Clinical trial NCT03233724
Conditions: Carcinoma, Non-Small-Cell Lung Lung Cancer Non-Small Cell Lung Cancer Carcinoma, Esophageal Malignant Pleural Mesotheliomas
Keywords: PD-L1 Demethylating Agents and Histone Deacetylase (HDAC) Inhibitors DNA Hypomethylating Agent Cytidine Deaminase Checkpoint Inhibitor
Sponsor: National Cancer Institute (NCI)
Start date: 2018-04-11; Completion date: 2021-10-26
Enrollment: 9 participants; Allocation: NON_RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
Background:

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Lung cancer is the leading cause of cancer-related death in the United States. Most people with lung cancer are already in the advanced stages of the disease by the time they see a doctor. Researchers want to see if combining an approved drug with two new drugs can help.

Objective:

To study if tetrahydrouridine-decitabine (THU-DAC) with pembrolizumab is safe and effective in people with non-small cell lung cancer that cannot be removed by surgery.

Eligibility:

People 18 years and older who have NSCLC that cannot be removed by surgery

Design:

Participants will be screened with

* Medical history
* Physical exam
* Blood and urine tests
* Tests of heart and lung function

They may have a small tumor sample taken (biopsy). They may have tumor scans.

Before starting treatment, participants will repeat the screening tests. They will also give a stool sample.

The study will be done in 3-week cycles for up to 6 cycles.

* Participants will take the 2 study drugs by mouth 3-5 days a week.
* Participants will get pembrolizumab in a vein for 30 minutes 1 day each cycle.

Participants will keep a study medication diary.

During cycle 1, participants will have blood taken multiple times on days 1 and 2.

Every 3 cycles, participants will repeat screening tests.

Participants will have a mandatory tumor biopsy.

When they finish treatment, participants will have a physical exam and blood tests.

Detailed description:
Background:

* Non-small cell lung cancers (NSCLC) esophageal cancer (EsC) and malignant pleural mesotheliomas (MPM) account for approximately 185,000 deaths annually in the United States, with over two thirds of patients presenting with advanced, incurable disease. 1st-line platinum-based chemotherapy for advanced NSCLC, esophageal cancer (ESCs) or malignant pleural mesothelioma (MPM) produces transient responses at best, with most patients succumbing to disease within 12-16 months following diagnosis.
* Recent randomized clinical trials have demonstrated response rates approximating 20% in unselected patients with advanced NSCLC or EsC, and nearly 45% in patients with tumors exhibiting high level expression of programmed death ligand 1 (PD-L1) following administration of pembrolizumab, a humanized monoclonal anti-Programmed cell death protein 1 (PD-1) antibody.
* Approximately 17% of unselected MPM patients have exhibited objective responses following administration of pembrolizumab or other PD-1 inhibitors.
* Preclinical studies have demonstrated that epigenetic drugs such as deoxyribonucleic acid (DNA) demethylating agents and histone deacetylase (HDAC) inhibitors can prime cancer cells and tumor microenvironments thereby enhancing efficacy of immune checkpoint inhibitors.
* Although a potent DNA demethylating agent, Decitabine has poor bioavailability and inconsistent distribution in solid tumors due to rapid inactivation by cytidine deaminase (CDA) which is present in high levels in many organs.
* Recent studies in rodents and nonhuman primates, as well as a Phase 1 clinical trial (NCT#01685515) in patients with sickle cell disease have demonstrated that oral tetrahydrouridine (THU), an inhibitor of CDA, significantly enhances bioavailability/solid-tissue-distribution of low dose oral Decitabine (DAC), thereby enhancing systemic DNA demethylation with acceptable toxicities.
* Preliminary results of recent clinical trial suggest that oral DAC-THU can increase the frequency and magnitude of responses to immune checkpoint inhibitors in lung cancer patients with low or absent intra-tumoral PD-L1 expression.
* These data support further evaluation of oral DAC-THU in combination with immune checkpoint inhibitors for therapy of thoracic malignancies.

Objectives:

Phase I

-To define pharmacokinetics, toxicities and maximum tolerated dose of oral DAC-THU in combination with pembrolizumab in patients with inoperable, or unresectable locally advanced or metastatic NSCLC, EsC, or MPM.

Phase II

-To determine clinical response by Response Evaluation Criteria in Solid Tumors (RECIST) criteria to oral DAC-THU in combination with pembrolizumab in patients with inoperable, or unresectable, locally advanced or metastatic NSCLC, EsC, or MPM.

Eligibility:

Inclusion Criteria

* Male or female, 18 years or older with histologically or cytologically proven, inoperable, or unresectable locally advanced, or metastatic NSCLC, EsC, or MPM.
* Measurable disease.
* Patients with high PD-L1 expression ((Bullet) 50%) and low PD-L1 expression (0-49%) in cancer cells by immunohistochemistry are eligible.
* NSCLC patients with no prior systemic treatment, or those with prior first line treatment including an immune checkpoint inhibitor are eligible for study.
* MPM patients who have received, refused, or are ineligible for first line chemotherapy are eligible.
* Patients with EsC including Seiwert-Stein Type I and Type II gastro-esophageal junction (GEJ) carcinomas who have received or refused standard of care first line therapy and/or targeted therapy are eligible.
* Patients who received DNA demethylating agents or PD-1/PD-L1 inhibitors for another malignancy may be eligible for study if there were no dose-limiting immune related events, and there has been either no clinical evidence of disease or minimal residual disease that has been stable for at least three years.
* Willingness to undergo tumor biopsies if safely accessible per PI discretion before and after treatment.
* Eastern Cooperative Oncology Group (ECOG) performance status 0 2.
* No evidence of unstable or decompensated myocardial disease; adequate pulmonary reserve.
* Adequate renal, hepatic and hematopoietic function.

Exclusion Criteria

* Patients with any targetable mutation for which there is approved first, or second line therapy.
* Serious cardiovascular conditions.
* Active Hepatitis A, Hepatitis B or Hepatitis C.
* Human immunodeficiency virus (HIV) or acquired immunodeficiency syndrome (AIDS)-related illness.
* Other active infection requiring systemic therapy.
* Pregnant or breastfeeding women.
* Patients who are receiving systemic corticosteroids.
* Patients receiving another investigational agent.
* Another malignancy.

Design:

* The Phase I component will be a standard 3+3 design combining high and low PD-L1 expressers with incremental dose escalation of oral DAC-THU to define maximum tolerated dose (MTD).
* Simon 2-stage design for Phase II studies will be used to determine clinical response at the MTD.
* Patients will receive oral DAC-THU- on T-W for two weeks out of every 3 for 9 weeks
* Pembrolizumab will be administered on Wednesday, Thursday or Friday at a fixed intravenous dose of 200 mg every 3 weeks.
* One cycle is three weeks; one course is 9 weeks. Treatment evaluation using RECIST 1.1 every 10 +/- 1 weeks.
* Those patients exhibiting disease progression or unacceptable toxicities will be removed from study. Patients exhibiting stable disease or disease regression will be offered an additional course of therapy followed by treatment evaluation. Treatment will continue in this manner until off-study criteria have been met.
* Once the MTD for DAC/THU has been identified, the MTD dose level will be expanded by 4 patients to confirm its safety. Then, including these 10 patients at the MTD, a total of 10 NSCLC patients with high (50% or greater) intratumoral PD-L1 expression and 10 NSCLC patients with low (0-49%) intratumoral PD-L1 expression will be accrued to the first stage of each of two separate Phase II cohorts using individual Simon optimal designs. If 5 or more patients of the 10 first stage NSCLC patients in the high PD-L1 cohort respond to treatment, the cohort will be expanded to 23 patients. If 11 of 23 of these patients respond to treatment, the trial will be deemed positive for NSCLC with high PD-L1 expression. If 2 or more of the 10 first stage NSCLC patients in the low PD-L1 expression cohort respond to treatment, the cohort will be expanded to 29 patients. If 6 or more of these 29 patients experience a response, the trial will be deemed positive for NSCLC with low PD-L1 expression. Up to 10 EsC patients, including those considered to be part of the Phase I component after the MTD has been identified, will be enrolled into a separate cohort to examine responses to DAC-THU/pembrolizumab at the MTD. If 2 or more of these 10 EsC patients respond to treatment, these findings may warrant an amendment or a separate Phase II trial to determine response rates to DAC-THU/pembrolizumab in EsC patients. Similarly, if 2 or more of 10 MPM patients respond to treatment, these findings may warrant an amendment or a separate Phase II trial to determine response rates to DACTHU/pembrolizumab in MPM.
* Biopsies of index lesions will be obtained at baseline and at treatment evaluation following the first course of therapy for analysis of pharmacodynamic endpoints.
* Patients will be followed for toxicity for at least 30 days after treatment has been discontinued, start of new anti-cancer treatment or until death, whichever occurs first.

Arms: arm 1: Decitabine (DAC)-Tetrahydrouridine (THU) + pembrolizumab at escalating doses arm 2: Decitabine (DAC)-Tetrahydrouridine (THU) + pembrolizumab at the dose established in Arm 1
Interventions: intervention 1: Decitabine (DAC) intervention 2: Tetrahydrouridine (THU) intervention 3: Pembrolizumab
Intervention descriptions: intervention 1: Administered orally on two consecutive days (preferably Tuesday and Wednesday) for two weeks out of three weeks x 9 weeks intervention 2: Administered orally on two consecutive days (preferably Tuesday and Wednesday) for two weeks out of three weeks x 9 weeks intervention 3: 200 mg intravenous (IV) once a day every Wednesday, Thursday or Friday every 3 weeks.

Protocol documents:
Prot_SAP_000.pdf: 
Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
1
Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
NIH Protocol #: 17C0140
Version Date: 08/01/2022
NCT Number: NCT03233724
Title: Phase I/II Evaluation of Oral Decitabine/Tetrahydrouridine as Epigenetic Priming for 
Pembrolizumab Immune Checkpoint Blockade in Inoperable Locally Advanced or Metastatic 
Non-Small Cell Lung Cancers, Esophageal Carcinomas, or Pleural Mesotheliomas
NCI Principal Investigator:
David S. Schrump, M.D., M.B.A.
Thoracic Surgery Branch (TSB), NCI, NIH
10 Center Drive, Room 4-3942
Bethesda, MD 20892-1502
Phone: 240-760-6239
Email: schrumpd@mail.nih.gov
Investigational Agents:
Drug Names:
Decitabine (DAC)
Tetrahydrouridine (THU)
Pembrolizumab
Sponsor:
Center for Cancer Research, NCI
IND Number:
126827
Manufacturer:
IriSys, LLC and 
DCTD, NCI
KP Pharmaceutical 
Technology Inc.
Generic
Supplier:
CC Pharmacy
CC Pharmacy
CC Pharmacy
Commercial Agents: None


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
2
PRÉCIS
Background:

Non-small cell lung cancers (NSCLC), esophageal carcinomas (EsC) and malignant pleural 
mesotheliomas (MPM) account for approximately 185,000 deaths annually in the United 
States, with over two thirds of patients presenting with advanced, incurable disease. 1st-line 
platinum-based chemotherapy for advanced NSCLC, EsC, or MPM produces transient 
responses at best, with most patients succumbing to disease within 12-16 months following 
diagnosis. 

Recent randomized clinical trials have demonstrated response rates approximating 20% in 
unselected patients with advanced NSCLC or EsC, and nearly 45% in patients with tumors 
exhibiting high level expression of programmed death ligand 1 (PD-L1) following 
administration of pembrolizumab, a humanized monoclonal anti-PD-1 antibody.

Approximately 17% of unselected MPM patients have exhibited objective responses 
following administration of pembrolizumab or other PD-1 inhibitors. 

Preclinical studies have demonstrated that epigenetic drugs such as DNA demethylating 
agents and histone deacetylase (HDAC) inhibitors can “prime” cancer cells and tumor 
microenvironments thereby enhancing efficacy of immune checkpoint inhibitors. 

Although a potent DNA demethylating agent, Decitabine has poor bioavailability and 
inconsistent distribution in solid tumors due to rapid inactivation by cytidine deaminase 
(CDA) which is present in high levels in many organs. 

Recent studies in rodents and nonhuman primates, as well as a Phase 1 clinical trial 
(NCT#01685515) in patients with sickle cell disease have demonstrated that oral 
tetrahydrouridine (THU), an inhibitor of CDA, significantly enhances bioavailability/solid-
tissue-distribution of low dose oral DAC, thereby enhancing systemic DNA demethylation  
with acceptable toxicities.

Preliminary results of a recent clinical trial suggest that oral DAC-THU can increase the 
frequency and magnitude of responses to immune checkpoint inhibitors in lung cancer 
patients with low or absent intra-tumoral PD-L1 expression.

These data support further evaluation of oral DAC-THU in combination with immune 
checkpoint inhibitors for therapy of thoracic malignancies.
Objectives:
Phase I

To define pharmacokinetics, toxicities and maximum tolerated dose of oral DAC-THU in 
combination with pembrolizumab in patients with inoperable, or unresectable, locally 
advanced or metastatic NSCLC, EsC, or MPM.
Phase II

To determine clinical response by RECIST criteria to oral DAC-THU in combination 
with pembrolizumab in patients with inoperable, or unresectable, locally advanced or 
metastatic NSCLC, EsC, or MPM.


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
3
Eligibility:
 Inclusion Criteria

Male or female, 18 years or older with histologically or cytologically-proven, inoperable, 
or unresectable locally advanced, or metastatic NSCLC, EsC, or MPM.

Measurable disease.

Patients with high PD-L1 expression (≥ 50%) and low PD-L1 expression (0-49%) in 
cancer cells by immunohistochemistry are eligible. 

NSCLC patients with no prior systemic treatment, or those with prior first line treatment 
including an immune checkpoint inhibitor are eligible for study.

MPM patients who have received, refused, or are ineligible for first line chemotherapy 
are eligible.

Patients with EsC including Seiwert-Stein Type I and Type II gastro-esophageal junction 
(GEJ) carcinomas who have received or refused standard of care first line therapy and/or 
targeted therapy are eligible.

Patients who received DNA demethylating agents or PD-1/PD-L1 inhibitors for another 
malignancy may be eligible for study if there were no dose-limiting immune related 
events, and there has been either no clinical evidence of disease or minimal residual 
disease that has been stable for at least three years.

Willingness to undergo tumor biopsies if safely accessible per PI discretion before and 
after treatment. 

ECOG performance status 0 – 2.

No evidence of unstable or decompensated myocardial disease; adequate pulmonary 
reserve. 

Adequate renal, hepatic and hematopoietic function.
Exclusion Criteria

Patients with any targetable mutation for which there is approved first or second line 
therapy.

Serious cardiovascular conditions.  

Active Hepatitis A, Hepatitis B or Hepatitis C. 

Human immunodeficiency virus (HIV) or acquired immunodeficiency syndrome (AIDS)-
related illness. 

Other active infection requiring systemic therapy.

Pregnant or breastfeeding women. 

Patients who are receiving systemic corticosteroids.

Patients receiving another investigational agent.

Another malignancy.
Design:

The Phase I component will be a standard 3+3 design combining high and low PD-L1 
expressers with incremental dose escalation of oral DAC-THU to define MTD.

Simon 2-stage design for Phase II studies will be used to determine clinical response at the 
MTD. 


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
4

Patients will receive oral DAC-THU on T-W for two weeks out of every 3 for 9 weeks. 

Pembrolizumab will be administered on Wednesday, Thursday or Friday at a fixed 
intravenous dose of 200 mg every 3 weeks. 

One cycle is three weeks; one course is 9 weeks. Treatment evaluation using RECIST 1.1 
every 10 +/- 1 weeks.

Those patients exhibiting disease progression or unacceptable toxicities will be removed 
from study. Patients exhibiting stable disease or disease regression will be offered an 
additional course of therapy followed by treatment evaluation. Treatment will continue in 
this manner until off-study criteria have been met. 

Once the MTD for DAC/THU has been identified, the MTD dose level will be expanded 
by 4 patients to confirm its safety. Then, including these 10 patients at the MTD, a total of 
10 NSCLC patients with high (50% or greater) intratumoral PD-L1 expression and 10 
NSCLC patients with low (0-49%) intratumoral PL-L1 expression will be accrued to the 
first stage of each of two separate Phase II cohorts using individual Simon optimal designs. 
If 5 or more patients of the 10 first stage NSCLC patients in the high PD-L1 cohort respond 
to treatment, the cohort will be expanded to 23 patients. If 11 of 23 of these patients respond 
to treatment, the trial will be deemed positive for NSCLC with high PD-L1 expression. If 
2 or more of the 10 first stage NSCLC patients in the low PD-L1 expression cohort respond 
to treatment, the cohort will be expanded to 29 patients. If 6 or more of these 29 patients 
experience a response, the trial will be deemed positive for NSCLC with low PD-L1 
expression. Up to 10 EsC patients, including those considered to be part of the Phase I 
component after the MTD has been identified, will be enrolled into a separate cohort to 
examine responses to DAC-THU/pembrolizumab at the MTD. If 2 or more of these 10 EsC 
patients respond to treatment, these findings may warrant an amendment or a separate 
Phase II trial to determine response rates to DAC-THU/pembrolizumab in EsC patients. 
Similarly, if 2 or more of 10 MPM patients respond to treatment, these findings may 
warrant an amendment or a separate Phase II trial to determine response rates to DAC-
THU/pembrolizumab in MPM.

Biopsies of index lesions will be obtained at baseline and at treatment evaluation following 
the first course of therapy for analysis of pharmacodynamic endpoints.

Patients will be followed for toxicity for at least 30 days after treatment has been discontinued, 
start of new anti-cancer treatment or until death, whichever occurs first. 


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
5
TABLE OF CONTENTS
PRÉCIS............................................................................................................................................2
TABLE OF CONTENTS.................................................................................................................5
STATEMENT of COMPLIANCE ................................................................................................10
1
INTRODUCTION..................................................................................................................10
1.1
Study Objectives ..................................................................................................................10
1.1.1
Primary Objectives ....................................................................................................10
1.1.2
Secondary Objectives ................................................................................................10
1.2
Background and Rationale ...................................................................................................10
1.2.1
Background and Significance:...................................................................................10
1.2.2
Pharmacokinetics of DAC Alone Following Intravenous (IV), Subcutaneous (SC) and 
Oral Administration in Non-Human Primates.......................................................................18
1.2.3
Identification of Dose and Timing of Oral THU Required to Increase Oral 
Bioavailability of DAC in Non-Human Primates..................................................................19
1.2.4
Effects of Oral THU on DAC Oral Bioavailability and Inter-Individual Variability in 
Non-Human Primates ............................................................................................................19
1.2.5
Effects of Repeat Administration of Oral THU-DAC on Blood Counts, DNA 
Methylation and Fetal Hemoglobin Levels in Non-Human Primates ...................................20
1.2.6
Clinical Pharmacokinetics: THU Alone ....................................................................24
1.2.7
Clinical Pharmacokinetics: DAC Alone....................................................................24
1.2.8
Clinical Safety: THU Alone ......................................................................................25
1.2.9
Clinical Safety and Efficacy: Oral THU and DAC in Combination, Repeat Dose Phase 
1/2 Study in SCD...................................................................................................................26
1.2.10
Pembrolizumab......................................................................................................38
1.2.11
Preliminary Experience with Oral DAC-THU and Immune Checkpoint Inhibitors 
in NSCLC ..............................................................................................................................44
2
ELIGIBILITY ASSESSMENTAND ENROLLMENT .........................................................49
2.1
Eligibility Criteria ................................................................................................................49
2.1.1
Inclusion Criteria .......................................................................................................49
2.1.2
Exclusion Criteria ......................................................................................................50
2.1.3
Recruitment Strategies...............................................................................................51
2.2
Screening Evaluation............................................................................................................52
2.3
Participant Registration and Status Update Procedures .......................................................52
2.3.1
Treatment Assignment Procedures............................................................................53
3
STUDY IMPLEMENTATION..............................................................................................53


