 
Baseline 
D1 
(+/- 
3 
days) 
D 
2 
D 
3 
D 
4 
D 
5 
D 
6 
D 
7 
D8 
D14 
 
D 
28 
D1 
(+/- 3 
days) 
 
D 
2 
 
D 
3 
 
D 
4 
 
D 
5 
 
D 
6 
 
D 
7 
 
D 
8 
 
 
D14 
 
 
D21 
D28 
Days 1-28 
(+/- 3 days ) 
Pregnancy 
Testg 
X 
 
 
 
 
 
 
 
 
 
 
 
Weight 
X 
 
 
X 
 
 
 
 
 
 
 
 
 
Concurrent 
Medications 
X 
X 
Performance 
status 
X 
 
Xh 
 
 
 
 
 
 
 
 
 
Comprehensi 
ve Serum 
Chemistry 
Paneli, k 
X 
X 
X 
X 
X 
 
Xx 
 
X 
 
 
 
 
 
 
 
 
 
X 
X 
CBC with 
Differentialj, 
k 
X 
X 
X 
X 
X 
 
Xx  
X 
 
 
 
 
 
 
 
 
 
X 
X 
Pregnancy 
Test – Urine 
or Serum β- 
HCGi 
X 
 
 
 
 
 
 
 
 
 
 
 
Urinalysis 
X 
X 
 
Xx 
 
 
 
 
 
 
 
 
 
 
X 
TSH; T3, 
T4k 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
Amylase and 
lipasef 
X 
 
 
 
 
 
 
 
 
 
 
 

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Pre- 
Study 
(21 
days) 
Cycle 1 
 
 
 
Cycle 2- 6 
Maintenance 
Therapy 
Cycles 7-30 
After 
1 
year 
Relaps 
e 
End 
of 
Study 
/ 
Progr 
essio 
n 
Post 
- 
Trea 
tme 
nt 
Foll 
ow- 
Upb 
Baseline 
D1 
(+/- 
3 
days) 
D 
2 
D 
3 
D 
4 
D 
5 
D 
6 
D 
7 
D8 
D14 
 
D 
28 
D1 
(+/- 3 
days) 
 
D 
2 
 
D 
3 
 
D 
4 
 
D 
5 
 
D 
6 
 
D 
7 
 
D 
8 
 
 
D14 
 
 
D21 
D28 
Days 1-28 
(+/- 3 days ) 
Bone 
marrow 
aspiration 
(FTL3 
mutations, 
karyotype) 
X 
 
 
 
 
 
 
 
 
 
 
 
Bone 
marrow 
aspiration in 
EDTA for 
MRDt,w 
X 
 
 
Xl 
 
 
 
 
 
 
 
 
 
 
X,m,x 
Xn,o 
X 
X 
Bone 
marrow 
aspiration in 
EDTAw 
X 
 
 
 
 
 
 
 
 
 
 
 
X,p,x 
X 
X 
X 
Blood in 
EDTAw 
X 
 
 
Xl 
 
 
 
 
 
 
 
 
 
 
X,m,x 
Xn, 
X 
X 
Blood in 
sodium 
heparinw 
X 
 
 
 
 
 
 
 
 
 
 
 
X,p.x 
X 
X 
X 
Blood in 
Streck tube w 
X 
 
 
Xl 
 
 
 
 
 
 
 
 
 
 
Xx 
X 
X 
X 

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Pre- 
Study 
(21 
days) 
Cycle 1 
 
 
 
Cycle 2- 6 
Maintenance 
Therapy 
Cycles 7-30 
After 
1 
year 
Relaps 
e 
End 
of 
Study 
/ 
Progr 
essio 
n 
Post 
- 
Trea 
tme 
nt 
Foll 
ow- 
Upb 
Baseline 
D1 
(+/- 
3 
days) 
D 
2 
D 
3 
D 
4 
D 
5 
D 
6 
D 
7 
D8 
D14 
 
D 
28 
D1 
(+/- 3 
days) 
 
D 
2 
 
D 
3 
 
D 
4 
 
D 
5 
 
D 
6 
 
D 
7 
 
D 
8 
 
 
D14 
 
 
D21 
D28 
Days 1-28 
(+/- 3 days ) 
Bone 
marrow core 
biopsyw 
Xo 
 
 
 