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
6
3.1
Study Design ........................................................................................................................53
3.1.1
Study Schema ............................................................................................................55
3.1.2
Definition of Dose Limiting Toxicity (DLT) ............................................................55
3.1.3
Dose Escalation .........................................................................................................56
3.2
Drug Administration ............................................................................................................56
3.2.1
DAC-THU .................................................................................................................56
3.2.2
Pembrolizumab..........................................................................................................59
3.2.3
Additional Modifications for Subjects with Grade 2 Infusion-Related Reactions....60
3.2.4
Severe Hypersensitivity Reactions and Flu-like Symptoms......................................60
3.2.3
CCR Self –Administered Study Drugs Policy...........................................................61
3.3
Dosing Delays and Modifications........................................................................................61
3.3.1
General Guidance for Dose Modifications................................................................61
3.3.2
Treatment Delays and Modifications for Medical Needs..........................................62
3.3.3
Dose Reductions ........................................................................................................62
3.3.4
Dosage Modification and Management of Immune-Related Adverse Events ..........62
3.3.5
Dose Modification of DAC-THU..............................................................................62
3.3.6
Dose Delay of Pembrolizumab..................................................................................64
3.4
On Study Protocol Evaluation (Appendix 3: Study Calendar) ............................................73
3.4.1
Baseline Assessments ................................................................................................73
3.4.2
Treatment Evaluation.................................................................................................74
3.4.3
Research Evaluations (Refer to Appendix 4, Unless Otherwise Indicated Below)...74
3.4.4
Follow-Up Evaluations..............................................................................................74
3.5
Costs and Compensation ......................................................................................................75
3.5.1
Costs ..........................................................................................................................75
3.5.2
Compensation ............................................................................................................75
3.5.3
Reimbursement..........................................................................................................75
3.6
Criteria for Removal from Protocol Therapy and Off Study Criteria..................................75
3.6.1
Criteria for Removal from Protocol Therapy ............................................................75
3.6.2
Off-Study Criteria......................................................................................................75
3.6.3
Lost to Follow-Up......................................................................................................76
4
CONCOMITANT MEDICATIONS/MEASURES................................................................76
4.1
Hematologic and Blood Product Support.............................................................................76
4.2
Antidiarrheals.......................................................................................................................76
4.3
Electrolyte Replacement ......................................................................................................76
4.4
Proton Pump Inhibitors ........................................................................................................76
5
CORRELATIVE STUDIES...................................................................................................77


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
7
5.1
Biospecimen Collection and Processing for Research.........................................................77
5.1.1
Pharmacokinetic (PK) Sampling ...............................................................................77
5.1.2
Studies on Tumor Tissue and Peripheral Blood ........................................................77
5.1.3
Immune Subset Analyses...........................................................................................77
5.1.4
Circulating Tumor Cells ............................................................................................77
5.2
Sample Storage, Tracking and Disposition..........................................................................78
5.2.1
Samples Managed by Dr. Figg’s Blood Processing Core (BPC) ..............................78
5.2.2
Procedures for Storage of Tissue Specimens in the Laboratory of Pathology ..........79
5.2.3
Thoracic Epigenetics Laboratory...............................................................................79
5.2.4
Trepel Laboratory ......................................................................................................79
5.2.5
Sample Destruction, End of Protocol Procedures......................................................80
6
DATA COLLECTION AND EVALUATION ......................................................................80
6.1
Data Collection.....................................................................................................................80
6.1.1
Routine Data Collection ............................................................................................80
6.1.2
Exclusions to Routine Data Collection......................................................................81
6.2
Data Sharing Plans ...............................................................................................................81
6.2.1
Human Data Sharing Plan .........................................................................................81
6.2.2
Genomic Data Sharing Plan.......................................................................................82
6.3
Response Criteria .................................................................................................................82
6.3.1
Confirmatory Measurement/Duration of Response...................................................82
6.3.2
Definitions .................................................................................................................82
6.3.3
Disease Parameters ....................................................................................................83
6.3.4
Methods for Evaluation of Measurable Disease........................................................84
6.3.5
Response Criteria for Radiographic Studies..............................................................85
6.4
Toxicity Criteria ...................................................................................................................87
7
NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN .....87
7.1
Definitions............................................................................................................................87
7.2
OHSRP Office of Compliance and Training/IRB Reporting...............................................87
7.2.1
Expedited Reporting ..................................................................................................87
7.2.2
IRB Requirements for PI Reporting Continuing Review..........................................87
7.3
NCI Clinical Director Reporting..........................................................................................87
7.4
NIH Required Data and Safety Monitoring Plan .................................................................88
7.4.1
Principal Investigator/Research Team.......................................................................88
8
SPONSOR SAFETY REPORTING.......................................................................................88
8.1
Definitions............................................................................................................................88
8.1.1
Adverse Event............................................................................................................88


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
8
8.1.2
Serious Adverse Event (SAE) ...................................................................................88
8.1.3
Life-threatening .........................................................................................................89
8.1.4
Severity......................................................................................................................89
8.1.5
Relationship to Study Product ...................................................................................89
8.2
Assessment of Safety Events................................................................................................89
8.3
Reporting of Serious Adverse Events ..................................................................................90
8.4
Waiver of Expedited Reporting to CCR ..............................................................................90
8.5
Reporting Pregnancy............................................................................................................90
8.5.1
Maternal Exposure....................................................................................................90
8.5.2
Paternal Exposure ......................................................................................................91
8.6
Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND........................91
8.7
Sponsor Protocol Deviation Reporting ................................................................................91
9
CLINICAL MONITORING...................................................................................................91
10
STATISTICAL CONSIDERATIONS...................................................................................92
10.1 Methods of Analysis.............................................................................................................94
11
HUMAN SUBJECTS PROTECTIONS.................................................................................95
11.1 Rationale for Subject Selection............................................................................................95
11.2 Participation of Children ......................................................................................................95
11.3 Participation of Subjects Unable to Give Consent...............................................................95
11.4 Evaluation of Benefits and Risks/Discomforts ....................................................................95
11.4.1
Risks ......................................................................................................................95
11.4.2
Benefits..................................................................................................................96
11.5 Consent Process and Documentation ...................................................................................97
11.5.1
Consent Process for Adults Who Lack Capacity to Consent to Research 
Participation...........................................................................................................................97
12
REGULATORY AND OPERATIONAL CONSIDERATIONS...........................................97
12.1 Study Discontinuation And Closure.....................................................................................97
12.2 Quality Assurance And Quality Control ..............................................................................98
12.3 Conflict Of Interest Policy ...................................................................................................98
12.4 Confidentiality and Privacy..................................................................................................99
13
PHARMACEUTICAL INFORMATION ..............................................................................99
13.1 Decitabine.............................................................................................................................99
13.1.1
Synonyms ..............................................................................................................99
13.1.2
Description...........................................................................................................100
13.1.3
Chemical Names..................................................................................................100
13.1.4
Chemical Identification .......................................................................................100
13.1.5
Formulation..........................................................................................................100


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
9
13.1.6
Stability................................................................................................................100
13.1.7
Storage .................................................................................................................101
13.1.8
Administration .....................................................................................................101
13.1.9
Toxicities .............................................................................................................101
13.2 Tetrahydrouridine...............................................................................................................102
13.2.1
Names ..................................................................................................................102
13.2.2
Mode of Action:...................................................................................................102
13.2.3
How Supplied: .....................................................................................................102
13.2.4
Storage:................................................................................................................102
13.2.5
Stability:...............................................................................................................102
13.2.6
Route of Administration: .....................................................................................102
13.2.7
Method of Administration: ..................................................................................102
13.2.8
Toxicities: ............................................................................................................103
13.3 Pembrolizumab...................................................................................................................103
13.3.1
Description:..........................................................................................................103
13.3.2
How Supplied: .....................................................................................................103
13.3.3
Reconstitution/Dose Preparation: ........................................................................103
13.3.4
Stability/Storage: .................................................................................................103
13.3.5
Administration:....................................................................................................103
13.3.6
Toxicities: ............................................................................................................104
14
REFERENCES.....................................................................................................................105
15
APPENDICES AND SUPPLEMENTARY TABLES.........................................................113
15.1 Supplementary Tables........................................................................................................113
15.1.1
Supplementary Table 1: Cancer/Testis Genes and Their Chromosomal Locations 
(Partial List).........................................................................................................................113
15.1.2
Supplementary Table 2: Frequency of CT Gene Expression in Various Human 
Cancers as Determined by Semi-Quantitative RT-PCR......................................................117
15.2 Appendices.........................................................................................................................120
15.2.1
Appendix 1: Performance Status Criteria............................................................120
15.2.2
Appendix 2: Sample Medication Self-Administration Record and Patient Diary
121
15.2.3
Appendix 3: Study Calendar................................................................................123
15.2.4
Appendix 4: Pharmacokinetic Samples and Research Bloods ............................126


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
10
STATEMENT OF COMPLIANCE
The trial will be carried out in accordance with International Conference on Harmonisation Good 
Clinical Practice (ICH GCP) and the following: 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 
CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812) 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training.
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study.  In addition, all changes to the consent form will be IRB-approved; an 
IRB determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form.
1
INTRODUCTION 
1.1
STUDY OBJECTIVES
1.1.1
Primary Objectives

To define pharmacokinetics, toxicities and maximum tolerated dose of oral DAC-THU in 
combination with pembrolizumab in patients with inoperable, or unresectable, locally 
advanced or metastatic NSCLC, EsC, or MPM. (Phase I Component)

To determine clinical response by RECIST criteria to oral DAC-THU in combination with 
pembrolizumab in patients with inoperable, or unresectable, locally advanced or metastatic 
NSCLC, EsC, or MPM. (Phase II Component)
1.1.2
Secondary Objectives

To examine if oral DAC-THU modulates DNA methylation, as well as gene, micro-RNA, 
and endogenous retroviral expression profiles of NSCLC, EsC, and MPM cells and alters 
the tumor micro-environment.

To examine if oral DAC-THU modulates plasma tumor DNA methylation, circulating 
tumor cells and peripheral immune subsets.
1.2
BACKGROUND AND RATIONALE
1.2.1
Background and Significance: 
Lung cancer is the leading cause of cancer-related mortality worldwide and presently accounts for 
~1.8 million annual deaths globally, of which nearly 160,000 occur in the United States (1, 2).  
The vast majority of these malignancies are directly attributable to cigarette smoking. Survival is 
stage dependent, and ranges from > 80% for patients with early stage tumors to less than 15% for 
patients with locally advanced or disseminated disease (3). The majority present with inoperable 
disease; as such median overall survival for lung cancer patients is 12-16 months. Approximately 
80% of pulmonary carcinomas are non-small cell lung cancers (NSCLC) comprised primarily of 


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11
adeno-, squamous cell and large cell undifferentiated carcinomas with distinct histologic and 
molecular features. The remaining 20% are highly lethal small cell lung cancers (SCLC) (4) which 
exhibit varying degrees of neuroendocrine differentiation, and are typically widely metastatic at 
presentation with a high propensity for recurrence despite initial, often dramatic responses to 
chemotherapy (5). 
Esophageal cancer (EsC) is the sixth leading cause of cancer-related mortality worldwide, 
accounting for ~509,000 deaths annually of which nearly 16,000 occur in the US (1, 6). Esophageal 
squamous cell cancers (ESCC) predominate in high incidence areas in Asia, South America, and 
the Middle East; these neoplasms typically arise in the upper and mid-esophagus, and are 
attributable to smoking, ethanol and cultural dietary practices. Esophageal adenocarcinomas 
(EAC) predominate in Western Europe and the US; these malignancies typically arise in the distal 
esophagus and gastro-esophageal junction and are attributable to obesity, gastro-esophageal reflux, 
Barretts esophagus, and to a lesser extent, cigarette smoking. Most patients (~70%) present with 
locally advanced or metastatic disease. Survival is stage dependent, ranging from ~40% for 
localized disease, to 20% for regional disease, and 5% for disseminated disease. With the exception 
of Her2 overexpression in a small percentage of cases, esophageal cancers typically have no 
actionable mutations (7, 8).
Malignant pleural mesotheliomas (MPM) are rare, lethal neoplasms attributable primarily to 
environmental or occupational exposure to asbestos and related fibers, influenced in some cases 
by predisposing germline mutations (9-12). An estimated 40,000 individuals succumb to 
mesotheliomas worldwide each year despite asbestos being banned in many industrialized 
countries (13, 14). Most patients present with advanced, inoperable disease; median survival of 
these individuals is approximately 6 months with supportive care alone, and 12 months with 
systemic chemotherapy (15, 16). Whereas patients with limited disease burden may benefit from 
cytoreductive surgery- typically in the context of multimodality therapy, the role and extent of 
surgery in patients with MPM remains controversial (16-18). MPM typically have no actionable 
mutations.
Immune checkpoint inhibitors are revolutionizing treatment of diverse malignancies including 
NSCLC, gastroesophageal carcinomas (2), and MPM. Humanized monoclonal antibodies such as 
pembrolizumab, which inhibit engagement of programmed death ligand -1 (PD-L1) on cancer cells 
and immune regulatory cells with programmed death-1 (PD-1) receptors on activated T cells (19), 
break tolerance and mediate objective responses in approximately 20% in patients with advanced 
NSCLC and gastroesophageal carcinomas when administered as second line therapy (20, 21). 
Response rates appear to be higher in smokers/former smokers due to increased mutational burden 
and neoantigen load (22-25), as well as patients whose tumors expressed high levels of PD-L1(20, 
25). The favorable toxicity profiles, impressive, durable response rates and survival benefit of 
pembrolizumab have led to FDA approval of this immune checkpoint inhibitor for second and 
third line therapy of NSCLC and gastroesophageal carcinomas, respectively, irrespective of tumor 
PD-L1 expression status, as well as first line therapy for non-squamous NSCLC with 50% or 
greater 
expression 
of 
PD-L1 
and 
no 
EGFR 
or 
ALK 
mutations 
(http://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm526430.htm). 
Although 
presently not approved for the treatment of MPM, pembrolizumab is listed in NCCN guidelines 
for second-line treatment of these neoplasms, with far fewer toxicities compared to chemotherapy.
Epigenetic Strategies for Cancer Therapy: Alterations in chromatin structure perturb gene 
expression during malignant transformation (26). DNA demethylation results in de-repression of 


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endogenous retroviruses, loss of imprinting, and activation of cancer-testis genes, which normally 
exhibit stage-specific expression during germ cell development in testes or placenta, and either no 
expression or highly restricted expression in normal somatic tissues.  In the context of genome 
wide DNA demethylation, numerous tumor suppressor genes are silenced by site specific DNA 
hypermethylation and/or polycomb repressive complexes. These alterations in chromatin 
structure, which recapitulate epigenomic states in normal germ cells provide the rationale for 
epigenetic therapy for cancer (27-29).
Aberrant activation of cancer-testis (CT) genes (also referred to as cancer-germline genes) in 
somatic cells during malignant transformation results in expression of highly restricted tumor 
antigens that induce serologic as well as cell-mediated immune responses in cancer patients; as 
such, cancer-testis antigens (CTAs) have emerged as attractive targets for cancer immunotherapy 
(30). To date, over 200 CTAs have been identified, approximately half of which are encoded on 
the X chromosome (31) (Figure 1). CT-X chromosome (CT-X) genes are normally expressed in 
spermatogonia, and typically comprise extended families associated with inverted DNA repeats. 
The non-X CT genes tend to be expressed during later stages of germ cell differentiation (i.e. 
spermatocytes), and do not appear to be associated with extended families or inverted repetitive 
DNA sequences (32, 33). Relative to autosomal CT genes, CT-X genes are more frequently 
activated in cancer cells, and particular gene families appear to be coordinately de-repressed in a 
tumor-specific manner.
Figure 1: Location of cancer testes (CT) genes on X chromosome, with corresponding number of family 
members in parenthesis. Copied from ref (34).
In general, the frequency and magnitude of CT-X gene de-repression in human cancers coincides 
with advanced stage of disease.  Typically, tumors with CT-X mRNA copy numbers > 10% of 
testes exhibit corresponding proteins detectable by immunohistochemistry techniques.  In contrast, 
tumors with CT-X mRNA levels < 1% of testes do not have detectable antigen expression. With 
the exception of synovial sarcomas or Hodgkin Lymphomas that tend to exhibit homogeneous 
expression of NY-ESO-1 and CT-45, respectively, the majority of human cancers exhibit 
extremely focal CTA expression.  These observations, together with recent reports demonstrating 
high level CT-X gene expression in cancer stem cells (35-37), raise the possibility that CT-X 
antigens are preferentially expressed in pluripotent tumor cells.  As such, pharmacologic 
interventions that enhance expression and recognition of CT-X antigens may be novel strategies 
to eradicate cancer stem cells contributing to systemic metastases. 


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Oral Decitabine and Tetrahydrouridine: Decitabine (5-AZA-2-deoxycytidine, DAC) was 
originally synthesized in 1964 by Pliml and Storm. This analogue of deoxycytidine has been 
extensively evaluated as an experimental anti-neoplastic and differentiation agent (38).  Unlike 
cytidine analogues such as cytarabine, the sugar moiety of decitabine is unmodified, enabling 
incorporation of the analogue into DNA without termination of chain elongation or cytotoxicity at 
low doses.  Through mechanisms that have not been fully delineated, the incorporated DAC 
induces rapid ubiquitination and proteosomal degradation of the “maintenance” DNA 
methyltransferase (DNMT) 1, but not “de-novo” DNMTs 3a or 3b (39, 40). In normal cells, 
DNMT1 functions primarily to restore DNA methylation following DNA replication; in cancer 
cells, DNMT1 is frequently over-expressed and contributes to de-novo DNA methylation and 
aberrant silencing of tumor suppressor genes; DNMT1 is indispensable for viability of cancer stem 
cells (41, 42). Depletion of DNMT1 in cancer cells by DAC results in passive DNA demethylation, 
and de-novo induction of a variety of genes including those encoding cancer-testis antigens, as 
well as epigenetically silenced tumor suppressors (43). At high concentrations DAC exhibits anti-
metabolite, DNA damaging, cytotoxic effects.
In published studies we have demonstrated induction of NY-ESO-1 and MAGE-A3 as well as p16 in 
NSCLC, EsC, and MPM cells but not normal respiratory epithelial cells or fibroblasts following 72 
hour exposure to DAC (44, 45); up-regulation of NY-ESO-1 and MAGE-A3 persisted for 
approximately one month following DAC treatment, and facilitated recognition and lysis of tumor 
cells by cytotoxic T-lymphocytes (CTL) or PBL expressing T cell receptors for these CT as shown in 
Figure 2 and Figure 3) (45-48).  We have also demonstrated eradication of pulmonary metastases 
in immunocompetent mice following systemic treatment with DAC and subsequent adoptive transfer 
of CTL specific for the murine CTA, P1A (Figure 4) (49).
Figure 2: qRT-PCR analysis of CT-X gene activation in H841 lung cancer cells, immortalized human 
bronchial epithelial cells (HBEC) and normal small airway epithelial cells (SAEC) following exposure to 
DAC, 3-deazaneplanocin (DZNep) or DAC +DZNep. DAC exposure markedly up-regulates CT-X gene 
expression, which can be further increased with DZNep in lung cancer cells and to a lesser extent in HBEC. 
Up-regulation of CT-X genes in SAEC is minimal following drug treatment.


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
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Figure 3: Chromium release assays demonstrating that up-regulation of NY-ESO-1 and MAGE-A3 
expression by DAC, DZNep and DAC+DZNep enhances lysis of lung cancer cells by PBMC expressing 
recombinant T-cell receptors (TCRs) recognizing these CTAs.
Figure 4: Systemic administration of DAC followed by adoptive transfer of P1A CTL eradicates pulmonary 
metastases in immunocompetent mice.
A Phase I dose escalation study was undertaken to examine the feasibility of using gene induction 
strategies to simultaneously augment immunogenicity and apoptosis in thoracic malignancies (50). 
Because DAC is an S-phase agent with a plasma t ½ alpha and t ½ beta of 7 and 35 minutes, 
respectively, following intravenous infusion (51), the study utilized a continuous 72-hour infusion 
to optimize the duration of DAC exposure in cells which are cycling at different rates within solid 
tumors, in an attempt to recapitulate treatment conditions in our preclinical experiments. The 
primary endpoints included identification of the MTD of DAC administered under these 
conditions, clinical response at the MTD, and analysis of NY-ESO-1, MAGE-A3 and p16 
expression in tumor tissues before and after DAC treatment. The MTD of DAC administered by 
72h continuous infusion was 60 mg/m2 for patients with three or more prior treatment regimens, 
and 75 mg/m2 for individuals with two or less prior treatments. Myelosuppression was the most 
common dose limiting toxicity. No objective responses were observed in 25 evaluable patients; 
however, stabilization of disease was observed in eight individuals. Two patients with advanced 
lung cancers refractory to prior therapy remained on study for approximately 12 months until being 
removed either because of inability to image residual disease in a previously radiated hilum, or 
slow disease progression. Pharmacokinetic analysis revealed average steady-state DAC plasma 
concentrations approximating 10 to 20 ng/ml (~50 nM), which corresponded with threshold 
concentrations for target gene induction in our preclinical tissue culture experiments. 


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
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Immunohistochemistry analysis revealed induction of NY-ESO-1, MAGE-A3, and p16 in 5 of 13, 
6 of 20, and 4 of 22 patients, respectively, whose tumor biopsies were sufficient for analysis. 
Virtually all of the non–small cell lung cancers that exhibited induction of NY-ESO-1, MAGE-
A3, and/or p16 had intense focal protein expression. In contrast, NY-ESO-1 or MAGE-A3 protein 
expression was markedly uniform in one small-cell lung cancer specimen following DAC 
exposure (representative results of NY-ESO-1, MAGE-A3, and p16 immunohistochemical 
analyses are depicted in Figure 5). 
Figure 5: Representative immunohistochemical analyses demonstrating up-regulation of NY-ESO-1, MAGE-
A3 and p16 lung cancer tissues following DAC treatment.
Four patients exhibited simultaneous up-regulation of NY-ESO-1 and MAGE-3; two patients 
exhibited p16 with either NY-ESO-1 or MAGE-A3 induction, and one lung cancer patient 
exhibited simultaneous induction of NY-ESO-1, MAGE-A3, and p16 in tumor tissues following 
DAC infusion. Post-treatment antibodies to NY-ESO-1 were observed in three patients who 
exhibited NY-ESO-1 induction in biopsy specimens following DAC infusion. The lack of purified 
recombinant MAGE-A3 protein precluded analysis of immune recognition of this CTA. Of 
particular interest was a lung cancer patient with recurrent endobronchial tumor that exhibited 
prolonged stabilization of disease following DAC treatment. This patient underwent numerous 
bronchoscopic biopsies, which were of sufficient quality to enable reliable qRT-PCR analysis of 
target gene expression. NY-ESO-1 as well as MAGE-A3 mRNA copy numbers increased 
dramatically, whereas p16 expression remained relatively stable over the course of DAC treatment 
(Figure 6). 


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
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Figure 6: qRT-PCR analysis depicting progressive increases in NY-ESO-1 and MAGE-A3 expression in an 
endobronchial lung tumor in a patient receiving monthly IV DAC infusions.
Immunohistochemistry and serum ELISA (performed in a blinded manner), revealed NY-ESO-1 
expression as well as NY-ESO-1 antibodies at the time of the fifth biopsy and thereafter. These 
data are consistent with a cumulative molecular treatment effect in this individual and suggest that 
chronic exposures to DNA demethylating agents are necessary to activate gene expression in solid 
tumors. 
As this trial was nearing completion, we attempted to perform more comprehensive evaluation of 
gene expression in lung cancer cells mediated by DAC exposure. In particular, we sought to 
ascertain the effects of DAC treatment relative to gene expression profiles detected in laser-
captured tumor cells and adjacent histologically normal bronchial epithelia from resected lung 
cancer specimens. Consistent with our observations regarding DAC treatment of lung cancer cells 
in vitro (data not shown), considerable interpatient heterogeneity was observed in baseline as well 
as post-treatment gene expression profiles of laser captured tumor cells. Data derived from eight 
resected specimens were sufficient for exploratory statistical analysis, allowing detection of 
induction/repression of gene expression with threshold of 1.2-fold and using P < 0.05 (two-tailed 
t test) to define potentially important modulation of gene expression. The limited number of arrays 
from DAC-treated patients precluded rigorous statistical analysis of gene modulation in these 
individuals. Seventy-five genes were induced, whereas 324 genes were repressed > 2-fold 
following DAC treatment. Interestingly, those genes that were induced or repressed > 2- fold by 
DAC seemed to be down-regulated or overexpressed, respectively, in resected primary lung 
cancers relative to adjacent, histologically normal bronchial epithelial cells (Figure 7). Sixty of 
the 75 genes induced, and 287 of 324 genes repressed, by DAC were mapped to the PANTHER 
database. Subsequent Gene Ontology analysis identified several potentially enriched functional 
groups, including signal transduction and protein modification. Although limited and exploratory 
in nature, these data suggested that prolonged DAC exposure can shift global gene expression 
profiles in lung cancer cells from a malignant signature to one more reflective of normal respiratory 
epithelial cells (50).


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
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Figure 7: Affymetrix array analysis of gene expression in laser captured lung cancer cells from patients 
receiving IV DAC, relative to gene expression in laser captured lung cancer cells and adjacent normal 
respiratory epithelia from patients undergoing resection of their tumors. Genes that were up-regulated in 
lung cancers relative to normal respiratory epithelia were repressed in lung cancers by DAC treatment. 
Conversely, genes that were down-regulated in lung cancers relative to normal respiratory epithelia were 
induced in lung cancer cells by DAC treatment. These data suggest that DAC therapy can “normalize” gene 
expression profiles in lung cancer cells.
Inhibition of gene-silencing chromatin modifying enzymes such as DNMT1 induces terminal 
differentiation of myeloid and other cancer cells in vitro (reviewed in ref (52)). These 
differentiation-mediated cell cycle exits, which are similar to those that occur during normal tissue 
differentiation, do not require p53, and are readily induced in p53/p16-null cancer cells (53). The 
same non-cytotoxic conditions also increase differentiation of normal progenitors, but in contrast, 
increase self-renewal of normal hematopoietic stem cells (52, 54). In cancer cells, DAC treatment 
facilitates differentiation-driving transcription factor-mediated up-regulation of maturation factors 
which antagonize MYC-function (e.g., CEBPE, p27/CDKN1B) and terminate proliferation 
independent of p53. In contrast, normal stem cells express master stem cell transcription factors 
(e.g., HLF, HOXB4), and activate stem cell genes and stem cell fate in response to DAC treatments 
(52). These observations suggest that dosing of DAC to avoid systemic toxicities may be a novel 
strategy to induce growth arrest, differentiation or apoptosis of pluripotent cancer cells (52, 54).
When administered by infusion or subcutaneous injection, DAC has pharmacologic limitations 
that impede translation to chronic epigenetic therapy for solid tumors. For example, DNMT-1 
depletion requires DAC levels to overlap with cellular S-phase entry, yet the plasma half-life of 
DAC is < 20 minutes, which severely curtails the probability of such overlap. This problem is not 
solved by increasing DAC dose, since off-target effects from higher Cmax cause cytotoxicity (i.e. 
myelosuppression) that paradoxically decreases efficacy by restricting the frequency of drug 
administration. Similarly, continuous infusion is likely not the answer, since toxic increases of 
DAC can occur in some tissues while inadequate exposure remains in others. This unbalanced 
distribution may be attributable to widely diverging tissue expression of cytidine deaminase 
(CDA), the enzyme that rapidly deaminates DAC and the related DNMT1-depleting cytidine 
analogue 5-azacytidine (5Aza) (55). Moreover, CDA expression is particularly high in the 
intestines and liver, suggesting that CDA could be a barrier to oral bioavailability of cytidine 
analogues (Figure 8).


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
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Figure 8: Cytidine deaminase (CDA) expression in normal tissues (RNA-sequencing).
One potential strategy to overcome these limitations is to combine oral DAC with a CDA inhibitor 
(oral tetrahydrouridine, THU), which has been used extensively with no appreciable toxicities in 
cancer patients. THU is a competitive inhibitor of CDA with Ki values of 3-5 x 10-8M (56, 57). 
The very high affinity of THU for CDA may be due to its resemblance to a tetrahedral intermediate 
of cytidine formed by addition of water across the 3, 4 double bond (57). CDA binds THU 
approximately 4-orders of magnitude more tightly than uridine, the substrate for the reverse 
reaction catalyzed by CDA (57). Slow onset of inhibition suggests that structural reorganization 
precedes the formation of a stable enzyme-inhibitor complex (57). Preclinical studies in mice and 
nonhuman primates (described in references (58, 59) and extensively summarized in the 
Investigators Brochure) have established that oral THU administered prior to oral DAC increases 
bioavailability of DAC approximately 10-fold, prolongs plasma t½ from < 20 minutes to > 3 hours, 
and produces significant in vivo pharmacodynamic effects at a DAC oral dosage of 2.5-5 mg/m2 
(59). Preclinical studies in non-human primates and results of a recent proof of concept Phase I 
clinical trial in sickle cell disease (SCD) are summarized below. 
1.2.2
Pharmacokinetics of DAC Alone Following Intravenous (IV), Subcutaneous (SC) 
and Oral Administration in Non-Human Primates 
The pharmacokinetics of DAC were assessed in female baboons following IV (n=2), SC (n=2), 
and oral (n=2) administration. The average initial concentration (C0) following IV DAC 10 mg/m2 
(0.5 mg/kg) in baboons PA7482 and PA7472 was > 3 µM (690 ng/mL), with mean alpha and beta 
half-lives 2.13 and 180.5 minutes respectively (Figure 9). SC DAC 10 mg/m2 in PA7258 and 
PA7254 produced peak drug levels < 0.5 µM (114 ng/mL) and a wider concentration-time profile 
than IV DAC at the same dose (Figure 9). Oral DAC 200 mg/m2 (10 mg/kg) in PA7470 and 
PA7484 produced peak levels of ~0.2 µM (46 ng/mL) and ~ 0.01 µM (2.3 ng/mL) respectively, 
and a wider concentration-time profile than SC administration (Figure 9).  


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Figure 9: Plasma concentration-time curves of decitabine following intravenous (i.v.), subcutaneous 
(s.c) and oral gavage (p.o) administration to baboons.  i.v. (0.5mg/kg), s.c. (0.5mg/kg) and p.o. 
(10mg/kg).  Blood was collected at 7 time-points and plasma concentrations were determined using 
an LC/MSMS method.  The horizontal green line indicates a concentration of 0.2 µM. 
1.2.3
Identification of Dose and Timing of Oral THU Required to Increase Oral 
Bioavailability of DAC in Non-Human Primates 
In two female baboons, PA7470 and PA7484, selected for high and low bioavailability of oral 
DAC alone respectively, THU 400 mg/m2 (20 mg/kg), 60 minutes before DAC 100 mg/m2 (5 
mg/kg), produced higher DAC concentrations than THU 40 mg/m2 (2 mg/kg) (Table 1) (59). In 
the same baboons after a wash-out period, THU 400 mg/m2 60 minutes before DAC produced 
higher DAC concentrations than THU 400 mg/m2 administered simultaneously or 30 minutes prior 
to DAC (Table 1) (59). 
1.2.4
Effects of Oral THU on DAC Oral Bioavailability and Inter-Individual Variability 
in Non-Human Primates
Eight female baboons were treated with oral DAC 200 mg/m2 (10 mg/kg) (59). The median AUClast 
with oral DAC alone was 463 min*ng/ml, with a range between 190 to 6279 min*ng/mL (33-
fold variation) (Table 1). After a wash-out period of > 2 weeks, the same animals were treated 
with oral THU 400 mg/m2 60 minutes prior to DAC 100 mg/m2 (5 mg/kg). The median AUClast 
with THU-DAC (decitabine at half the dose) was 2284 min*ng/ml, with a range between 534 to 
7515 min*ng/mL (14-fold variation) (Table 1). The increase in AUClast in the THU group was 
statistically significant (p=0.02, Wilcoxon test) (Table 1), even though AUClast was calculated 
over 25% more time in the DAC alone group (240 minutes versus 180 minutes for THU-DAC). 
Furthermore, in many animals in the THU-DAC group, the concentration-time trends suggested 
DAC levels may have continued to increase beyond the last measured time-point at 180 minutes. 


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Hence, AUClast values for THU-DAC are underestimates. The coefficient of variation (as a 
measure of inter-individual variability) of AUClast was 141 in the DAC alone group and 97 in the 
THU-DAC group. The increase in AUClast and accompanied by decrease in coefficient of variation 
with THU-DAC was because the largest increases in DAC AUClast occurred in animals which had 
the poorest response to decitabine alone (Table 1): this converged the pharmacokinetic profiles in 
low oral bioavailability animals towards the animals with higher intrinsic oral bioavailability 
(Table 1). The average decitabine Cmax was 0.05 µM (10.85 ng/mL) for DAC alone and 0.12 µM 
(26.98 ng/mL) for THU-DAC (decitabine at half the dose).
Table 1: The largest increases in AUClast produced by co-administration of oral THU occurred in baboons 
which had the lowest AUClast with DAC alone
1.2.5
Effects of Repeat Administration of Oral THU-DAC on Blood Counts, DNA 
Methylation and Fetal Hemoglobin Levels in Non-Human Primates 
To evaluate pharmacodynamic effects with repeat dose administration, oral THU-DAC 2X/week 
for 8 weeks was administered to baboons PA7484 and PA7472 (59). DAC doses were 10 and 5 
mg/m2 respectively, administered by oral gavage 60 minutes after oral THU 400mg/m2, dosages 
that were expected to produce Cmax within the range (0.005-0.5 µM) that was demonstrated to 
deplete DNMT1 in normal hematopoietic precursors without causing significant DNA damage or 
cytotoxicity (see Investigators Brochure). In PA7484, platelet counts increased during the first 6 
AUClast (min*ng/mL)#
Baboon Number
Weight 
(kg)
Decitabine 10 
mg/kg alone
Decitabine 5 
mg/kg alone
THU 20 mg/kg 60 mins 
before Decitabine 5 
mg/kg
PA7482
12.3
49.98
Not done
Not quantifiable
PA7484
11.5
190.35
252.73
5621.05
PA7256
19.8
299.02
Not done
760.27
PA7472
10.7
327.25
Not done
444.825
PA7254
14.4
463
Not done
2587
PA7255
19.9
807.8
Not done
533.47
PA7258
12.6
2863.6
Not done
2284.48
PA7470
9.6
6278.78
1219.98
7515.32
MeanSD
1604  2262
2821  2749
MedianIQR
463  2565
2284  5088
MedianIQR per 
mg of decitabine*
160  226 
457  1017
Fold-variation
30-fold
14-fold
Coefficient of 
Variation
141
97
#AUClast calculated over 240 minutes for decitabine alone, 180 minutes for THU-
decitabine.  *p=0.02. Wilcoxon test. SD=standard deviation. IQR = inter-quartile 
range  


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21
weeks of drug administration, with a decrease during the last 2 weeks (but not below the lower 
limit of normal) (Figure 10A). In the same animal, neutrophil counts decreased during the first 3 
weeks of drug administration (but not below the lower limit of normal), and increased during the 
last 2 weeks (Figure 10B). In PA7472, platelet counts increased during drug administration 
(Figure 10A), while neutrophil counts decreased but not below the lower limit of normal (Figure 
10B). These alterations in blood counts are characteristic of the shifts in hematopoietic 
differentiation produced by DNMT1 depletion by DAC (60, 61). In PA7472, a bone marrow 
aspirate 96 hours after THU-DAC administration provided sufficient cells for analysis of DNA 
damage/repair by H2AX staining. There was a small increase in H2AX compared to negative 
control that was not suggestive of major DNA damage, although early DNA damage induction 
would have been missed (Figure 10C). Oral THU-DAC was also administered 3X/week with 50% 
of the above DAC doses, again with concurrent increases in platelet and decreases in neutrophil 
counts (Figure 11).  
One potential application of long-term DNMT1-depleting therapy is to increase fetal hemoglobin 
(HbF) in red blood cells as a means to treat hemoglobinopathies such as sickle cell disease (as 
envisaged in the proposed clinical trial). HbF levels increased progressively during treatment in 
both PA7484 and PA7472, although at late time-points in PA7484, there was a partial reverse of 
this trend (Figure 10D). In repeat dose experiments with 50% of the above DAC doses 
administered 3X/week, progressive HbF increases were again noted (Figure 11). One intended 
biological effect of therapy is to hypomethylate promoter CpG that regulate expression of target 
genes (e.g., -globin gene [HBG] promoter CpG). HBG promoter CpG that are hypomethylated at 
the developmental stages associated with HbF expression (and which therefore potentially play a 
role in regulating HBG expression) were identified by comparing methylation of -globin gene 
cluster CpG in DNA isolated from baboon fetal liver, fetal bone marrow, and adult bone marrow 
erythroid precursors (Figure 10E) (methylation levels of embryonic [HBE] and -globin gene 
[HBB] CpG were similar in fetal and adult erythroid precursors; in both fetal and adult erythroid 
precursors, CpG in the locus control region, an enhancer for the -globin genes, were 
hypomethylated compared to the methylation level in white blood cells). The developmentally 
responsive HBG promoter CpG, but not other CpG such as HBE and HBB gene promoter CpG, 
were hypomethylated by up to 20% by the 2X/week oral THU-DAC administration to PA7484 
and PA7472, and by up to 50% by 10 days of daily administration of SC decitabine 5 mg/m2 in 
PA7002 (Figure 10E). The SC treatment with DAC alone was used as a positive control and 
increased HbF levels up to 81.3% (data not shown). 


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Figure 10: Pharmacodynamic effects of repeat dose oral THU-DAC in non-human primates. DAC 10 mg/m2 
(PA7484) or 5 mg/m2 (PA7472) 60 minutes after THU 400 mg/m2 was administered 2X/week for 8 weeks. First 
dose Day 1. Last day of treatment Day 51 in PA7484, Day 50 in PA7472. A) Platelet counts during treatment. 
B) Absolute neutrophils counts during treatment. C) Phospho-H2AX (H2AX) labeling of bone marrow cells 
96 hours after THU-DAC administration in week 8 to PA7472. Positive control Hela cells treated with 
camptothecin 10 µM. Negative control vehicle treated Hela cells. D) HbF expression during treatment. E) 
Specific hypomethylation of CpG sites in the -globin gene (HBG) promoter in erythroid precursors. Top panel: 
Methylation levels of CpG in the beta globin cluster in erythroid precursors isolated from baboon fetal liver 
(FL), fetal bone marrow (FBM), adult bone marrow (ABM) measured by LCMSMS to identify CpG 
hypomethylated at the developmental stages associated with HbF expression, and to compare methylation of 
these CpG in erythroid versus non-erythroid cells (white blood cells, WBC). Bottom panel: Methylation levels 
of these CpG before and after 8 weeks of 2X/week oral THU-DAC administration in PA7484 and PA7472, and 
before and after 10 days of daily treatment with SC DAC 5 mg/m2 in PA7002. 


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Figure 11: Pharmacodynamic effects of repeat dose oral THU-DAC in non-human primates. DAC 5 mg/m2 
(PA7484) or 2.5 mg/m2 (PA7472) 60 minutes after THU 400 mg/m2 was administered 3X/week for 5 weeks 
(PA7484) or 3X/week for 3 weeks (PA7472). First dose Day 1. Last day of treatment Day 31 in PA7484, Day 19 
in PA7472. A) Platelet counts in PA7484. B) Absolute neutrophil counts (ANC) in PA7484. C) HbF% in PA7484. 
D) Platelet counts in PA7472. E) ANC in PA7472. F) HbF% in PA7472.