 
 
 
 
 
 
 
 
Xo,p,x 
Xo 
Xo 
X 
Bone 
marrow 
biopsy as 
part of 
standard of 
care 
X 
 
 
Xr, l 
 
 
 
 
 
 
 
 
 
 
Xr,x 
Skin Punch 
Biopsyw 
X 
 
 
 
 
 
 
 
 
 
 
 
Stool,w 
X 
 
X(+/ 
- 3 
days 
) 
 
 
 
 
 
 
 
 
 
 
Adverse 
Events 
X 
X 

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Pre- 
Study 
(21 
days) 
Cycle 1 
 
 
 
Cycle 2- 6 
Maintenance 
Therapy 
Cycles 7-30 
After 
1 
year 
Relaps 
e 
End 
of 
Study 
/ 
Progr 
essio 
n 
Post 
- 
Trea 
tme 
nt 
Foll 
ow- 
Upb 
Baseline 
D1 
(+/- 
3 
days) 
D 
2 
D 
3 
D 
4 
D 
5 
D 
6 
D 
7 
D8 
D14 
 
D 
28 
D1 
(+/- 3 
days) 
 
D 
2 
 
D 
3 
 
D 
4 
 
D 
5 
 
D 
6 
 
D 
7 
 
D 
8 
 
 
D14 
 
 
D21 
D28 
Days 1-28 
(+/- 3 days ) 
Survival 
Statusq 
 
 
 
 
 
 
 
 
 
 
 
X 
a If the patient does not have a CR/CRi before starting this part of the study, the patient should go off study. 
b 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. 
c Administered at 75 mg/mL2 IV over 10-40 min daily or SubQ. Treatment cycle begins on Day 1 of azacitidine administration. Days 5-2-2 schedule allowed if weekend administration not available. Allow at least 1 hour after 
Pembrolizumab completion when given on same day. 
d Administered daily at 400 mg orally. Can be interrupted for 14 days to allow for morphologic leukemia and count recovery. From cycle 2+, should be given from either Days 1-21 or Days 1-28 depending on count recovery. 
e Only for patients randomized to the intervention arm at 200 mg. Started on Cycle 1 Day 8, then Q3W (+/- 3 days). During the maintenance phase, MK-3457 is administered Q3W (+/- 3 days) for up to 2 years for a maximum of 36 doses. 
f As clinically indicated after Pre-study assessment. 
g 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 treatment arms. If the urine test is positive or cannot be confirmed as negative, a 
serum pregnancy test will be required. 
h Note: Performance status evaluations are based on a 4-week cycle. At minimum, performance status should be evaluated at the beginning of every cycle. 
i Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, LDH, phosphorus, potassium, total protein, SGOT [AST], SGPT [ALT], sodium, uric acid. 
j 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. 
k CBC, CMP, TSH should be performed at baseline and <72 hours prior to each dose of MK-3475 (pembrolizumab) for those on Intervention arm. Additional T3 and T4 tests should be performed as clinically indicated, if TSH is 
abnormal, or if symptoms are suggestive of thyroid dysfunction. 
l To be collected ideally between Day 21 and 28, and as close to day 21 as possible. However, if this was not done for any reason at this time, it must be collected before starting Cycle 2. Bone marrow exam must be done between day 21 
and day 42 of cycle 1 even if there is no count recovery with the exact timing of evaluation up to investigator, as long as bone marrow exam is done prior to starting cycle 2. 
m To be collected after 2nd, 4th, and 6th cycles. 
n To be collected every 3 months during maintenance phase. 
o Optional. 
p Collected after 4th and 6th cycles 
q Information will be collected via questionnaire conducted with a phone call. 
r Standard of care bone marrow biopsy will be collected after 1st, 2nd, 4th, and 6th cycles 
s Azacitidine will be administered on Days 1-7 or days 5-2-2 (to avoid weekend administration). 
t. Bone marrow aspirate is required to be submitted for central MRD testing as outlined in Section 5. Submit to Brent Wood Lab, 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. 
w. Refer to Section 5.1 Summary Table for Specimen Collection 
x. +/- 3 days 

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12 MEASUREMENT OF EFFECT 
 
Responses in AML patients will be assessed using ELN 2017 response criteria (Dohner et al., 
2017) (Appendix D) given shortcomings of the International Working Group 2003 criteria in the 
era of MRD assessment (Cheson et al., 2003; Bloomsfield et al., 2018). 
 