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1.2.6
Clinical Pharmacokinetics: THU Alone
As previously discussed, THU is a competitive inhibitor of CDA, the enzyme that is largely 
responsible for the metabolism of cytidine analogues (62).  THU has no therapeutic utility as a 
separate entity, instead, it is frequently used in clinical studies to inhibit CDA and block the 
metabolism of cytidine analogues which are co-administered as the active therapeutic agent.  The 
clinical pharmacokinetics of THU have been extensively characterized (63).    
In these studies, with the exception of time-to-peak THU levels, the pharmacokinetics of 
intravenous, SQ and oral THU at a dose of 200 mg/m2 were essentially identical (63).  By each 
route of administration, the initial and secondary half-lives were ~8 and 80 minutes, respectively 
with a plasma half-life or approximately 7 hours.  THU elimination was primarily in the urine and 
with intact drug but no metabolites identified in urine or plasma.
These observations were extended through the use of [14C]-THU (64).  In this study, 50 mg/kg 
[14C]-THU was rapidly cleared from the blood with a plasma half-life of about 1 hour following 
intravenous administration.  Approximately 10% of the same dose was absorbed from the GI tract 
following oral administration.   The main pathway of excretion was through the kidneys with 
approximately 50% of the administered drug excreted within 24h and 100% elimination within 
48h.THU at 10, 25, and 50 mg/kg given 15 minutes before [3H]-cytosine arabinoside (ara-C) at a 
dose of 0.003 mg/kg produced an approximately two-fold increase in ara-C blood levels at all 
times measured from 5 minutes to 4 hours, with only slight increases in the half-life of ara-C (64).  
A dose-related effect of THU upon the deamination of ara-C was obvious only during the time 
from 15 minutes to 1 hour after the injection of 3H-ara-C. The inhibitory effect of THU upon CDA 
was also reflected in a considerably increased ratio of ara-C/uracil arabinoside in the urine.
1.2.7
Clinical Pharmacokinetics: DAC Alone 
The clinical pharmacokinetics of three doses of DAC administered as 6-hour infusions are shown 
in Figure 12 (65).  In this study, steady state concentrations are reached within 1-2h of the onset 
of the infusion, depending upon the dose administered, and are maintained throughout the infusion 
cycle.  Upon cessation of the infusion, decitabine is rapidly removed from circulation (t ½α = 7 
minutes) with a slower β-elimination phase (t ½β = 35 minutes). 
Figure 12: Mean peak plasma levels of DAC were 83, 185.4, and 342.8 ng/mL and a t1/2 of 7 minutes and t1/2 
β of 35 minutes (data extracted from (2))


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1.2.8
Clinical Safety: THU Alone 
Administration of THU to humans by the IV, SQ (SQ), and oral (PO) routes, in combination with 
various cytidine analogues, in studies spanning over 30 years, suggest it has a benign toxicity 
profile (63, 64, 66-70) (summarized in Table 2).  Reports of adverse events following treatment 
are infrequent.  In a study of THU alone administered by IV, SQ and PO routes at a single dose of 
200 mg/m2 to 5 patients with malignant melanoma, there was no mention of toxicities (63).  
Similarly, THU administered at a daily dose of approximately 900-1800 mg/m2 for 5 consecutive 
days together with cytosine arabinoside did not produce toxicities other than those expected with 
cytosine arabinoside alone (70).  A similar experience is described when THU was administered 
at 700 mg/m2 daily for 4 consecutive days together with cytosine arabinoside (68).  Currently, 
THU is being used in three NIH-sponsored clinical trials: at 350-500 mg/m2 administered daily for 
3-5 consecutive days in combination with 5-fluoro-2’-deoxycytidine (e.g. NCT00077051), or daily 
for 7 or more days in combination with other oncotherapeutics (e.g. NCT00378807, 
NCT00521183).
Table 2: Clinical trials with tetrahydrouridine (published data).
THU dose
THU 
Route
Combination drug
Number 
of 
patients
Toxicity attributed to THU?
Reference
350 
mg/m2 
Q12 
hours x 4 days
IV
IV Ara-C 100-200 
mg/m2 Q12 hours x 4 
days
32
None reported
(68)
350 mg/m2 daily x 5 
days
IV
IV 
5-fluoro-2’-
deoxycytidine 
5-80 
mg/m2 daily x 5 days
11
None reported
 (71)
2,100 
– 
2,800 
mg/m2 total dose
IV
IV Ara-C 1,200 - 
1,600 
mg/m2 
and 
carboplatin 
900 
mg/m2 total dose
8
Myelosuppression, 
unacceptable hepatotoxicity, 
diarrhea.  No attribution to 
direct THU effect
(72)
2 - 4 mg/kg (approx 
75-150 mg/m2) for 
3 consecutive days 
every week
oral
Oral 
5-azacytidine 
0.2mg/kg 
for 
3 
consecutive 
days 
every week
2
None reported
(69)
25 – 50 mg/kg 
(approx 900 – 1800 
mg/m2) daily x 5 
days
IV
IV Ara-C 0.1 – 0.2 
mg/kg (approx 4 – 8 
mg/m2) daily x 5 days
None reported.  Toxicities 
were as expected from Ara-C 
alone
(70)
200 mg/m2 x 1
IV, SQ 
and 
oral
none
5
None reported
(63)
350 mg/m2 daily x 5 
days every 21 days 
OR for 5 days in 
each 
of 
2 
consecutive weeks 
every 28 days 
IV
IV 
5-fluoro-2’-
deoxycytidine 
2.5-
180 mg/m2/day x 5 
days every 21 days 
OR for 5 days in each 
of 
2 
consecutive 
weeks every 28 days 
58
None reported
(73)


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1.2.9
Clinical Safety and Efficacy: Oral THU and DAC in Combination, Repeat Dose 
Phase 1/2 Study in SCD 
A recent Phase I clinical trial was performed to evaluate the toxicities and potential efficacy of 
oral DAC in combination with oral THU in patients with SCD (Saunthararajah, et al, submitted 
for publication). The objectives of the study were to (i) extend DAC half-life/Tmax to hours instead 
of minutes, to increase S-phase dependent DNMT1-depletion (ii) without high DAC Cmax that 
causes off-target effects and cytotoxicity (intended Cmax > 5nM and < 200nM), (iii) decrease within 
and between patient variability in DAC distribution (that is, to produce stable distribution of DAC 
into tissues that otherwise provide sanctuary to myeloid cancer cells from DAC treatment effects), 
(iv) increase HbF to clinically meaningful levels in patients with SCD. 
The timings of administration between oral THU and oral DAC, and the dosages of THU and DAC 
in this clinical trial, were selected based on the above pharmacologic/pharmacodynamics 
objectives and studies in mice, and non-human primates (58, 59).
Pharmacokinetic results: For the purposes of non-cytotoxic DNMT1-depletion, a wide 
concentration-time profile with low peak concentration or Cmax but extended exposure time or 
extended Tmax is desired (Figure 13) (59). To evaluate effects of different routes of administration 
on the DAC concentration-time profile, plasma DAC levels were compared after intravenous (IV), 
subcutaneous (SQ) and oral administration to non-human primates (baboons). IV DAC 0.5 mg/kg 
produced peak plasma drug concentrations (Cmax) > 600 nM and plasma t½ < 5 minutes (Figure 
13). SQ DAC at the same dose produced Cmax ~300 nM and t½ ~40 minutes. Oral DAC at 20-fold 
greater dose (10 mg/kg) produced widely differing Cmax of ~220 and 10 nM in two baboons, with 
t½ > 100 minutes (Figure 13A). Thus, DAC oral absorption is very limited and very variable, 
although it does produce the wide concentration-time profile desired for non-cytotoxic DNMT1-
depletion. 
Pre-clinical studies in mice and baboons suggested that combining oral THU with oral DAC could 
address the very poor oral bioavailability and very wide inter-individual variability, creating the 
rationale for this first-in-man clinical trial of oral THU-DAC (Figure 13) (59). Samples for PK 
analyses were obtained at 0, 2, 4 and 24 hours in 12 of the 15 subjects who received study drug 
(dictated by venous access). DAC levels were measurable even in subjects receiving the lowest 
DAC dose of 0.01 mg/kg. A dose-dependent increase in plasma DAC was observed. The highest 
dose of oral Dec, 0.16 mg/kg, produced DAC plasma concentrations at 2 hours between 39-54 nM. 
The dose level below this, 0.08 mg/kg, produced concentrations at 2 hours of between 9-21 nM. 
These concentrations of DAC were in the range predicted to have non-cytotoxic epigenetic 
therapeutic effects (59), and accordingly, these dose levels were the only ones also associated with 
clinical pharmacodynamic effects, as described below. The model fit suggested Cmax was at 
approximately 1 hour, prior to the 2 hour time-point measured in this study. At all dose levels, by 
4 hours, DAC plasma levels had decreased to < 50% of the level measured at 2 hours. 


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Figure 13: Pharmacokinetics (PK) of DAC and oral THU-DAC. A)  DAC (Dec in this figure) PK after 
intravenous (IV), subcutaneous (SQ) and oral (PO) administration to baboons. B) DAC PK in human 
subjects from this clinical trial. Samples for PK analysis were obtained in 12 of the 15 subjects who received 
study drug. Stated times are hours after administration of oral Dec, data-points are measured values while 
curves were fitted by a global fit model using the R package PKLMfit.R. The inset shows a close up for hours 
0-4, to facilitate comparison to the 4 hour time-frame of the PK studies in baboons. Dec was quantified by a 
validated LC-MS/MS method. C) Dec AUC estimations in the baboons and in the different cohorts of the 
human clinical trial (model-dependent AUC estimates PKLMfit.R). The objectives with combination with THU 
were a wider concentration-time profile (that is low Cmax but extended Tmax or t½) and Dec distribution 
through CDA rich organs such as the intestines and liver.
Adverse events: Non-cytotoxic modification of hematopoietic differentiation by DAC is expected 
to simultaneously increase platelet counts and decrease absolute neutrophil counts (shown in vitro 
and in clinical studies with DAC alone (60, 61, 74, 75)). This expected pattern in blood counts was 
seen in subjects receiving oral DAC 0.08 and 0.16 mg/kg (dose levels 4 and 5) with the steepest 


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blood count changes observed at the highest dose (Figure 14). Platelet and neutrophil counts on 
therapy did not reach the a priori defined triggers for dose modification, that is, neither a platelet 
count of > 1200 x 109/L, nor an absolute neutrophil count < 1.5 x 109/L, were reached (the highest 
platelet count on therapy was 1122 x 109/L in a subject with a pre-treatment count of 733 x 109/L, 
and the lowest absolute neutrophil count on therapy was 1.6 x 109/L in a subject with a pre-
treatment count of 1.8 x 109/L) (Figure 14).  Of particular relevance to this protocol, absolute 
lymphocyte counts remained stable during and subsequent to the eight-week THU-DAC therapy 
(Figure 15). The pattern of adverse events was similar between subjects receiving placebo or oral 
THU-DAC in all dose cohorts (Table 3A). There were no significant adverse events, including no 
significant gastro-intestinal adverse events, attributed to study drug (Table 3B). The most frequent 
adverse event in placebo and study drug treated subjects was pain from vaso-occlusive sickle cell 
crisis (Table 3A). There were no Grade 4 adverse events in placebo or study drug treated subjects 
(Table 3). No subjects discontinued placebo or study drug because of adverse events. 
Figure 14: Platelet counts increased and absolute neutrophil counts simultaneously decreased during repeat 
dose administration, the blood count profile expected from non-cytotoxic DNMT1 depletion. A) Study schema 
showing repeat dose administration 2X/week for 8 weeks. B) Platelet counts. C) Absolute neutrophil counts 
(ANC).


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Figure 15: Absolute lymphocyte counts in SCD patients treated with increasing doses of THU-DAC.
Efficacy/Pharmacodynamic Effects: Hemoglobin F levels were measured by HPLC through the 
Clinical Pathology laboratory and also by flow cytometric quantification of HbF in individual red 
cells. Significant increases in HbF were observed by both these measurements at dose levels 4 and 
5 (DAC 0.08 and 0.16 mg/kg respectively), with the greatest increases occurring at dose level 5 
(Figure 16). These HbF increases were associated with trends for increasing total hemoglobin, 
decreasing LDH and total bilirubin (as biomarkers of hemolysis) and decreasing D-dimer levels 
(as a biomarker of coagulation pathway activation and vaso-occlusion) (Figure 17). Increases in 
HbF were not sustained once study drug was stopped in Week 8 (Figure 16).
Hematologic side-effects and toxicity: There were no hematologic toxicities warranting treatment 
hold (please see above).  
Non-hematologic toxicity: No significant non-hematologic toxicities were observed (please see 
above).
In summary, results from the aforementioned Phase 1 clinical trial of oral THU-DC demonstrate 
that the non-human primate data essentially mapped to subjects with high risk SCD: combining a 


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mini-dosage of oral DAC (0.16 mg/kg; ~5 mg/m2) with THU 10 mg/kg produced major 
improvement in DAC tissue distribution and exposure, creating a wide DAC concentration-time 
profile that is desired for DNMT1-depletion without cytotoxicity in vivo. This DAC oral dosage 
compared very favorably to parenteral dosages FDA approved for administration to humans (20-
45 mg/m2/day for 3-5 days every 4-6 weeks). The THU-DAC was very well-tolerated without 
significant gastro-intestinal or other drug-related adverse events with repeat administration 
2X/week for 8 weeks. Changes in hematologic parameters were those expected from non-cytotoxic 
DNMT1-depletion by DAC, with simultaneous increases in platelet counts and hemoglobin and 
decreases in absolute neutrophil counts. The platelet and absolute neutrophil count changes did 
not cross thresholds that would have triggered treatment holds (> 1,200 x 109/L for platelets, < 1.5 
x 109/L for neutrophils). Increases in HbF that would be considered clinically significant if 
generated by standard of care HU therapy were observed at DAC dosage of 0.08 and 0.16 mg/kg. 
The HbF increases were associated with favorable trends in total hemoglobin and in peripheral 
blood markers of hemolysis and coagulopathy/vaso-occlusion – serum LDH, total bilirubin and 
D-dimer.  


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Table 3: Adverse events in subjects treated with placebo and with oral THU-DAC at the various dose levels 
(Cohorts 1-5). A) All adverse events as per CTCAE v 4.0. There were no Grade 4 adverse events. B) Adverse 
events with attribution other than unrelated to study drug per the judgement of the treating clinical team.


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Figure 16: Efficacy. A) Increase in HbF% measured by HPLC was observed in subjects receiving THU-DAC 
0.08 and 0.16 mg/kg (fixed dose THU 10 mg/kg 60 mins prior to DAC). B) Change in HbF% from pre-
treatment to Week 8 or 10 was most prominent at THU-Dec 0.08 and 0.16 mg/kg. C) Absolute HbF levels. 


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Horizontal grey line shows 0.5 g/dL threshold associated with better survival. D) Proportion of RBCs 
expressing high levels of HbF (F-cells) in subjects receiving THU-DAC 0.08 or 0.16 mg/kg. E) Raw F-cell flow 
cytometry data in Cohort 4 and 5 subjects.
Figure 17: Efficacy: hematologic and chemistry parameters impacted by changes in HbF expression in 
subjects receiving DAC 0.08 and 0.16 mg/kg. A) Total hemoglobin. B) LDH (biomarker of hemolysis). C) 
Total bilirubin (biomarker of hemolysis). D) D-dimer (biomarker of coagulation pathway activation and 
vaso-occlusion).
Because FOXP3 is regulated by epigenetic mechanisms (76), it is possible that DAC-THU therapy 
will mediate expansion and enhanced immunosuppressive function of Tregs in cancer patients. In-
vitro treatment of human Tregs with DAC induced hypomethylation of the Treg specific DNA 
demethylated region (TDMR), which is required for stable expression of FOXP3, and increased 
expression of Treg specific genes; however, the cells did not exhibit full suppressive function (77). 
On the other hand, 5-Aza which is converted to DAC before incorporation into DNA increases 
Tregs and significantly reduces airway inflammation in mice (78). Furthermore, adoptive transfer 
of Tregs treated in vitro with DAC mediates resolution of lung inflammation in a murine ARDS 
model (79).  Collectively these data suggest that DNMT depletion may enhance Treg function in 
humans; as such strategies to inhibit Treg activity may facilitate more effective antitumor effector 
responses in cancer patients receiving DAC-THU therapy.


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Rationale to Combine DAC-THU with Immune Checkpoint Inhibitors for Treatment of Thoracic 
Malignancies:
Although substantial preclinical studies including those cited above as well as a recent report by 
Kim et al (80) support evaluation of epigenetic regimens in combination with immune checkpoint 
inhibitors, observations that 3 of 5 lung cancer patients previously treated with sequential 
azacytidine/etinostat without success exhibited objective responses to anti-PD-1 or anti-PD-L1 
therapy (81-83) provided the clinical impetus to develop combinatorial epigenetic 
immunotherapies for cancer. Numerous trials have been initiated to evaluate azacytidine with or 
without etinostat or other HDAC inhibitors and either pembrolizumab or nivolumab in cancer 
patients who have failed first-line cytotoxic or TKI therapies (84).  
Several recent studies have further evaluated potential mechanisms by which DNA demethylating 
agents augment antitumor immunity Figure 18. Wrangle et al (85) examined gene expression 
profiles in several lung cancer lines following treatment with low dose 5-azacytidine (AZA)  which 
is converted to DAC upon incorporation into DNA, and observed that AZA up-regulated cancer-
testis antigen expression as well as numerous pathways mediating innate and adaptive immunity. 
In addition, AZA up-regulated PD-L1, which could dampen cellular immune responses to cancer 
cells. The effects of AZA on immune pathways in lung cancer cells were very similar to those 
observed in HCT116 colon cancer cells following knock-out of DNMT1 and DNMT3b. 
Figure 18: DNA demethylating agents augment antitumor immunity
Li et al (86) examined gene expression profiles in a large panel of breast, colorectal and ovarian 
cancer lines following treatment with low dose AZA, and observed up-regulation of cancer-testis 
antigens as well as significant enrichment for immunomodulatory pathways including antigen 


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processing and presentation, cytokines/chemokines, and interferon signaling. Based on expression 
profiling, an AZA-IMune (AIM) gene set was defined, which appeared to be up-regulated in 
primary tumor tissues from cancer patients receiving AZA treatment. More recently, Chiappinelli 
et al (87) observed that low dose AZA de-represses endogenous retroviruses (ERV) which trigger 
the double stranded RNA (dsRNA) sensing machinery to induce a type I interferon response 
similar to a viral response. Analysis of melanoma, breast, ovarian, colon and lung cancers in TCGA 
revealed that cancers with high levels of viral defense genes were more likely to have better 
outcomes. Furthermore, high-level intratumoral expression of viral defense genes was associated 
with improved outcomes in melanoma patients receiving the immune checkpoint inhibitor, anti-
CTLA-4. Roulois et al (88) observed that low dose AZA targets colorectal cancer initiating cells 
(CICs) by induction of “viral mimicry” (Figure 8) secondary to up-regulation of ERV, activation 
of the MDA5/MAVS RNA recognition pathway and up-regulation of IRF7. Knock-down 
ofMDA5/MAVS, or IRF7 significantly attenuated the inhibitory effects of AZA on proliferation 
and clonogenic potential of CICs.
A series of experiments have been performed in the Thoracic Epigenetics Laboratory, TSB to 
examine endogenous retroviral (ERV) expression in lung cancer cells, and to evaluate the 
feasibility of using DNA hypomethylating agents to augment ERV expression as a potential 
adjunct to immune check point inhibitor therapy for lung cancer. We also sought to identify 
potential PD endpoints that could be incorporated into our epigenetic-immunotherapy protocols. 
Briefly, SYBR Green quantitative RT-PCR techniques were used to examine expression of nine 
ERV family sequences (MLTA10, MLT1B, MER21C, ERVL, MLT1C627, MER4D, MER57B1, 
MTE2B4) and cancer-germline genes in normal human bronchial epithelial (NHBE) cells, normal 
small airway epithelial cells (SAEC), 13 NSCLC lines, 20 SCLC lines, and 5 NSCLC lines and 
NHBE treated with 5-aza-2’-deoxycytidine (DAC; 0.1 uM x 72 hours). ERV expression patterns 
in lung cancer lines and normal respiratory epithelial cells were primer specific with no consistency 
in SCLC or NSCLC lines (Figure 19). Lung cancer lines generally showed higher ERV expression 
than normal cells.  MLT1B, ERVL, and MLT1C627 were up-regulated more than two-fold in 
82%, 73%, and 70% of lung cancer lines, respectively, compared to normal respiratory epithelial 
cells. Activation of ERV in lung cancer lines did not appear to coincide with de-repression of 
cancer-testis genes.  DAC mediated relatively modest (two-fold average) up-regulation of ERV, 
with MLTA10 (2.7 fold), MER21C (3.6 fold) and ERVL (2.6 fold) showing highest induction. 
Collectively these data demonstrate that cultured lung cancer cells exhibit heterogeneous 
endogenous ERV expression profiles, as well as variable patterns of ERV induction following 
exposure to DNA demethylating agents. 


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Figure 19: Top panel: qRT-PCR analysis of endogenous ERV expression in a panel of NSCLC specimens and 
matched normal lung tissues; middle panel: ERV expression levels in NSCLC vs SCLC lines; lower panel: 
effects of DAC on ERV expression in cultured NSCLC cells.
Figure 20: Top left: survival of mice with Lewis lung cancer metastases treated with PBS, DAC, or DAC-
THU. Top right: immunoblot demonstrating depletion of DNMT1 and MYC in lung cancer tissues following 
DAC-THU exposure. Lower left: qRT-PCR demonstrating effects of DAC-THU on cancer-testis gene 
expression in-vivo lung cancers. Lower right: THU enhances DAC-mediated induction of IFNγ in lung cancer 
tissues.
β-actin 
p27
c-MYC
DNMT1
PBS                   DAC            DAC-THU                  


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Recently a series of preclinical experiments have been performed by Drs. Vamsi Velchetti and 
Yogen Saunthararajah at the Cleveland Clinic to examine potential efficacy of DAC-THU and 
anti-PD-1 therapy for lung cancer. Briefly, immunocompetent C57/BL6 mice were inoculated via 
tail vein with Lewis lung cancer cells. Eighteen days later, mice were imaged to confirm lung 
colonization and then randomized to receive PBS SC, DAC 0.2mg/kg SC, or DAC (0.1mg/kg) SC/ 
THU (10mg/kg IP) q M-W-F. On Day 32, mice were bled for flow cytometry experiments and 
subsequently euthanized when ill-appearing. DAC-THU therapy significantly prolonged survival 
relative to DAC alone or PBS (Figure 20). Combination treatment markedly decreased DNMT1 
and to a lesser extent, MYC protein levels while increasing p27 expression, consistent with the 
aforementioned p53- independent cell cycle exit effect of DNMT1 depletion. DAC-THU therapy 
also significantly up-regulated intratumoral MAGE-A1 and MAGE-A3 cancer-testis gene 
expression, as well as intratumoral interferon-γ levels. Furthermore, relative to PBS controls, mice 
treated with either DAC or DAC-THU exhibited significantly higher peripheral CD4 lymphocytes 
(approaching those in untreated, nontumor bearing mice), as well as significantly decreased MDSC 
in peripheral blood (Figure 21).
               
Figure 21: Effects of systemic DAC and DAC-THU on peripheral T cell subsets and MDSC in mice bearing 
Lewis lung cancer metastases.
In additional experiments, C57/BL6 mice with Lewis lung cancer metastases were randomized to 
receive PBS SC, DAC (0.1 mg/kg) SC/THU (10 mg/kg IP) q M-W-F, a murine anti-PD-1 antibody 
(100 ug) q 5 days, or a combined DAC-THU/ anti-PD-1 regimen. Both DAC-THU and anti-PD-1 
alone prolonged survival; a combinatorial effect was observed, with 40% of mice receiving DAC-
THU-anti-PD-1 therapy appeared to be cured of their disease (Figure 22; left panel). Relative to 
tumor bearing mice treated with PBS alone, mice receiving epigenetic treatment, immunotherapy, 
or combination DAC-THU and anti-PD-1 therapy exhibited significantly higher peripheral CD4 
and CD8 lymphocyte counts (Figure 22; right panel). Collectively these findings, together with 
recent observations that DNA demethylating agents and other epigenetic drugs enhance the 
efficacy of immune checkpoint inhibitors by blocking immunosuppressive activities of Tregs and 
MDSC in the tumor microenvironment provide strong rationale for evaluation of DAC-THU and 
pembrolizumab for cancer therapy.


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Figure 22: Left: survival of mice with Lewis lung cancer metastases following treatment with PBS, DAC-THU, 
anti-PD-1, or combined DAC-THU/anti-PD-1 therapy. Right: effects of therapy on peripheral T cell subsets in 
mice with metastatic Lewis lung cancers.
1.2.10 Pembrolizumab
The importance of immune surveillance in controlling outgrowth of neoplastic transformation was 
initially postulated by Ehrlich (89) nearly a century ago, and subsequently revised by Burnet (90).  
Recent accumulating evidence shows a correlation between tumor-infiltrating lymphocytes (TILs) 
in cancer tissue and favorable prognosis in various malignancies (91).  In particular, the presence 
of CD8+ T-cells and the ratio of CD8+ effector T-cells / FoxP3+ regulatory T-cells seems to 
correlate with improved prognosis and long-term survival in many solid tumors.
The programmed death (PD)-1 receptor-ligand interaction is a major pathway utilized by tumors 
to establish equilibrium and ultimately escape immune surveillance (92). PD-1 (encoded by the 
gene Pdcd1) is an Ig superfamily member related to CD28 and CTLA-4 that is normally expressed 
on the surface of activated T-cells, and functions to negatively regulate antigen receptor signaling 
upon engagement of its ligands (PD-L1 and/or PD-L2). The mechanism by which PD-1 attenuates 
T-cell responses is similar to, but distinct from that of CTLA-4 as both molecules regulate an 
overlapping set of signaling proteins.  PD-1 is expressed on activated lymphocytes including 
peripheral CD4+ and CD8+ T-cells, B-cells, T regs and natural killer cells. The ligands for PD-1 
(PD-L1 and PD-L2) are constitutively expressed or can be induced in a variety of cell types, 
including non-hematopoietic tissues as well as in various tumors. Both ligands are type I 
transmembrane receptors containing both IgV- and IgC-like domains in the extracellular region 
and contain short cytoplasmic regions with no known signaling motifs.  Binding of either PD-1 
ligand to PD-1 inhibits T-cell activation triggered through the T-cell receptor.  PD-L1 is normally 
expressed at low levels on various non-hematopoietic tissues, most notably on vascular 
endothelium, whereas PD-L2 protein is only detectably expressed on antigen-presenting cells in 
lymphoid tissue or chronic inflammatory environments.  PD-L2 is thought to control immune T-
cell activation in lymphoid organs, whereas PD-L1 serves to dampen unwarranted T-cell function 
in peripheral tissues.  Although healthy organs express little (if any) PD-L1, a variety of 
malignancies including lung cancers express abundant levels of this T-cell inhibitor; PD-L1 
expression is also elevated in tumor associated macrophages and fibroblasts.  As a result, the PD-
1/PD-L1 pathway has emerged as an attractive target for therapeutic intervention in a wide variety 
of human malignancies (19, 84).


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
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Pembrolizumab is a potent and highly selective humanized monoclonal antibody (mAb) of the 
IgG4/kappa isotype which directly inhibits interaction of PD-1 with its ligands, PD-L1 and PD-L2 
(20).  In 2015, pembrolizumab was approved in the US for second-line therapy of NSCLC (93), 
and in 2016, this immune checkpoint inhibitor was approved as first-line therapy for NSCLC with 
high level (≥ 50%) PD-L1 expression. In 2017, the FDA approved pembrolizumab for recurrent 
locally advanced, or metastatic gastroesophageal cancer with PD-L1 expression (CPS) greater than 
or equal to 1. In a Phase I trial (Keynote 001), 495 patients with advanced NSCLC were assigned 
to either training or validation groups, and received pembrolizumab at 2 mg/kg or 10 mg/kg every 
three weeks, or 10 mg/kg every two weeks (94). Fatigue, decreased appetite and pruritus were the 
most common side effects (single digit numbers); severe pneumonitis was observed in 1.8% of 
patients, including one death. The ORR, median PFS and median OS for treatment naïve patients 
were 24.8%, 6.0 months and 16.2 months, respectively, vs 18%, 3.0 months, and 9.3 months, 
respectively, for previously treated patients. Subsequent analysis demonstrated ORR and median 
PFS of 43.9% and 6.1 months vs 50.0% and 12.5 months, respectively, for treatment naïve and 
previously treated patients whose tumors had a proportion score (PS) of at least 50% cancer cells 
showing positive membrane staining for PD-L1 (94). 
In a Phase III trial (Keynote 024), Reck et al (95) randomly assigned 305 patients with untreated, 
advanced NSCLC with no sensitizing EGFR or ALK mutations and  PD-L1 expression on 50% or 
more tumors cells to receive either pembrolizumab (200 mg Q3W) or platinum-based 
chemotherapy. Cross-over from the chemotherapy to pembrolizumab was allowed. The objective 
response rate was 44.8% in the pembrolizumab arm compared to 27.8% in the chemotherapy arm. 
Median progression free survival was 10.3 months (95% CI: 6.7 months to not reached) compared 
to 6.0 months (95% CI: 4.2 to 6.2 months) in the chemotherapy arm. Median duration of response 
was longer (not reached; range 1.9 to 14.5 months) in the pembrolizumab arm compared to 6.3 
months (range: 2.1 to 12.6 months). Grade 3, 4, or 5 treatment related adverse events were 
significantly lower in the pembrolizumab arm compared to the chemotherapy arm (26.6% vs 
53.3%). 


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
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Table 4: Systemic Toxicities of Pembrolizumab vs Chemotherapy in Treatment Naïve NSCLC Patients.
In a Phase 2, open-label, single-arm, multicohort study (KEYNOTE-059), 259 patients with 
recurrent gastric or gastroesophageal cancer received pembrolizumab, 200 mg intravenously every 
3 weeks until disease progression, investigator or patient decision to withdraw, or unacceptable 
toxic effects (96). The primary end points were objective response rate and safety. Secondary end 
points included response duration. Expression of PD-L1 was assessed by immunohistochemistry. 
Tumors were considered positive if the combined positive score (CPS) of PD-L1 positive cells 
(tumor are well as lymphoid cells) per total number of cells was ≥ 1%. The objective response rate 
was 11.6% (95% CI, 8.0%-16.1%; 30 of 259 patients), with complete response in 2.3% (95% CI, 
0.9%-5.0%; 6 of 259 patients). Median (range) response duration was 8.4 (1.6+ to 17.3+) months 


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(+ indicates that patients had no progressive disease at their last assessment). Objective response 
rate and median (range) response duration were 15.5% (95% CI, 10.1%-22.4%; 23 of 148 patients) 
and 16.3 (1.6+ to 17.3+) months and 6.4% (95% CI, 2.6%-12.8%; 7 of 109 patients) and 6.9 (2.4 
to 7.0+) months in patients with PD-L1-positive and PD-L1-negative tumors, respectively. Forty-
six patients (17.8%) experienced 1 or more Grade 3 to 5 treatment-related adverse events. Two 
patients (0.8%) discontinued because of treatment-related adverse events, and 2 deaths were 
considered related to treatment.
A subsequent analysis compared outcomes for three patient cohorts in this trial (97). Cohort 1 
patients received pembrolizumab alone after ≥ 2 prior lines of therapy. Cohort 2 patients received 
pembrolizumab + cisplatin (80 mg/m2 Day 1) + 5-fluorouracil (800 mg/m2 Days 1-5 Q3W) or 
capecitabine (in Japan only, 1000 mg/m2 twice daily) as first-line. Cohort 3 patients received 
pembrolizumab alone as first-line. Patients were enrolled in Cohorts 1 and 2 regardless of tumor 
PD-L1 expression; only patients with PD-L1-positive tumors (combined positive score of ≥ 1%) 
were enrolled in Cohort 3. In all cohorts, pembrolizumab was given at 200 mg Q3W for up to 2 
years. Median (range) follow-up was 6 (1-25), 14 (2-24), and 18 (2-21) months for Cohorts 1 (259 
patients), 2 (25 patients) and 3 (31 patients), respectively. Confirmed ORR (95% CI) were 16% 
(11-23) in PD-L1-positive, and 6% (3-13) in PD-L1-negative tumors in Cohort 1, 73% (45-92) in 
PD-L1-positive, and 38% (9-76) in PD-L1-negative tumors in Cohort 2, and 26% (12-45) in 
Cohort 3. Median PFS (95% CI) were 2 (2-2), 7 (6-11), and 3 (2-6) months in Cohorts 1, 2, and 3, 
respectively. Median OS (95% CI) were 6 (4-7) and 14 (9-not estimable) months for Cohorts 1 
and 2 respectively, and not reached (9-21) in Cohort 3. In Cohorts 1, 2 and 3, Grade 3-5 treatment-
related adverse events (TRAE) incidence occurred in 46 (18%), 19 (76%), and 7 (23%) patients in 
Cohorts 1, 2, and 3, respectively. 
In a recent randomized Phase III trial, 395 patients with previously treated advanced gastric or 
gastro-esophageal junction cancers with PD-L1 CPS ≥ 1% were randomized to receive 
pembrolizumab 200 mg q 3 weeks for up to 2 years or standard dose paclitaxel (98). Median 
overall survival was 9.1 months (95% CI 6.2–10.7) with pembrolizumab and 8.3 months (7.6–
9.0) with paclitaxel (hazard ratio [HR] 0.82, 95% CI 0.66–1.03; one-sided p=0.0421). Median 
progression-free survival was 1.5 months (95% CI 1.4–2.0) with pembrolizumab and 4.1 months 
(3.1–4.2) with paclitaxel (HR 1.27, 95% CI 1.03–1.57).  Grade 3–5 treatment related adverse 
events occurred in 42 (14%) of the 294 patients treated with pembrolizumab and 96 (35%) of the 
276 patients treated with paclitaxel.
Whereas pembrolizumab did not significantly improve overall survival compared with paclitaxel 
as second-line therapy for advanced gastric or gastro-oesophageal junction cancer with PD-L1 
CPS of 1 or higher, it is noteworthy that the survival curves for the treatment cohorts crossed at 8 
months in favor of the pembrolizumab arm suggesting a delayed and sustained benefit from the 
immunotherapy (Figure 23). The authors stated that this violation of the proportional hazards 
assumption suggests that alternative statistical methods such as a weighted log-rank test should be 
used to evaluate potential efficacy of immunotherapy in clinical trials. Post-hoc analysis using a 
weighted log-rank test that placed more weight on the events that occurred around the middle of 
the follow-up duration (i.e., around the time at which the overall survival curves crossed) and less 
weight on events that occurred early or late resulted in a one-sided p-value of 0.0009. The survival 
curves and results of the weighted log-rank test, as well as the relative toxicity profiles of 
pembrolizumab and paclitaxel clearly suggest that pembrolizumab is preferable to chemotherapy 
for second-line treatment of gastric or gastroesophageal junction carcinomas.


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
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Figure 23: Overall survival of patients receiving pembrolizumab or paclitaxel in Keynote-061.
In a multi-institutional phase 1b trial, 25 previously treated MPM patients received pembrolizumab  
10 mg/kg q2 weeks (99). Five (20%) patients experienced Grade 3 treatment-related adverse 
events. Three (12%) patients required dose interruption because of immune-related adverse events. 
No treatment-related deaths or discontinuations were observed. Five patients (20%) had a partial 
response; objective response: 20% (95% CI 6·8-40·7); 13 patients (52%) had stable disease. The 
responses were durable (median response duration 12·0 months [95% CI 3·7 to not reached]). 
These preliminary findings demonstrated safety and potential efficacy of pembrolizumab in MPM 
patients.
In a subsequent study, Metaxas et al (100) reported results of 93 MPM patients who received 
pembrolizumab off-label in Switzerland and Australia. 48 patients (52%) received pembrolizumab 
as second-line treatment. 68 patients (73%) had epithelioid MPM, and 67 (72%) had an Eastern 
Cooperative Oncology Group performance status of 0 or 1. 66 of the 93 patients (71%) had PD-
L1 expression analysis: 68% were negative, 18% were intermediate, and 14% had high intra-
tumoral PD-L1 expression. The overall response rate (ORR) was 18%, with a median progression-
free survival (mPFS) of 3.1 months (Figure 24); median overall survival was 7.2 months. ORR 
was 37%, mPFS was 3.7 months, and the median overall survival was 10.2 months in patients with 
ECOG PS of 0 or 1 and only one previous systemic treatment (n = 35), Patients with nonepitheloid 
histological subtype showed an improved ORR (24% versus 16% [p = 0.54) and mPFS (5.6 versus 
2.8 months [p = 0.02]). Compared with intermediate and negative PD-L1 expression, high PD-L1 
expression was associated with an improved ORR (44% versus 42% versus 11% [p = 0.01]) and 
mPFS (6.2 versus 3.9 versus 2.7 months [p = 0.04]). Toxicities in MPM patients were similar to 
those observed in patients with other malignancies. 


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Figure 24: Progression-free survival (PFS) and overall survival (OS) in the entire cohort. Kaplan-Meier curves 
of survival in the entire cohort (A), PFS according to line of treatment and performance status (PS) (B), and 
OS according to line of treatment and PS (C) (see the text).
In an abstract presented at ESMO 2019, Popat and colleagues (101) reported preliminary results 
of the Phase III European Thoracic Oncology Platform (ETOP 9-15) PROMISE-meso trial. 144 
patients with relapsed MPM following platinum-based chemotherapy were randomized 1:1 to 
receive 200 mg of pembrolizumab q 3 weeks (n = 73) or physician’s choice of gemcitabine or 
vinorelbine at standard doses (n = 71). Treatment was administered until disease progression by 
RECIST1.1 criteria up to 2 years, and patients were assessed every 9 weeks for 6 months, followed 
by every 12 weeks thereafter. Upon disease progression, those in the chemotherapy arm were 
permitted to cross over to the PD-1 inhibitor arm. The primary endpoint of the trial was PFS; 
secondary endpoints were ORR, time to treatment failure (TTF), overall survival (OS), 
investigator-assessed PFS, and adverse events (AEs). Outcomes by PD-L1 status served as a 
correlative 
endpoint. 
Baseline characteristics were similar between arms. The median age was 70 years, most patients 
had epithelioid histology (88.9%), and the majority had an ECOG performance status of 1 (75.1%). 
Prior therapies included carboplatin/pemetrexed, cisplatin/pemetrexed, platinum or without 
pemetrexed, 
and 
cisplatin/pemetrexed 
plus 
carboplatin/pemetrexed.
The ORR was 22% with pembrolizumab compared with 6% in those who received chemotherapy 
(P = .004). The median DOR was 4.6 months for pembrolizumab and 11.2 months with 
chemotherapy (95% CI, 2.2-10.3; 95% CI, 6.2-15.3). Median PFS was 2.5 months for 
pembrolizumab compared with 3.4 months for chemotherapy (HR, 1.06; 95% CI, 0.73-1.53; P = 
.76). The 6-month PFS rates were 25.0% and 27.4% with pembrolizumab and chemotherapy, 
respectively. At a median follow-up of 11.8 months, the median OS was 10.7 months in the 
pembrolizumab arm versus 11.7 months in the chemotherapy arm (HR, 1.04; 95% CI, 0.66-
1.67; P = .85). Even when censoring for crossover, there was no OS benefit observed with 
pembrolizumab (HR, 1.44; 95% CI, 0.77-2.67; P = .25), nor for inverse probability weighting (HR, 
1.07; 95% CI, 0.67-1.71; P = .79). PFS was similar in both PD-L1–positive and –negative subsets. 
Toxicities of pembrolizumab therapy were similar to those previously reported.
Collectively, the above results indicate that pembrolizumab has activity in MPM patients. 
Although pembrolizumab is not approved for MPM, it is listed in the current NCCN guidelines 
for palliative therapy of this disease. In light of the profound immunosuppressive 


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
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microenvironment associated with MPM (102), there is clear rationale to utilize epigenetic priming 
regimens as a strategy to enhance the efficacy of immune checkpoint inhibitors for MPM therapy.
1.2.11 Preliminary Experience with Oral DAC-THU and Immune Checkpoint Inhibitors 
in NSCLC 
1.2.11.1 DAC-THU and Nivolumab
A Phase II trial of oral DAC-THU and Nivolumab as second line therapy for NSCLC 
(NCT02664181) was recently conducted at the Cleveland Clinic and the NCI (D. Schrump, PI). 
Patients were randomized 2:1 to receive oral DAC/THU on two consecutive days each week with 
Nivolumab every two weeks, or Nivolumab alone. Two of five (40%) evaluable patients on the 
experimental arm had objective responses, one of which was a CR (Figure 25). Interestingly, both 
of these patients were never smokers (1 ALK positive (PDL1 unknown) patient and 1 HER2 
insertion site mutation (PDL1- 1-49%). 1 other patient who had PD-L1 0% had SD after 2 
cycles. Typically, patients who are never smokers exhibit low mutational burden, and low or no 
detectable PD-L1 expression; response rates following immune checkpoint inhibitor therapy in 
patients with these lung cancers range from 0-5% (103, 104). These preliminary findings suggest 
that oral DAC-THU modulates the tumor and/or microenvironment to enhance efficacy of immune 
checkpoint blockade in NSCLC patients. In light of current controversies regarding the accuracy 
of determining PD-L1 expression in NSCLC based on single biopsies (105), and inconclusive data 
pertaining to the validity of PD-L1 expression as a biomarker of response to immune checkpoint 
inhibitors (106, 107), these findings support the use of this oral immune-modulatory regimen in 
conjunction with immune checkpoint inhibitors for the treatment of thoracic malignancies.
Figure 25: CT Images of Lung Cancer Patients Before and After DAC-THU/Nivolumab Therapy.
1.2.11.2 DAC-THU and Pembrolizumab


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
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Four patients were treated on this protocol before enrollment was voluntarily halted (without 
formal suspension) to allow for revision of the dosing regimen. Briefly, patient #1 with medically 
inoperable, treatment naïve, synchronous primary NSCLC commenced oral DAC-THU (0.22 
mg/kg) 3 days per week on 4/16/2018. During Week 4 she developed precipitous Grade 4 
neutropenia after receiving a total DAC dose of 300 mg (2.7 mg/kg).  She progressed to complete 
agranulocytosis several days later and was transferred to the intensive care unit for neutropenic 
fever and commenced broad-spectrum intravenous antibiotics. Due to ongoing agranulocytosis as 
well as mild anemia and thrombocytopenia, a bone marrow aspirate was performed which 
demonstrated hematopoietic stem cells with erythrocyte and platelet differentiation but no myeloid 
precursors. Although these findings were suggestive of anticipated reprogramming of 
hematopoietic stem cell differentiation (Section 1.2.9), the magnitude of the granulocytopenia in 
the context of the investigational treatment regimen raised the possibility of autoimmune 
agranulocytosis. As such, the patient received IVIG, high-dose steroids, cyclosporine, and 
granulocyte infusions, ultimately recovering her blood counts approximately three weeks later. 
She commenced a gradual taper of her prednisone and cyclosporine, and was removed from 
treatment on 5/31/2018, and removed from study on 6/8/2018. Subsequently, she received 
stereotactic XRT and cryoablation for local control of her cancers. She remains alive with no 
clinical evidence of active disease as of June 2019.
Patient #2 with inoperable, treatment naïve NSCLC also received DAC-THU (0.22 mg/kg) 3 days 
per week, commencing on 5/7/2018.  When patient #1 developed severe neutropenia, DAC-THU 
was held for 1 week (Week 3; Cycle 1) for patient #2 despite normal blood counts.  The following 
week, patient #2 received DAC-THU for 2 days instead of three without hemato-toxicity. She 
developed Grade 4 ANC during Week 5 after receiving a total DAC dose of 160 mg (1.80 mg/kg). 
Because her clinical picture was similar to that of patient #1, she was hospitalized for observation; 
bone marrow aspirate revealed hematopoietic stem cells with very few myeloid precursors but 
preservation of erythrocyte and platelet lineages. Once again, given concerns regarding possible 
autoimmune agranulocytosis and ongoing neutropenia in patient #1, patient #2 received IVIG, 
high-dose steroids, and cyclosporine, without granulocyte infusions with resolution of her Grade 
4 neutropenia within 1 week.  She continued her steroid taper for approximately six weeks until 
she was removed from treatment and taken off protocol on 6/21/2018. Thereafter, she received 
XRT for new brain metastases and initiated standard of care palliative chemotherapy at an outside 
institution, ultimately succumbing to her disease on 2/9/2019. 
As a result of the aforementioned hematologic toxicities, no patients were accrued to the trial until 
we had a better understanding of their etiology, and subsequent patients received DAC-THU at 
dose level -1 per protocol. On 1/7/2019, patient #3 with treatment naïve stage IV NSCLC 
commenced DAC-THU (0.2 mg/kg) twice-weekly. During Week 6 after receiving a total DAC 
dose of 150 mg (2.01 mg/kg), the patient developed Grade 4 neutropenia. Bone marrow aspirate 
demonstrated significantly decreased myeloid precursors with preservation of hematopoietic stem 
cells and erythroid and platelet lineages. IVIG, steroids, and cyclosporine were initiated.  Four 
days later, the patient developed febrile neutropenia with pseudomonal bacteremia. He 
commenced a prolonged course of IV antibiotics due to the bacteremia and associated ecthyma 
gangrenosum involving the dorsum of the right hand. Within 8 days of initiating 
immunosuppression, the Grade 4 neutropenia had resolved to Grade 2. Steroids and cyclosporine 
were tapered over the course of two months. CT scans obtained as part of the evaluation for sepsis 
demonstrated a major response to therapy (Figure 26). Following consultation with the CCR-SD 
and immunotherapy experts at the NCI, Cleveland Clinic, and NYU Medical Center, the patient 


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
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resumed pembrolizumab without DAC-THU. He now continues pembrolizumab q3 weeks per 
protocol with ongoing major response as of May 2019.
 