12.1 
Definitions 
 
Evaluable for toxicity. All patients will be evaluable for toxicity from the time of their first 
treatment with azacitidine + venetoclax 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). To account for the 
expected drop-out that might occur during induction before primary end point assessment, we 
would add another 20% patients to enroll in each arm, for a total planned enrollment of 76 
patients. 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 
 
12.2 
Hematologic Response 
 
12.2.1 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 
 
12.2.2  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 
 
12.2.3 CR with incomplete count recovery (CRh or CRi): All CR criteria except for residual 
neutropenia (<1.0 × 109/L [1000/μL]) or thrombocytopenia (<100 × 109/L [100,000/μL] 
 
12.2.4 CR with partial recovery count (CRh): All CR criteria with residual neutropenia > 0.5 
x 109/L [1000/μL]) and thrombocytopenia (>50 × 109/L [100,000/μL] (DiNardo et al., 
2018b). 
 
12.2.5. Hematologic improvement: Erythroid response (E) (pretreatment Hgb <11 g/dL) Hgb 
increase by >/=1.5 g/dL, Platelet response (P) (pretreatment platelets <100 x109/L) Absolute 
increase of >/=30 x 109/L for patients starting with > 20 x 109/L platelets Increase from < 20 
x 109/L to > 20 x 109/L and by at least 100% 
 
12.2.6. Morphologic leukemia-free state (MLFS): Bone marrow blasts <5%; absence of blasts 
with Auer rods; absence of extramedullary disease; no hematologic recovery required. 

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(Marrow should not merely be “aplastic”; at least 200 cells should be enumerated or 
cellularity should be at least 10%) 
 
12.2.7. 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: 
x Neutrophil count ≥1.0 × 109/L. 
x Platelet count ≥100 × 109/L. 
x Reduced hemoglobin concentration or hematocrit has no bearing on remission status. 
x Leukemic blasts must not be present in the peripheral blood. 
 
Bone Marrow Aspirate and Biopsy: 
x Cellularity of bone marrow biopsy must be >20% with maturation of all cell lines. 
x <5% blasts 
x Auer rods must not be detectable. 
 
Extramedullary leukemia, such as CNS or soft tissue involvement, must not be present. 
 
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 
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 with stable disease at 
their discretion if they judge the patient to be deriving clinical benefit on study, that is defined by 
transfusion independence (defined as at least 8 consecutive weeks without transfusions of either 
red blood cells or platelets for patients who were receiving transfusions prior to or during 
therapy) or by hematologic improvement criteria as listed in section 12.2.5 of the protocol. 
 
12.2.8 MRD Response 
Refer to section 9.1.1 
12.3 
Other Response Parameters 
 
x 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. 

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13 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). 
 
13.1. 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. 
 
 
During the Phase 2 portion of the study, the Protocol Principal Investigator will have, at a 
minimum, quarterly conference calls with the Study Investigators and the CTEP Medical 
Officer(s) to review accrual, progress, and pharmacovigilance. 
 
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. 
 
13.2. 
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: 
 
x 
Site staff will need to be registered with CTEP and have a valid and active CTEP-IAM 
account, and 
x 
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 

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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. 
 
13.2.1. 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. 
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. 
 
 
 
13.2.2. 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 

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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. 
 
An End of Study CRF is to be completed by the PI, and is to include a summary of study 
endpoints not otherwise captured in the database, such as (for phase 1 trials) the recommended 
phase 2 dose (RP2D) and a description of any dose-limiting toxicities (DLTs). 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). 
 
13.3. 
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. 

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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. 
 
13.4. 
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: 
 
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. 
 
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”): 

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a. 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. 
 
b. 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. 
 
c. 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 approval, and commercialization of its own 
Agent. 
 
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. 
 
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. 
 
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. 
 
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 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 

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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. 
 
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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 
x 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. 
x 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. 
x 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 and keep 
it with you in case you go to 
the emergency room. 
Patient Name: 
Diagnosis: 
Study Doctor: 
Study Doctor Phone #: 
NCI Trial #: 10334 
Study Drug(S): MK-3475 (pembrolizumab), 
venetoclax, and azacitidine 
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 hematologic recovery (CRi): All CR criteria except for residual neutropenia 
(<1.0 × 109/L [1000/μL]) or thrombocytopenia (<100 × 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 
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 

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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 
“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 
PATIENT DIARY 
 
 
PATIENT’S MEDICATION DIARY – Venetoclax – CYCLE 1 
 
Today’s date  
 
Agent 
 
Patient Name 
(initials acceptable)  Patient Study ID 
 
 
INSTRUCTIONS TO THE PATIENT: 
1. 
You will take your dose of venetoclax every single day at the same time for the first 28 days of the first cycle (Days 1-28). On 
Day 1 you will take 100mg daily dose. On Day 2 you will take 200 mg daily dose. On Days 3-28 you will take 400 mg daily 
dose. 
2. Take venetoclax with a meal.. You should swallow the tablets whole. Do not chew. 
3. 
If you have any comments or notice any side effects, please record them in the Comments column. 
4. 
Please return the forms to your physician when you go for your next appointment. 
 
 
Day 
 
 
Date 
 
What time was 
dose taken? 
# of 100mg tablets taken 
Comments 
1 
 
 
 
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CTEP-assigned Protocol #_10334 
 
Local Protocol #  
  

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Physician’s Office will complete this section: 
1. 
Date patient started protocol treatment  
 
2. 
Date patient was removed from study  
 
3. 
Patient’s planned total daily dose  
 
 
4. 
Total number of pills taken this month (each size)  
 
5. 
Physician/Nurse/Data Manager’s Signature  
 
 
Patient’s signature:  
 

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PATIENT’S MEDICATION DIARY – Venetoclax – CYCLE 2+ 
 
Today’s date  
 
Agent 
 
Patient Name 
(initials acceptable)  Patient Study ID 
 
 
INSTRUCTIONS TO THE PATIENT: 
1. 
Complete one form for each month. 
2. 
You will take your dose of venetoclax on Days 1-28 or Days 1-21 depending on your blood count of Cycle 2 onwards. . 
3. Take venetoclax with a meal.. You should swallow the tablets whole. Do not chew. 
4. 
If you have any comments or notice any side effects, please record them in the Comments column. 
5. 
Please return the forms to your physician when you go for your next appointment. 
 
Day 
 
Date 
What time was 
dose taken? 
# of100mg tablets taken 
Comments 
 
1 
 
 
 
2 
 
 
 
3 
 
 
 
4 
 
 
 
5 
 
 
 
6 
 
 
 
7 
 
 
 
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28 
 
(-) 
 
Physician’s Office will complete this section: 
1. 
Date patient started protocol treatment  
 
2. 
Date patient was removed from study  
 
3. 
Patient’s planned total daily dose 
CTEP-assigned Protocol #_10334 
 
Local Protocol #  
 

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Patient’s signature:  
 
 
4. 
Total number of pills taken this month (each size)  
 
5. 
Physician/Nurse/Data Manager’s Signature  
 

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APPENDIX F 
CUTTING AND EMBEDDING SKIN 
 
Skin Specimens - General Considerations for FFPE tissue block preparations: It is crucial to 
maintain vertical orientation at all times in the sections. 
A. Ink can be used to highlight areas of the specimen, including how to orient it when 
embedding (e.g., inked dermal/subcutaneous margins). Black or blue inks are 
preferred. 
B. Diagrams should be used for any difficult or complicated biospecimens. 
 
Skin Shave Biopsies 
A. Principle: Ink the dermal/subcutaneous margins of all skin specimens. 
 