Figure 26: Baseline PET scan (left) and PET scan following two cycles of DAC-THU/ Pembrolizumab therapy 
(right) in a patient with NSCLC.
Patient #4 who had received chemotherapeutic pleurodesis but no systemic therapy for stage IV 
NSCLC commenced twice weekly DAC/THU on 1/28/2019. Because of her small size (59.7 kg) 
and lymphopenia requiring intermittent interruptions in drug administration, the patient’s DAC 
dose (0.17 mg/kg) was less than the other patients, and she only received a total dose of 70 mg 
(1.17 mg/kg) during the 9-week course of therapy. She experienced only one Grade 3 ANC, and 
developed thrombocytosis requiring ASA prophylaxis suggestive of reprogramming of 
hematopoietic stem cells due to DNMT1 depletion. At treatment evaluation, staging studies 
demonstrated modest disease progression. Molecular profiling of tissue obtained from a post-
treatment excisional biopsy suggested the presence of an atypical EGFR mutation not detected by 
NGS of her pretreatment biopsy tissue; Sanger sequencing confirmed the mutation. The patient 
was removed from treatment on 4/3/2019 and is now experiencing a major response to osimertinib 
evidenced by resolution of palpable chest wall masses and complete cessation of narcotics. Staging 
studies have not as yet been performed. These findings are provocative given observations that a 
patient with EGFR mutant NSCLC refractory to EGFR TKI had a near CR after resuming TKI 
therapy following treatment with DAC-THU and nivolumab on protocol NCT02664181.
In light of the severe neutropenias observed in the first three patients, and following notification 
of the CCR Director of Clinical Sciences, accrual to this trial was voluntarily (but not formally) 
suspended in order to revise the DAC-THU dosing as well as the window for assessing DLT. Data 
pertaining to the aforementioned patients are summarized in Table 5. The severe 
granulocytopenias observed in these patients have now been attributed to reprogramming of 
hematopoietic stem cells due to DNMT1 depletion rather than autoimmunity, suggesting that the 
PD effect has been occurring albeit too dramatically following DAC-THU therapy. Consistent 
with this experience all eight patients treated with DAC-THU and Nivolumab on NCT 
NCT02664181 experienced Grade 3 or greater neutropenia; 5 patients experienced Grade 4 
neutropenia, including one patient who was hospitalized for neutropenic fever and Grade 4 
pneumonia/pneumonitis. Consequently, the dose regimen is now modified such that drug will be 
administered on two consecutive days (preferably Tuesday and Wednesday to facilitate bloodwork 
on Mondays and Thursdays) two out of every three weeks (instead of every week) for 9 weeks. 
Extrapolation of data from Table 5, demonstrates that the same doses of DAC (and THU) over 9 


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vs 4-6 weeks would result in > 30% decreases in intensity of DAC-THU dosing. Additionally, the 
revised DAC dosing regimen will be based on the total amount of DAC (mg/kg) administered per 
course. As such, the revised target of 2.5 mg/kg DAC over a 9-week course will be the new DL1, 
which corresponds to a 54% reduction from the dose initially attempted in this protocol and a 15% 
reduction from the exposures in SCD patients (108). The dosing per weight has been tightened 
(i.e.; q 10 kg rather than q 20 kg) to avoid relative under- or overdosing of patients at the extremes 
of each weight range. Lastly, the window for assessment of DLT is now revised to include Cycles 
1 and 2, rather than only Cycle 1 of Course 1 to better monitor toxicities, and modify drug dosing, 
accordingly, while optimizing epigenetic priming for immune checkpoint blockade.
Patient
Weight 
(kg)
BSA 
(M2)
Number 
of Caps 
(5 mg 
each 
pill)
DAC 
per 
Day 
(mg)
Days 
per 
Week
Total 
Number 
of DAC 
Days
Total 
DAC 
Dose 
(mg)
Total 
DAC 
(mg/kg)
DAC 
per BSA 
(mg/M2)
1
112.8
2.19
5
25
3
12
300
2.66
15.2
2
88.8
1.99
4
20
3
8
160
1.8
17.3
3
74.3
1.93
3
15
2
10
150
2.01
11.5
4
59.7
1.59
2
10
2
7
70
1.17
20.9
Table 5: Summary of DAC Administered with THU and Pembrolizumab.
Table 6: Summary of DAC Exposures in Various Protocols.
Two additional patients have been treated on the protocol since the dosing was amended. The first 
patient with extensive tobacco and ethanol exposure and underlying CAD presented initially with 
potentially resectable stage III distal esophageal adenocarcinoma. Restaging after induction 
chemo-XRT revealed new cervical lymph node disease that was biopsy proven to be EsC. He 
commenced DAC-THU and Pembro at the modified dose and exhibited no treatment related 
toxicities. Restaging after the first course of treatment revealed regression of disease not reaching 
PR. He commenced his second course of therapy and prior to his first DAC infusion he experienced 
a non-STEMI, which was deemed to be unrelated to DAC-THU or pembrolizumab. He underwent 
uneventful multivessel stenting. Because of the MI, he was no longer eligible to continue therapy. 
The second patient was a 35 year old male with stage IV esophageal cancer who was referred for 
study after progressing on standard chemotherapy and Herceptin. He had disease within the 
esophagus extending from the thoracic inlet to the cardia with bulky nodal disease in the neck and 
mediastinum.  Due to bulk of disease and stricture, a pediatric endoscope could barely pass through 
  Cumulative DAC Exposures over Nine-Week Treatment 
Dose/day (mg/kg)
# Days
Total Dose (mg/kg) % Change
DAC-THU/Pembro
0.2
27
5.4
DAC-THU/Nivo
0.2
18
3.6
-33
DAC-THU (SCD)
0.16
18
2.9
-47
DAC/THU/Pembro (rev)
0.21
12
2.5
-54


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the esophagus. He also tolerated the first course of therapy well with no significant treatment 
related toxicities. Staging studies following Course 1 revealed stable disease. Endoscopy revealed 
a more pliable and distensible esophagus allowing easy passage of an adult scope. The patient was 
now able to eat solid foods. Because of incapaciting pain, he received a palliative course of XRT 
to the cervical nodes. Repeat imaging demonstrated complete resolution of the left cervical 
lymphadenopathy (Figure 27). Although the regression of the lymphadenopathy could have been 
attributable to priming of the microenvironment by the palliative radiation that enhanced the 
efficacy of the immune checkpoint blockade, the magnitude of the response (CR in the neck) 
exceeded that which one would have anticipated from sublethal XRT and pembrolizumab alone. 
Furthermore, consistent with the endoscopic findings, there was less FDG uptake in the esophagus. 
Additionally, there was metabolic response in the retroperitoneal lymph nodes.
Figure 27: PET/CT scan images at Baseline and Post Course 1 of DAC-THU/Pembro and palliative XRT to left 
cervical region.
While at home in West Virginia as the COVID-19 pandemic started to hit the US, he developed 
line sepsis. His port had been used for all blood draws at home as well as his pembro infusions. 
The port was removed, and he commenced IV antibiotics. Since he could not undergo TEE to rule 
out endocarditis, Infectious Disease consultants mandated six weeks of IV antibiotics. Because 
this would have significantly impacted his ability to receive investigational therapy and in light of 
the evolving restrictions regarding patient care at the NIH Clinical Center, he was removed from 
study so that he could commence cytotoxic chemotherapy while completing his antibiotic regimen.
Pretreatment                                                Post-Course 1 and Palliative XRT to left cervical region


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2
ELIGIBILITY ASSESSMENTAND ENROLLMENT
2.1
ELIGIBILITY CRITERIA
2.1.1
Inclusion Criteria
2.1.1.1
Histologically or cytologically confirmed, inoperable or unresectable locally advanced, 
or metastatic NSCLC, esophageal cancers including Seiwert-Stein Type I and Type II 
gastro-esophageal junction (GEJ) carcinomas, or MPM.
2.1.1.2
NSCLC patients with no prior systemic treatment, or those with prior first line treatment 
including an immune checkpoint inhibitor, are eligible for study.
2.1.1.3
Patients with esophageal and gastro-esophageal junction (GEJ) cancers are potentially 
eligible for study if they have received or refused first line standard of care cytotoxic 
therapy, and subsequent targeted therapy if appropriate. 
2.1.1.4
Patients with MPM are eligible for study if they have received, refused or are ineligible 
for first line chemotherapy.
2.1.1.5
Patients who received DNA demethylating agents or PD-1/PD-L1 inhibitors for another 
malignancy may be eligible for study if there were no dose-limiting immune related 
events, and there has been either no clinical evidence of disease or minimal residual 
disease that has been stable for at least three years.
2.1.1.6
Patients must have analysis of PD-L1 expression in cancer cells quantitated by 
immunohistochemistry analysis. 
2.1.1.6.1 Patients in Cohort 1 (Dose Escalation) may have any level of expression.
2.1.1.6.2 Patients in Cohort 2 (Dose Expansion: NSCLC with high PD-L1) must have ≥ 50% 
expression in cancer cells.
2.1.1.6.3 Patients in Cohort 3 (Dose Expansion: NSCLC with low PD-L1) must have 0-49% 
expression. Note: Patients in this cohort must have been offered and refused standard of 
care platinum-based chemotherapy.
2.1.1.6.4 Patients in Cohort 4 (Dose Expansion: EsC) may have any level of expression.
2.1.1.6.5 Patients in Cohort 5 (Dose Expansion: MPM) may have any level of expression.
2.1.1.7
Measurable disease, per RECIST 1.1. (see Section 6.3.3 for the evaluation of measurable 
disease).
2.1.1.8
Willingness to undergo tumor biopsies if safely accessible per PI discretion before and 
after treatment. 
2.1.1.9
Age ≥18 years. Because no dosing or adverse event data are currently available on the 
use of Decitabine (DAC) and Tetrahydrouridine (THU) in combination with 
Pembrolizumab in patients < 18 years of age, children are excluded from this study, but 
will be eligible for future pediatric trials. 
2.1.1.10 ECOG performance status of ≤ 2 (see Appendix 1). 
2.1.1.11 Patients must be without evidence of unstable or decompensated myocardial disease; and 
must have adequate pulmonary reserve evidenced by FEV1 and DLCO ≥ 35% predicted; 
oxygen saturation equal to or greater than 90% on room air by pulse oximetry or ABG (to 
be drawn if pulse oximetry < 90% on room air).
2.1.1.12 No immunosuppressive medications except non-systemic corticosteroids.


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2.1.1.13 Patients must have normal organ and marrow function as defined below: 
leukocytes
≥ 3,000/mcL
absolute neutrophil count
≥ 1,500/mcL (without transfusion or cytokine 
support)
absolute lymphocyte count          > 800/mcL
platelets
≥ 100,000/mcL
PT 
no more than 2 seconds above the ULN
total bilirubin
< 1.5 X institutional upper limit of normal
OR
direct bilirubin
  ≤ ULN for patients with total bilirubin > 1.5 
ULN
serum albumin
≥ 2.0 mg/dL
AST(SGOT)/ALT(SGPT)
≤ 2.5 X institutional ULN
creatinine
≤ 1.6 mg/ml
OR
creatinine clearance (eGFR)
≥ 60 mL/min/1.73 m2 for patients with 
creatinine levels above institutional normal 
at the time DAC-THU and pembrolizumab 
treatment commences.
2.1.1.14 Patients with history of brain metastases except those with meningeal carcinomatosis or 
leptomeningeal disease may be eligible for treatment a minimum of 1 week following 
completion of gamma knife or whole brain radiotherapy, or 4 weeks following surgical 
resection of brain metastasis provided post-treatment MR scan reveals no evidence of 
active disease, and no ongoing need for systemic steroids. 
2.1.1.15 Patients with laboratory evidence of autoimmune disease (e.g., positive ANA or lupus 
anticoagulant) without associated symptoms; vitiligo, or mild autoimmunity not impacting 
the function of organs, such as Hashimoto or psoriasis may be eligible for study.
2.1.1.16 The effects of DAC-THU and pembrolizumab on the developing human fetus are 
unknown.  For this reason and because antineoplastic agents as well as other therapeutic 
agents used in this trial are known to be teratogenic, women of child-bearing potential 
and men who engage in sexual activity must agree to use 2 forms of contraception at least 
1 of which must be highly effective (intrauterine device [IUD], hormonal, tubal ligation; 
not highly effective includes barrier method) prior to study entry, for the duration of study 
participation and for 60 days after completion of the study treatment.  Should a woman 
become pregnant, or suspect she is pregnant, while she or her partner is participating in 
this study the study participant should inform the study physician immediately. 
2.1.1.17 Ability of subject to understand and the willingness to sign a written informed consent 
document.
2.1.2
Exclusion Criteria
2.1.2.1
Patients with cancers harboring targetable mutations for which there is approved first or 
second line therapy, unless standard of care therapy refused.
2.1.2.2
Clinically significant cardiovascular / cerebrovascular disease as follows: cerebral 
vascular accident / stroke (< 6 months prior to enrollment), myocardial infarction (< 6 


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
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51
months prior to enrollment), unstable angina, congestive heart failure (New York Heart 
Association Classification Class ≥ II), serious cardiac arrhythmia, clinically significant 
bleeding or clinically significant pulmonary embolism. 
2.1.2.3
Active Hepatitis A, Hepatitis B (e.g., HBsAg reactive) or Hepatitis C (e.g., HCV RNA 
[qualitative] is detected).
2.1.2.4
Human immunodeficiency virus (HIV) or acquired immunodeficiency syndrome 
(AIDS)-related illness due to unknown effects of DAC-THU on systemic immunity.
2.1.2.5
Other active infection requiring systemic therapy.
2.1.2.6
Pregnant women are excluded from this study because DAC-THU may have 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 DAC-
THU, breastfeeding should be discontinued if the mother is treated with DAC-THU.  
These potential risks may also apply to other agents used in this study. 
2.1.2.7
Other severe acute or chronic medical or psychiatric conditions or laboratory abnormality 
that may increase the risk associated with study participation or study drug 
administration, or may interfere with the interpretation of study results, and in the 
judgment of the investigator would make the patient inappropriate for entry into this 
study.
2.1.2.8
Patients who are receiving systemic corticosteroids.
2.1.2.9
Patients with history of or active autoimmune disease including thyroiditis, colitis, 
nephritis, neuropathy or pneumonitis.
2.1.2.10 Patients receiving another investigational agent.
2.1.2.11 An additional malignancy that is progressing or requires active treatment.  Exceptions 
include basal cell carcinoma of the skin or squamous cell carcinoma of the skin that has 
undergone potentially curative therapy, or in-situ cervical or anal cancer, or ductal 
carcinoma in-situ.
2.1.2.12 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. 
2.1.2.13 Psychiatric or substance abuse disorders that would interfere with cooperation with the 
requirements of the trial.
2.1.2.14 Thrombocytosis defined as platelet count > 1,200,000/mcL. 
2.1.3
Recruitment Strategies
This study will be posted on NIH websites and on NIH social media forums. Participants may also 
be recruited through self-referrals, physician referrals, and referrals from the NIH Clinical Center 
(CC) Office of Patient Recruitment. 
Patients may also be referred to this study based on confirmation of histology (done by the 
Laboratory of Pathology) under protocol 06C0014.


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2.2
SCREENING EVALUATION
Note: Screening evaluation testing/procedures are conducted under the separate screening protocol 
06C0014 OR the consent for study 01C0129 (provided the procedure is permitted on that study) 
per PI discretion. Assessments and procedures to confirm study eligibility should be completed as 
follows:
Within 4 Weeks Prior to Protocol Enrollment:
a.
Medical history and physical exam including vital signs and performance status.
b. Confirmation of histology (archival or fresh tissue if archival tissue is insufficient), 
PD-L1 expression and mutation status (EGFR and ALK) by the Laboratory of 
Pathology (any time prior to enrollment). This confirmation will be done under 
protocol 06C0014 (patients will be co-enrolled).
c.
Blood tests:
i. CBC with differential
ii. Acute Care Panel: Sodium, potassium, chloride, bicarbonate, creatinine, 
glucose, BUN, eGFR
iii. Hepatic Panel: Alkaline phosphatase, ALT, AST, total and direct bilirubin
iv. PT/PTT
d. Viral Markers (anti-HIV, anti-HCV, HBsAg, anti-HAV IgM); HCV RNA if 
needed.
e.
12-lead EKG.  Patients with EKG evidence of cardiac ischemia (ST 
depression/elevation greater than or equal to 2 mm), or arrhythmia will undergo 
cardiology evaluation to determine eligibility. 
f.
Echocardiogram
g. X-rays, contrast enhanced CT scans of chest, abdomen, pelvis, as well as brain MR, 
and fused PET-CT scans to evaluate the status of disease (all imaging must be 
obtained within 4 weeks prior to starting therapy).
h. Pulmonary function tests (PFTs) with and/or without bronchodilators.
i.
Arterial Blood Gas test (ABG) on room air (to be drawn if pulse oximetry < 90% on 
room air).
j.
Lupus Anticoagulant and ANA
Within 3 Days Prior to Protocol Enrollment:
a.
Women of child-bearing potential will have a urine or serum hCG pregnancy test.
2.3
PARTICIPANT REGISTRATION AND STATUS UPDATE PROCEDURES
Registration and status updates (e.g. when a participant is taken off protocol therapy and when a 
participant is taken off-study) will take place per CCR SOP ADCR-2: CCR Participant 
Registration & Status Updates found here. 