 
 
 
B. Processing the Specimen 
1. The specimen type (shave) dimensions (including depth and surface appearance) 
are described. The specimen is usually oval and relatively flat. The edges may 
curl secondary to retraction of the dermis. Specimens larger than 4 mm in 
diameter should be bisected, larger than 6 mm in diameter should be trisected, 
larger than 8 mm should be quadrisected or serially sectioned, and end sections 
submitted in a separate cassette. The vertical orientations should be maintained 
by making cuts perpendicular to the surface at 2-3 mm intervals. 
 
2. For small shaves < 4mm, the intact specimen should be placed on edge (aka: cut 
side down) in a cassette. 
 
3. For larger shaves > 4mm and < 8 mm, the sections should be placed in a single 
cassette on edge. 
 
4. For shaves > 8 mm, the sections should be placed in 2 or more cassettes with the 
ends in 1 cassette. 

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Skin Punch Biopsies 
A. Principle: Punch biopsies are performed to completely excise small lesions or to sample 
large lesions. Punches can range from 2-8 mm in diameter. 
 
 
 
B. Processing the specimen 
 
1. The type of specimen (punch biopsy) is described including diameter, depth, and 
skin color. Lesions are described including size, type (macular, papular, 
vesicular, plaque), borders (well-circumscribed, irregular), color, shape 
(verrucous, lobulated, bosselated). 
 
2. Apply ink to the cut surfaces of all punch biopsies. 
 
3. See below instructions for specific sectioning details 

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Punch Biopsy < 4mm in diameter
Lay specimen on its side intact in a cassette
Punch Biopsy > 4mm in diameter
Bisect punch lengthwise and place both halves with cut 
surface from middle of specimen face down in cassette.
Embedding – Summary
Skin shave biopsies
Orientation: Embed the cut surface of the skin sliver to be positioned perpendicularly so that all layers 
will be displayed. Shave biopsies often will resemble “crescent” shapes when embedded. Use the 
epidermal surface to assist orientation.

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When a specimen is inked, all inked edges should face the same direction in the block face. 
 
 
Skin punch biopsies - not bisected: 
Orientation: Embed with the long side with epidermal disc on edge to show epidermis, dermis and 
subcutaneous fat layer in cross-section. The skin pieces should be placed at a slight angle with the 
epidermis facing all in the same direction (multiple pieces). The specimen and the epidermis must be 
pressed to lie in the same plane. The entire epidermal edge must be present on the completed slides. 
 
Skin punch biopsies - bisected 
Orientation: Embed cut surfaces down, long side with epidermal disc on edge to show epidermis, dermis 
and subcutaneous fat layer in cross-section. 
 
Notes: 
1. Many times, special instructions for embedding are noted, especially if the histology laboratory 
has a specific SOP for embedding skin lesions. 
2. Identify the tissue in the cassette. Use submitted descriptions regarding the gross appearance of 
the biopsied specimen (e.g., features of the lesion may include color, ulceration, polypoid 
configuration). 
3. Check if the specimen was inked and if there is a description of the reason for inking (e.g., 
identify the cut surface or the dermal/subcutaneous margin). If the specimen is inked be sure to 
follow embedding instructions (e.g., inked side down). 
4. Choose a mold that is of adequate size for the dimensions of the tissue. 
5. If you re embedding very small samples, use a magnifying glass to arrange them on edge. 
6. If the edges of the skin specimen curl due to retraction (commonly seen with shave biopsies), 
uncurl the tissue gently with forceps and warm them on the embedding center before embedding 
on edge. Skin shave biopsies should be straightened before paraffin hardens completely. 
 
Slide preparation - The FFPE tissue block will be trimmed or “faced” until the full outline of the tissue 
is visible. Cut at 3-4 microns. 
 
For layered skins (e.g., skin shave biopsies >4 mm), the epidermis should be facing toward the 
embedding unit, cassette number facing to the right. The skin pieces should be placed at a slight angle 
with the epidermis facing all in the same direction (multiple pieces). The specimen and the epidermis 
must be pressed to lie in the same plane. The entire epidermal edge must be present on the completed 
slides. 

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APPENDIX G 
BONE MARROW CORE AND SKIN PUNCH BIOPSY SAMPLES 
o Tissue must be fixed in neutral-buffered formalin (no acid-based products). 
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