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2.3.1
Treatment Assignment Procedures
Cohorts
Number
Name
Description
1
Dose-Escalation
Subjects enrolled to dose escalation cohorts
2
Dose Expansion: NSCLC 
with high PD-L1
NSCLC subjects with high PD-L1 tumor expression 
enrolled at the MTD after the MTD is established
3
Dose Expansion: NSCLC 
with low PD-L1
NSCLC subjects with low PD-L1 tumor expression 
enrolled at the MTD after MTD is established
4
Dose Expansion: EsC
EsC subjects enrolled at the MTD after MTD is 
established
5
Dose Expansion: MPM
MPM patients enrolled at the MTD after MTD is  
established
Arms
Number
Name
Description
1
Dose Escalation
DAC-THU + pembrolizumab at escalating doses
2
Dose Expansion
DAC-THU + pembrolizumab at the dose established in 
Arm 1
Arm Assignment
Subjects in Cohort 1 will be assigned to Arm 1.
Subjects in Cohort 2, Cohort 3, Cohort 4 and Cohort 5 will be assigned to Arm 2.
3
STUDY IMPLEMENTATION 
3.1
STUDY DESIGN
This trial is unique in that it is intended to establish the rationale and conditions for the use of oral 
DAC-THU and pembrolizumab therapy for NSCLC, EsC, and MPM. The goal is to establish the 
basis for subsequent evaluation of this treatment regimen in combination with other agents 
targeting MDSC, or adoptive immunotherapy regimens in patients with thoracic malignancies.
Patients potentially eligible for trial will be evaluated, and undergo subsequent eligibility 
assessment in the Thoracic Surgery Clinic/NCI.  Patients meeting eligibility criteria will undergo 
baseline (if fresh tumor biopsy was not performed at screening) and post treatment tumor biopsies 
either by percutaneous image-guided FNA techniques in Interventional Radiology (Clinical 
Center/NIH) or endoscopic/thoracoscopic techniques in the CC operating room by Thoracic 
Surgery staff using standard of care practice guidelines. Tumor biopsies may be omitted at the 
discretion of the PI if risks outweigh potential research benefits.
The Phase I component will be a standard 3+3 design starting with oral DAC-THU on two 
consecutive days (preferably Tuesday and Wednesday) for two weeks out of three weeks for 9 


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54
weeks with incremental dose escalation as shown in Section 3.1.3. Patients with high PD-L1 tumor 
expression will be combined with patients with low PD-L1 tumor expression to minimize accrual 
numbers required for the Phase I component of the study. Pembrolizumab will be administered on 
Wednesday, Thursday or Friday at a fixed intravenous dose of 200 mg q 3 weeks. One cycle is 
three weeks; one course is 9 weeks. Treatment evaluation including repeat tumor biopsy will be 
performed after one course of therapy (Week 10 +/- one week). Those patients exhibiting disease 
progression or unacceptable toxicities will be removed from study. Patients exhibiting stable 
disease or disease regression will be offered an additional course of therapy followed by treatment 
evaluation (without mandatory biopsy). Treatment will continue in this manner until off-study 
criteria have been met. 
Once the MTD for DAC-THU has been identified, the MTD dose level will be expanded by 4 
patients to confirm safety. Then, including these 10 patients at the MTD, a total of 10 NSCLC 
patients with high intratumoral PD-L1 expression and 10 NSCLC patients with low intra-tumoral 
PD-L1 expression will be evaluated in the first stage of two separate Phase II cohorts each using a 
Simon optimal design (Phase II; Stage I). If 5 or more of 10 NSCLC patients with high PD-L1 
expression respond to treatment, the cohort will be expanded to 23 patients (Phase II; Stage II) to 
determine ORR at the MTD. If 2 or more of 10 patients with low PD-L1 expression respond to 
treatment, the cohort will be expanded to 29 patients (Phase II; Stage II) to determine ORR at the 
MTD. Up to 10 EsC patients including any patients treated in the expansion cohort of the Phase I 
component of the trial will be accrued to a separate cohort at the MTD. If 2 or more of these 10 
EsC patients respond to therapy, the protocol may be amended to enable further evaluation of the 
investigational treatment regimen in EsC patients. Similarly, up to 10 MPM patients including any 
patients treated in the expansion cohort of the Phase I component of the trial will be accrued to a 
separate cohort at the MTD. If 2 or more of these 10 MPM patients respond to therapy, the protocol 
may be amended to enable further evaluation of the investigational treatment regimen in these 
patients.


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
55
3.1.1
Study Schema
Treatment evaluation including staging studies and biopsy will be performed on Days 64 and 65 
+/- 7 days. DAC-THU will be given on two consecutive days as indicated, with 
Tuesday/Wednesday optimal but not absolute. Pembrolizumab may be administered on 
Wednesday, Thursday, or Friday for administrative convenience. 
3.1.2
Definition of Dose Limiting Toxicity (DLT)
DLT is defined as:

Any Grade 3 or greater toxicity that cannot be attributed to a cause other than study 
treatment during the first two cycles of Course 1 of DAC-THU and/or pembrolizumab 
therapy such as disease progression or intercurrent illness.

Any toxicity that cannot be attributed to a cause other than study treatment (DAC-THU 
and/or Pembrolizumab) that results in study treatment discontinuation.
Week
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
1
Day 0
1
2
3
4
5
6
2
7
8
9
10
11
12
13
3
14
15
16
17
18
19
20
4
21
22
23
24
25
26
27
5
28
29
30
31
32
33
34
6
35
36
37
38
39
40
41
7
42
43
44
45
46
47
48
8
49
50
51
52
53
54
55
9
56
57
58
59
60
61
62
10
63
64
65
66
67
68
69
DAC/THU
Pembro
Treatment 
Evaluation
DAC-THU/Pembro Treatment: Course 1 (and subsequent courses if indicated)


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
56
3.1.3
Dose Escalation
The revised dosing regimen selected for this trial is based on pharmacodynamic evidence of 
systemic hypomethylation without significant systemic toxicities mediated by oral DAC-THU in 
patients with sickle cell disease in a recent Phase 1 clinical trial (108), as well as experience with 
DAC-THU/Pembrolizumab in NSCLC (Section 1.2.11.2). The frequency of DAC-THU dosing 
will increase using a conventional 3+3 Phase I study design attempting to maximize systemic 
DNMT1 depletion while avoiding Grade 3 or greater systemic toxicities (primarily 
neutropenia/thrombocytopenia). Due to the uncertainty regarding cumulative toxicities, no intra-
patient escalation will be allowed.  
Dose escalation will proceed as indicated in Table 7 using three patients per dose level 
(irrespective of Tumor PD-L1 expression) until DLT within the first 6 weeks (two cycles) of DAC-
THU therapy is observed. If one of three patients at any given dose level experiences DLT, up to 
three additional patients will be treated at this dose level. If only one of six patients exhibit DLT, 
subsequent patients will be enrolled into the next higher dose level. As soon as two patients at any 
given dose level develop DLT, no additional patients will be entered at that level. Subsequent 
patients will be accrued into the preceding dose level; if DLT is observed in less than two of six 
patients treated at this lower level, this dose will represent maximum tolerated dose (MTD). 
Table 7: DAC-THU Dosing Schema for High (50% or Greater) and Low (0-49%) PD-L1 
Expression Groups
Dose Level
DAC
DAC
THU
THU
Frequency^
mg/kg/course
mg/kg/day*
mg/kg/course
mg/kg/day*
wk 1,2,4,5,7,8
-1
2.0
0.17
120
10
T;W
1
2.5
0.21
120
10
T;W
2
3.0
0.25
120
10
T;W
*on days administered
^ T: Tuesday; W: Wednesday
3.2
DRUG ADMINISTRATION
3.2.1
DAC-THU 
Timing between THU and decitabine: Oral THU capsules should be ingested within two hours 
prior to or two hours after a meal followed 60 minutes later by oral decitabine capsules. The 
capsules are to be ingested twice weekly on consecutive days as indicated in Table 7 (‘Smart Caps’ 
that can remind patients about timing of administration should be considered for use). 


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
57
3.2.1.1
THU Dosing
Weight 
 (kg)
THU 
(caps/dose)
40-59
2
60-79
3
80-99
4
100-119
5
120-139
6


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
58
3.2.1.2
DAC Dosing
Note: Dose is to be adjusted based on weight changes during the treatment only if patients fall into 
a different weight category bracket.
Dose Level -1: DAC 2 mg/kg/course
 DAC Pills/week
Mass (kg)
Total # Pills
wk1 (T;W) wk2 (T;W) wk3(T;W) wk4(T;W) wk5(T;W) wk6(T;W) wk7(T;W) wk8(T;W) wk9(T;W)
40-49
18
2;1
2;1
none
2;1
2;1
none
2;1
2;1
none
50-59
22
2;2
2;2
none
2;2
2;1
none
2;2
2;1
none
60-69
26
3;2
2;2
none
3;2
2;2
none
2;2
2;2
none
70-79
30
3;2
3;2
none
3;2
3;2
none
3;2
3;2
none
80-89
34
3;3
3;3
none
3;3
3;2
none
3;3
3;2
none
90-99
38
4;3
3;3
none
4;3
3;3
none
3;3
3;3
none
100-109
42
4;3
4;3
none
4;3
4;3
none
4;3
4;3
none
110-119
46
4;4
4;4
none
4;4
4;3
none
4;4
4;3
none
120-129
50
5;4
4;4
none
5;4
4;4
none
4;4
4;4
none
130-139
54
5;5
5;4
none
5;4
5;4
none
5;4
4;4
none
Dose Level 1: DAC 2.5 mg/kg/course
DAC Pills/week
Mass (kg)
Total # Pills
wk1 (T;W) wk2 (T;W) wk3(T;W) wk4(T;W) wk5(T;W) wk6(T;W) wk7(T;W) wk8(T;W) wk9(T;W)
40-49
22
2;2
2;2
none
2;2
2;1
none
2;2
2;1
none
50-59
27
3;2
2;2
none
3;2
2;2
none
3;2
2;2
none
60-69
32
3;3
3;3
none
3;3
3;3
none
3;2
3;2
none
70-79
37
4;3
3;3
none
3;3
3;3
none
3;3
3;3
none
80-89
42
4;3
4;3
none
4;3
4;3
none
4;3
4;3
none
90-99
47
4;4
4;4
none
4;4
4;4
none
4;4
4;3
none
100-109
52
5;4
5;4
none
5;4
4;4
none
5;4
4;4
none
110-119
57
5;5
5;4
none
5;5
5;4
none
5;5
5;4
none
120-129
62
6;5
5;5
none
6;5
5;5
none
5;5
5;5
none
130-139
67
6;6
6;5
none
6;6
6;5
none
6;5
5;5
none
Dose Level 2: DAC 3.0 mg/kg/course
DAC Pills/week
Mass (kg)
Total # Pills# wk1 (T;W) wk2 (T;W) wk3(T;W) wk4(T;W) wk5(T;W) wk6(T;W) wk7(T;W) wk8(T;W) wk9(T;W)
40-49
27
3;2
2;2
none
3;2
2;2
none
3;2
2;2
none
50-59
33
3;3
3;2
none
3;3
3;2
none
3;3
3;2
none
60-69
39
4;3
3;3
none
4;3
3;3
none
4;3
3;3
none
70-79
45
4;4
4;3
none
4;4
4;3
none
4;4
4;3
none
80-89
51
5;4
4;4
none
5;4
4;4
none
5;4
4;4
none
90-99
57
5;5
5;4
none
5;5
5;4
none
5;5
5;4
none
100-109
63
6;5
5;5
none
6;5
5;5
none
6;5
5;5
none
110-119
69
6;6
6;5
none
6;6
6;5
none
6;6
6;5
none
120-129
75
7;6
6;6
none
7;6
6;6
none
7;6
6;6
none
130-139
81
7;7
7;6
none
7;7
7;6
none
7;7
7;6
none


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
59
3.2.2
Pembrolizumab  
Pembrolizumab will be administered as a 30 minute IV infusion every three weeks at a fixed dose 
of 200 mg/infusion. Subjects may be dosed no less than 12 days from the previous dose of drug. 
There are no pre-medications recommended for pembrolizumab on the first cycle. 
Treatment of Pembrolizumab Related Infusion Reactions: 
Since pembrolizumab contains only human immunoglobulin protein sequences, it is unlikely to be 
immunogenic, and induce infusion or hypersensitivity reactions. However, if such a reaction were 
to occur, it might manifest with fever, chills, rigors, headache, rash, pruritus, arthralgias, hypo- or 
hypertension, bronchospasm, or other symptoms. All Grade 3 or 4 infusion reactions should be 
reported within 24 hours to the study sponsor as an SAE if it meets the criteria. Infusion reactions 
should be graded according to NCI CTCAE (Version 5.0) guidelines. 
Treatment recommendations are provided below and may be modified based on local treatment 
standards and guidelines, as appropriate:
Table 8: Treatment Modification for Pembrolizumab
NCI-CTCAE Grade
Treatment Modification for Pembrolizumab
Grade 1 – mild
Mild transient reaction; infusion interruption 
not indicated; intervention not indicated.
Decrease the pembrolizumab infusion rate 
by 50%; remain at bedside and monitor 
closely for any worsening.
The total infusion time for pembrolizumab 
should not exceed 120 minutes.
Grade 2 – moderate
Therapy or infusion interruption indicated but 
responds promptly to symptomatic treatment 
(e.g., antihistamines, NSAIDs, narcotics, IV 
fluids); prophylactic medications indicated for
 24 hours.
Stop pembrolizumab infusion.
Resume infusion at 50% of previous rate once 
infusion-related reaction has resolved or 
decreased to at least Grade 1 in severity, and 
monitor closely for any worsening.
Grade 3 or Grade 4 – severe or 
life-threatening
Grade 3: Prolonged (e.g., not rapidly 
responsive to symptomatic medication and/or 
brief interruption of infusion); recurrence of 
symptoms following initial improvement; 
hospitalization indicated for clinical sequelae.
Grade 4: Life-threatening consequences; urgent 
intervention indicated.
Stop pembrolizumab infusion immediately 
and disconnect infusion tubing from the 
subject. Subjects have to be withdrawn 
immediately from pembrolizumab treatment 
and must not receive any further 
pembrolizumab treatment.


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
60
NCI-CTCAE Grade
Treatment Modification for Pembrolizumab
- Once the pembrolizumab infusion rate has been decreased by 50% or interrupted 
due to an infusion-related reaction, it must remain decreased for all subsequent 
infusions.
- If the subject has a second infusion-related reaction Grade ≥ 2 on the slower infusion rate, the 
infusion should be stopped and the subject should be removed from study treatment.
- If hypersensitivity reaction occurs, the subject must be treated according to the best available 
medical practice.
IV=intravenous, NCI-CTCAE=National Cancer Institute-Common Terminology Criteria for 
Adverse Event, NSAIDs=nonsteroidal anti-inflammatory drugs.
3.2.3
Additional Modifications for Subjects with Grade 2 Infusion-Related Reactions
If, in the event of a Grade 2 infusion-related reaction that does not improve or worsens after 
implementation of the modifications indicated in Table 8 (including reducing the infusion rate 
by 50%), the investigator may consider treatment with corticosteroids and the infusion should 
be stopped for that day. At the next cycle, the investigator may consider the addition of H2-
blocker antihistamines (e.g., famotidine), in addition to the mandatory premedication, for 
select subjects. However, prophylactic steroids are NOT permitted.
3.2.4
Severe Hypersensitivity Reactions and Flu-like Symptoms
A. Symptoms

Impaired airway

Decreased oxygen saturation (< 92%)

Confusion

Lethargy

Hypotension

Pale/clammy skin

Cyanosis
B. Management
1. Epinephrine injection and dexamethasone infusion;
2. Patient should be placed on monitor immediately;
3. Alert intensive care unit (ICU) for possible transfer if required.
For prophylaxis of flu-like symptoms, 25 mg indomethacin or comparable NSAID dose (e.g., 
ibuprofen 600 mg, naproxen sodium 500 mg) may be administered 2 hours before and 8 hours after 
the start of each dose of pembrolizumab IV infusion. Alternative treatments for fever (e.g., 
paracetamol) may be given to subjects at the discretion of the investigator.
For Grade 1 Symptoms: (Mild reaction; infusion interruption not indicated; intervention 
not indicated): Remain at bedside and monitor subject until recovery from symptoms. The 
following prophylactic pre-medications are recommended for future infusions: diphenhydramine 
50 mg (or equivalent) and/or paracetamol 325 to 1000 mg (acetaminophen) at least 30 minutes 
before additional pembrolizumab administrations.
For Grade 2 Symptoms: (Moderate reaction requires therapy or infusion interruption but 
responds promptly to symptomatic treatment [e.g., antihistamines, non-steroidal anti-


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inflammatory drugs, narcotics, corticosteroids, IV fluids]; prophylactic medications 
indicated for 24 hours):  Stop the pembrolizumab infusion, begin an IV infusion of normal saline, 
and treat the subject with diphenhydramine 50 mg IV (or equivalent) and/or paracetamol 325 to 
1000 mg (acetaminophen); remain at bedside and monitor subject until resolution of symptoms to 
Grade 1 or less. Resume infusion at 50% rate.
3.2.3
CCR Self –Administered Study Drugs Policy
All oral self-administered investigational agents will be properly accounted for, handled, and 
disposed in accordance with existing federal regulations and principles of Good Clinical Practice. 
All oral study drugs will be recorded in the patient diary found in Appendix 2.  This will be used 
as a memory aide for subjects. The clinical research team will maintain the primary source record.  
Subjects should be asked to bring the diary as well as unused study agent and empty containers 
with them at each study visit.  If a subject goes off study while at home, the research nurse will 
ensure and document the return of the unused oral investigational agents from the participant.
Unused investigational study agents will be disposed and destroyed per CC pharmacy SOPs.
3.3
DOSING DELAYS AND MODIFICATIONS
This study is intended to evaluate the safety and potential efficacy of oral DAC-THU and 
pembrolizumab as epigenetic immunotherapy in patients with NSCLC, EsC and MPM.  The 
proposed oral DAC dose of ~0.17 mg/kg to be combined with THU ~10 mg/kg administered 60 
minutes before oral DAC, is based on non-human primate studies and the Phase 1 clinical trial in 
SCD which had an identical pharmacologic objective of DAC distribution through tissues with 
high CDA expression (intestines, liver etc.), and the wide concentration-time profile desired for 
non-cytotoxic DNMT1-depletion. This dose of DAC administered orally is 10-20% of the FDA-
approved dosages administered intravenously and is < 50% of the dose of oral decitabine 
administered together with another CDA-inhibitor (a THU-analogue) in clinical trials by 
Astex/Otsuka.
Although it is anticipated that this therapy will be well tolerated at the revised doses, 
myelosuppression resulting from reprogramming of hematopoietic stem cell differentiation may 
become dose limiting (50). If dose limiting myelosuppression is observed during the first 2 cycles 
of Course 1, DAC-THU will be held until toxicities have resolved to Grade 2 or less, and DAC-
THU therapy will recommence at the next lower dose, or at the SAME dose if deemed appropriate 
by the PI, as indicated in the dose escalation schema (Table 7). Only one dose reduction will be 
allowed. 
3.3.1
General Guidance for Dose Modifications
3.3.1.1
Patients may continue to receive therapy provided the following criteria are met on Day 1 
of each cycle:
Non-hematologic toxicity recovered to < Grade 2 (or tolerable Grade 2 or baseline).
No evidence of progressive disease.
3.3.1.2
In the event of an adverse event at least possibly related to the investigational agent, the 
dose of the investigational agent should be adjusted according to the parameters 
articulated above and guidelines listed in the Dose Delays / Dose Modifications tables 
shown below (Table 9 and Table 10). If an adverse event is not covered in such tables, 
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3.3.1.3
If patients fail to recover to CTCAE Grade 0-1, tolerable Grade 2 (or within 1 grade of 
starting values for pre-existing laboratory abnormalities), or baseline from a treatment-
related adverse event attributable to DAC-THU within 42 days (2 cycles), they cannot 
receive additional DAC-THU. Patients may continue pembrolizumab monotherapy at the 
investigator discretion provided that discontinuation criteria for pembrolizumab toxicity 
have not been met.
3.3.2
Treatment Delays and Modifications for Medical Needs
Brief interruptions and delays up to 3 weeks may be required due to patients experiencing 
complications of their disease or other medical illness not attributable to disease progression, or 
protocol therapy. A patient that interrupts therapy for more than 3 weeks will be taken off 
treatment.
3.3.3
Dose Reductions
No dose reductions of Pembrolizumab are permitted on this study.
3.3.4
Dosage Modification and Management of Immune-Related Adverse Events
Since inhibition of PD-L1 stimulates the immune system, irAEs may occur. Treatment of irAEs 
is mainly dependent upon severity (NCI-CTCAE grade):
Grade 1 to 2: treat symptomatically or with moderate dose steroids, more frequent 
monitoring.
Grade 1 to 2 (persistent): manage similar to high grade AE (Grade 3 to 4).
Grade 3 to 4: treat with high dose corticosteroids.
 Treatment of irAEs should follow accepted guidelines set forth in Table 10.
3.3.5
Dose Modification of DAC-THU
Table 9 lists standard laboratory values that should trigger dose modifications of the 
investigational drug.
Table 9: Trigger Values that Result in Dose Modification
General Adverse Events
Action
Non-hematological, Grade 1 or 2
(excluding hemorrhage)

Continue DAC-THU therapy at full dose prescribed. 

Apply maximum supportive care recommendations.

If prolonged duration of Grade 2 adverse event (≥ 7 days) 
is affecting quality of life, start the next treatment cycle at 
DL-1.

If event persists and continues to affect quality of life for 
≥ 7 more days following initial dose reduction, 
discontinue DAC-THU.
Grade 3 ALT and AST

Hold DAC-THU therapy until recovery to Grade  1. 

If ALT and AST resolve to Grade  1 within 10 days, 
maintain current dose level.

If ALT and AST do NOT resolve to Grade  1 within 10 
days, dose reduce 1 level.

If Grade 3 ALT or AST occurs after dose reduction, 
discontinue DAC-THU.


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General Adverse Events
Action
Grade 4 ALT and AST

Hold DAC-THU therapy until recovery to Grade  1. 

Resume DAC-THU at DL-1.

If Grade 4 toxicities occur after dose reduction, 
discontinue DAC-THU.
Non-hematological, Grade 3 or 4
(excluding ALT, AST,  hemorrhage 
and QT prolongation)

Apply maximum supportive care recommendations. Hold 
DAC-THU until recovery to Grade  1 and resume DAC-
THU at the next lower dose level. 

If symptoms continue to persist at Grade 3 or 4, following 
dose reduction, discontinue drug.
QT prolongation, Grade 3 or 4

Cardiology consultation will be obtained to determine 
whether any cardiac functional assessment is warranted or 
if therapy should be delayed or discontinued. When 
warranted, ECGs will subsequently be reviewed by the 
NCI consulting cardiologist.

If therapy is continued, dose reduce 1 level for Grade 4 
events.

If Grade 3 or 4 toxicities occur after dose reduction, 
discontinue DAC-THU.
Thrombocytosis

If platelet count > 500/ mcL but < 1.2M/mcL, continue 
DAC-THU at current dose. Begin ASA 325mg PO daily.

If platelet count 1.2M /mcL or more, hold DAC-THU until 
platelet count < 1.2 M/mcL, begin ASA 325 mg PO daily 
and resume DAC-THU at DL-1 if within the first two 
Cycles of Course 1; or at SAME dose or DL-1 per PI 
discretion in subsequent cycles.
Hemoglobin

Hgb > 18 g/dL: hold DAC-THU until Hgb WNL. Resume 
DAC-THU at DL-1.
Hematological Adverse Events
Grades 1 and 2 events

Continue DAC-THU therapy at full dose prescribed. 
Apply maximum supportive care recommendations.
Grade 3 events

Apply supportive care.

If toxicities occur in the first two cycles of therapy and 
resolve to Grade 1 or less, in 10 days, resume DAC-THU 
at current dose.

If toxicities occur in the first two cycles of therapy and 
resolve to Grade 1 or less > 10 days following drug hold, 
resume DAC-THU at next lower dose.

If toxicities occur in subsequent cycles and resolve to 
Grade 1 or less in 21 days following drug hold, resume 
DAC-THU at SAME dose. If toxicities recur within two 
cycles of resuming DAC-THU, hold until toxicities 
resolve to Grade 1 or less, and resume DAC-THU at next 
lower dose.

If toxicities occur in subsequent cycles and resolve to 


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General Adverse Events
Action
Grade 1 or less > 21 days following drug hold, resume 
DAC-THU at next lower dose. 

If toxicities recur within two cycles after maximal dose 
reduction, discontinue DAC-THU. 
Grade 4 events

Apply maximum supportive care recommendations.

Hold DAC-THU therapy until patient meets hematologic 
criteria for retreatment and resume DAC-THU at next 
lower dose.

If recurrence of hematological adverse event after drug 
hold/ interruptions is observed with treatment at DL-1, in 
the setting of maximum supportive care measures applied, 
hold drug once again until patient meets hematologic 
criteria for retreatment, further dose reduce DAC-THU if 
possible or discontinue DAC-THU per PI discretion.
3.3.6
Dose Delay of Pembrolizumab
No dose modification for pembrolizumab is allowed. Because of the potential for clinically 
meaningful pembrolizumab-related AEs requiring early recognition and prompt intervention, 
management algorithms have been developed for suspected AEs of selected categories. Dose delay 
criteria apply for all drug-related adverse events (regardless of whether or not the event is attributed 
to pembrolizumab). If AE is felt to be immune related and attributable to pembrolizumab, the 
patient can continue DAC-THU while pembrolizumab is held. Dose delay criteria apply for all 
drug-related AEs. Pembrolizumab must be delayed until treatment can resume. 
3.3.6.1
Pembrolizumab Administration Should be Delayed for the Following:
Table 10: Management of Immune-Related Adverse Events
Gastrointestinal irAEs
Severity of Diarrhea/Colitis
(NCI-CTCAE v5)
Initial Management
Follow-up Management
Grade 1
Diarrhea: < 4 stools/day over 
Baseline
Colitis: asymptomatic 
Continue Pembrolizumab
Symptomatic treatment (e.g. 
loperamide)
Close monitoring for worsening 
symptoms
Educate subject to report worsening 
immediately
If worsens:
Treat as Grade 2, 3 or 4. 


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Gastrointestinal irAEs
Severity of Diarrhea/Colitis
(NCI-CTCAE v5)
Initial Management
Follow-up Management
Grade 2 
Diarrhea: 4 to 6 stools per day over 
Baseline; IV fluids indicated < 24 
hours; not interfering with ADL
Colitis: abdominal pain; blood in 
stool 
Withhold Pembrolizumab
Symptomatic treatment
If improves to Grade ≤ 1: 
Resume Pembrolizumab therapy
If persists > 5-7 days or recurs:
Treat as Grade 3 or 4.
Grade 3 to 4
Diarrhea (Grade 3): ≥ 7 stools per 
day over Baseline; incontinence; IV 
fluids ≥ 24 h; interfering with ADL
Colitis (Grade 3): severe abdominal 
pain, medical intervention indicated, 
peritoneal signs
Grade 4: life-threatening, 
perforation
Withhold Pembrolizumab for Grade 3.
Permanently discontinue 
Pembrolizumab for Grade 4 or 
recurrent Grade 3.
1.0 to 2.0 mg/kg/day prednisone IV or 
equivalent
Add prophylactic antibiotics for 
opportunistic infections 
Consider lower endoscopy 
If improves: 
Continue steroids until Grade ≤ 1, 
then taper over at least 1 month; 
resume Pembrolizumab therapy 
following steroids taper (for initial 
Grade 3). 
If worsens, persists > 3 to 5 days, or 
recurs after improvement:
Add infliximab 5mg/kg (if no 
contraindication). Note: infliximab 
should not be used in cases of 
perforation or sepsis. 
Dermatological irAEs
Grade of Rash
(NCI-CTCAE v5)
Initial Management
Follow-up Management
Grade 1 to 2 
Covering ≤ 30% body surface area 
Continue Pembrolizumab 
Symptomatic therapy (for example, 
antihistamines, topical steroids) 
If persists > 1 to 2 weeks or recurs: 
Withhold Pembrolizumab therapy 
Consider skin biopsy 
Consider 0.5-1.0 mg/kg/day 
prednisone or equivalent. Once 
improving, taper steroids over at least 
1 month, consider prophylactic 
antibiotics for opportunistic 
infections, and resume 
Pembrolizumab therapy following 
steroids taper.


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Dermatological irAEs
Grade of Rash
(NCI-CTCAE v5)
Initial Management
Follow-up Management
If worsens: 
Treat as Grade 3 to 4. 
Grade 3 to 4
Grade 3: Covering > 30% body 
surface area; 
Grade 4: Life threatening 
consequences 
Withhold Pembrolizumab for Grade 3.
Permanently discontinue for Grade 4 
or recurrent Grade 3. 
Consider skin biopsy 
Dermatology consult 
1.0 to 2.0 mg/kg/day prednisone or 
equivalent
Add prophylactic antibiotics for 
opportunistic infections 
If improves to Grade ≤ 1: 
Taper steroids over at least 1 month; 
resume Pembrolizumab therapy 
following steroids taper (for initial 
Grade 3).
Pulmonary irAEs
Grade of Pneumonitis
(NCI-CTCAE v5)
Initial Management
Follow-up Management
Grade 1
Radiographic changes only 
Consider withholding Pembrolizumab 
therapy 
Monitor for symptoms every 2 to 3 
days 
Consider Pulmonary and Infectious 
Disease consults 
Re-assess at least every 3 weeks
If worsens: 
Treat as Grade 2 or Grade 3 to 4. 
Grade 2
Mild to moderate new symptoms 
Withhold Pembrolizumab therapy
Pulmonary and Infectious Disease 
consults
Monitor symptoms daily; consider 
hospitalization 
1.0 to 2.0 mg/kg/day prednisone or 
equivalent
Add prophylactic antibiotics for 
opportunistic infections 
Consider bronchoscopy, lung biopsy 
Re-assess every 1 to 3 days 
If improves: 
When symptoms return to Grade ≤ 1, 
taper steroids over at least 1 month, 
and then resume Pembrolizumab 
therapy following steroids taper 
If not improving after 2 weeks or 
worsening: 


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Pulmonary irAEs
Grade of Pneumonitis
(NCI-CTCAE v5)
Initial Management
Follow-up Management
Treat as Grade 3 to 4. 
Grade 3 to 4
Grade 3: Severe new symptoms; 
New/worsening hypoxia;
Grade 4: Life-threatening 
Permanently discontinue 
Pembrolizumab therapy.
Hospitalize.
Pulmonary and Infectious Disease 
consults.
1.0 to 2.0 mg/kg/day prednisone or 
equivalent 
Add prophylactic antibiotics for 
opportunistic infections 
Consider bronchoscopy, lung biopsy 
If improves to Grade ≤ 1: 
Taper steroids over at least 1 month 
If not improving after 48 hours or 
worsening: 
Add additional immunosuppression 
(for example, infliximab, 
cyclophosphamide, IV 
immunoglobulin, or mycophenolate 
mofetil) 
Hepatic irAEs
Grade of Liver Test Elevation
(NCI-CTCAE v5)
Initial Management
Follow-up Management
Grade 1
Grade 1 AST or ALT > ULN to 3.0 
x ULN and/or Total bilirubin > 
ULN to 1.5 x ULN 
Continue Pembrolizumab therapy
Continue liver function monitoring 
If worsens: 
 Treat as Grade 2 or 3 to 4. 
Grade 2
AST or ALT > 3.0 to ≤ 5 x ULN 
and/or total bilirubin > 1.5 to ≤ 3 x 
ULN 
Withhold Pembrolizumab therapy
Increase frequency of monitoring to 
every 3 days.
If returns to Grade ≤ 1:
Resume routine monitoring; resume 
Pembrolizumab therapy.
If elevation persists > 5 to 7 days or 
worsens: 
Treat as Grade 3 to 4.
Grade 3 to 4
AST or ALT > 5 x ULN and/or total 
bilirubin > 3 x ULN 
Permanently discontinue 
Pembrolizumab therapy 
Increase frequency of monitoring to 
every 1 to 2 days 
1.0 to 2.0 mg/kg/day prednisone or 
equivalent
If returns to Grade ≤ 1: 
Taper steroids over at least 1 month 
If does not improve in > 3 to 5 days, 
worsens or rebounds: 


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Hepatic irAEs
Grade of Liver Test Elevation
(NCI-CTCAE v5)
Initial Management
Follow-up Management
Add prophylactic antibiotics for 
opportunistic infections 
Consult gastroenterologist/ 
hepatologist
Consider obtaining MRI/CT scan of 
liver and liver biopsy if clinically 
warranted 
Add mycophenolate mofetil 1 gram 
(g) twice daily 
If no response within an additional 3 
to 5 days, consider other 
immunosuppressants per local 
guidelines.
Renal irAEs
Grade of Creatinine Increased 
(NCI-CTCAE v5)
Initial Management
Follow-up Management
Grade 1
Creatinine increased > ULN to 1.5 
x ULN
Continue Pembrolizumab therapy
Continue renal function monitoring
If worsens:
Treat as Grade 2 to 3 or 4.
Grade 2 to 3
Creatinine increased > 1.5 and ≤ 6 x 
ULN
Withhold Pembrolizumab therapy
Increase frequency of monitoring to every 
3 days and consider Nephrology consult
1.0 to 2.0 mg/kg/day prednisone or 
equivalent.
Add prophylactic antibiotics for 
opportunistic infections
Consider renal biopsy
If returns to Grade ≤ 1:
Follow Nephrology recommendations 
if consult obtained; alternatively, 
taper steroids over at least 1 month, 
and resume Pembrolizumab therapy 
following steroids taper.
If worsens: 
Treat as Grade 4.
Grade 4
Creatinine increased > 6 x ULN
Permanently discontinue Pembrolizumab 
therapy
Monitor creatinine daily
1.0 to 2.0 mg/kg/day prednisone or 
equivalent.
Add prophylactic antibiotics for 
opportunistic infections
Consider renal biopsy
Nephrology consult
If returns to Grade ≤ 1:
Follow Nephrology 
recommendations; alternatively, taper 
steroids over at least 1 month.


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Cardiac irAEs
Myocarditis
Initial Management
Follow-up Management
New onset of cardiac signs or 
symptoms and / or new laboratory 
cardiac biomarker elevations (e.g., 
troponin, CK-MB, BNP) or cardiac 
imaging abnormalities suggestive 
of myocarditis. 
Withhold Pembrolizumab therapy.
Hospitalize.
In the presence of life threatening cardiac 
decompensation, consider transfer to a 
facility experienced in advanced heart 
failure and arrhythmia management.
Cardiology consult to establish etiology 
and rule-out immune-mediated 
myocarditis. 
Guideline based supportive treatment as 
per cardiology consult. *
Consider myocardial biopsy if 
recommended per cardiology consult.
If symptoms improve and 
immune-mediated etiology is ruled 
out, re-start Pembrolizumab therapy.
If symptoms do not 
improve/worsen, viral myocarditis 
is excluded, and immune-mediated 
etiology is suspected or confirmed 
following cardiology consult, 
manage as immune-mediated 
myocarditis.
Immune-mediated myocarditis
Permanently discontinue 
Pembrolizumab.
Guideline based supportive treatment as 
appropriate as per cardiology consult. *
1.0 to 2.0 mg/kg/day prednisone or 
equivalent.
Add prophylactic antibiotics for 
opportunistic infections.
Once improving, taper steroids over 
at least 1 month.
If no improvement or worsening, 
consider additional 
immunosuppressants (e.g. 
azathioprine, cyclosporine A).
*Local guidelines, or e.g., ESC or AHA guidelines 
ESC guidelines website: https://www.escardio.org/Guidelines/Clinical-Practice-Guidelines
AHA guidelines website: 
http://professional.heart.org/professional/GuidelinesStatements/searchresults.jsp?q=&y=&t=1001
Endocrine irAEs
Endocrine Disorder
Initial Management
Follow-up Management
Grade 1 or Grade 2 
endocrinopathies 
(hypothyroidism, 
hyperthyroidism, adrenal 
insufficiency, type I diabetes 
mellitus)
Continue Pembrolizumab therapy
Endocrinology consult if needed
Start thyroid hormone replacement 
therapy (for hypothyroidism), 
anti-thyroid treatment (for 
hyperthyroidism), corticosteroids (for 
adrenal insufficiency) or insulin (for 
Type I diabetes mellitus) as 
appropriate. 
Continue hormone 
replacement/suppression and 
monitoring of endocrine function as 
appropriate.


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Endocrine irAEs
Endocrine Disorder
Initial Management
Follow-up Management
Rule-out secondary endocrinopathies 
(i.e., hypopituitarism / hypophysitis)
Grade 3 or Grade 4 
endocrinopathies 
(hypothyroidism, 
hyperthyroidism, adrenal 
insufficiency, type I diabetes 
mellitus)
Withhold Pembrolizumab therapy
Consider hospitalization
Endocrinology consult
Start thyroid hormone replacement 
therapy (for hypothyroidism), 
anti-thyroid treatment (for 
hyperthyroidism), corticosteroids (for 
adrenal insufficiency) or insulin (for 
type I diabetes mellitus) as 
appropriate. 
Rule-out secondary endocrinopathies 
(i.e. hypopituitarism / hypophysitis)
Resume Pembrolizumab once 
symptoms and/or laboratory tests 
improve to Grade ≤ 1 (with or 
without hormone 
replacement/suppression).
Continue hormone 
replacement/suppression and 
monitoring of endocrine function as 
appropriate. 
Hypopituitarism/Hypophysitis 
(secondary endocrinopathies)
If secondary thyroid and/or adrenal 
insufficiency is confirmed (i.e. 
subnormal serum FT4 with 
inappropriately low TSH and/or low 
serum cortisol with inappropriately 
low ACTH):

Refer to endocrinologist for 
dynamic testing as indicated and 
measurement of other hormones 
(FSH, LH, GH/IGF-1, PRL, 
testosterone in men, estrogens in 
women) 

Hormone 
replacement/suppressive therapy 
as appropriate 

Perform pituitary MRI and visual 
field examination as indicated
If hypophysitis confirmed:

Continue Pembrolizumab if mild 
symptoms with normal MRI. 
Repeat the MRI in 1 month
Resume Pembrolizumab once 
symptoms and hormone tests 
improve to Grade ≤ 1 (with or 
without hormone replacement).
In addition, for hypophysitis with 
abnormal MRI, resume 
Pembrolizumab only once shrinkage 
of the pituitary gland on MRI/CT 
scan is documented. 
Continue hormone 
replacement/suppression therapy as 
appropriate.


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Endocrine irAEs
Endocrine Disorder
Initial Management
Follow-up Management

Withhold Pembrolizumab if 
moderate, severe or life-
threatening symptoms of 
hypophysitis and/or abnormal 
MRI. Consider hospitalization. 
Initiate corticosteroids (1 to 2 
mg/kg/day prednisone or 
equivalent) followed by 
corticosteroids taper during at 
least 1 month.

Add prophylactic antibiotics for 
opportunistic infections.
Other irAEs (not described above)
Grade of other irAEs 
(NCI-CTCAE v5)
Initial Management
Follow-up Management
Grade 2 or Grade 3 clinical signs 
or symptoms suggestive of a 
potential irAE
Withhold Pembrolizumab therapy 
pending clinical investigation
If irAE is ruled out, manage as 
appropriate according to the 
diagnosis and consider re-starting 
Pembrolizumab therapy
If irAE is confirmed, treat as Grade 2 
or 3 irAE.
Grade 2 irAE or first occurrence 
of Grade 3 irAE
Withhold Pembrolizumab therapy
1.0 to 2.0 mg/kg/day prednisone or 
equivalent 
Add prophylactic antibiotics for 
opportunistic infections 
Specialty consult as appropriate
If improves to Grade ≤ 1: 
Taper steroids over at least 1 month 
and resume Pembrolizumab therapy 
following steroids taper.
Recurrence of same Grade 3 
irAEs
Permanently discontinue 
Pembrolizumab therapy
to 2.0 mg/kg/day
prednisone or equivalent
Add prophylactic antibiotics for 
opportunistic infections 
Specialty consult as appropriate
If improves to Grade ≤ 1: 
Taper steroids over at least 1 month.
Grade 4 
Permanently discontinue 
Pembrolizumab therapy
to 2.0 mg/kg/day
If improves to Grade ≤ 1: 
Taper steroids over at least 1 month


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Other irAEs (not described above)
Grade of other irAEs 
(NCI-CTCAE v5)
Initial Management
Follow-up Management
prednisone or equivalent and/or other 
immunosuppressant as needed
Add prophylactic antibiotics for 
opportunistic infections 
Specialty consult.
Requirement for 10 mg per day 
or greater prednisone or 
equivalent for more than 12 
weeks for reasons other than 
hormonal replacement for 
adrenal insufficiency
Persistent Grade 2 or 3 irAE 
lasting 12 weeks or longer
Permanently discontinue 
Pembrolizumab therapy
Specialty consult
ADL=activities 
of 
daily 
living, 
ALT=alanine 
aminotransferase, 
AST=aspartate 
aminotransferase, irAE=immune-related adverse event, IV=intravenous, LLN=lower limit of 
normal, MRI=magnetic resonance imaging, NCI-CTCAE=National Cancer Institute-Common 
Terminology Criteria for Adverse Event, NSAIDs=nonsteroidal anti-inflammatory drugs, 
T4=thyroxine, 
TSH=thyroid-stimulating 
hormone, 
ULN=upper 
limit 
of 
normal, 
IVIG=intravenous immunoglobulins, CPK=creatine phosphokinase.
Note: Subjects who require delay of pembrolizumab should be re-evaluated closely as 
clinically indicated and resume pembrolizumab dosing when re-treatment criteria are met.
3.3.6.2
Criteria to Resume Pembrolizumab Treatment
Subjects may resume treatment with study drug when the drug-related AE(s) resolve to Grade ≤ 1 
or baseline value, with the following exceptions:

Subjects may resume treatment in the presence of Grade 2 fatigue. 

Subjects who have not experienced a Grade 3 drug-related skin AE may resume treatment in 
the presence of Grade 2 skin toxicity. 

Subjects 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.

Subjects with combined Grade 2 AST/ALT AND total bilirubin values meeting 
discontinuation parameters should have treatment permanently discontinued.

Drug-related pulmonary toxicity, diarrhea, or colitis, must have resolved to baseline before 
treatment is resumed. Subjects with persistent Grade 1 pneumonitis after completion of a 
steroid taper over at least 1 month may be eligible for retreatment.

Drug-related endocrinopathies adequately controlled with only physiologic hormone 
replacement may resume treatment.


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If the criteria to resume treatment are met, the subject should restart treatment at the next scheduled 
time point per protocol. However, if the treatment is delayed past the next scheduled time point 
per protocol, the next scheduled time point will be delayed until dosing resumes.
If treatment is delayed or interrupted for > 6 weeks, the subject must be permanently discontinued 
from study therapy unless there is evidence (stable disease or disease regression) of clinical benefit.
3.4
ON STUDY PROTOCOL EVALUATION (APPENDIX 3: STUDY CALENDAR)
3.4.1
Baseline Assessments
Note: Baseline assessments do not need to be repeated if performed during screening in the 
designated time frame.
Any Time Prior to Initiation of Treatment:
a.
HLA class I and class II analysis of PBMC.
b. Tumor biopsy if fresh sample from previous biopsy is not available. Note: Samples 
collected on 06C0014 or any other NCI protocol in which the patient is co-enrolled 
can be used.
Within 10 Days Prior to Commencing DAC-THU/Pembrolizumab Therapy:
a.
Physical examination including assessment of vital signs and ECOG status.
b. Chemistries
i. Acute Care Panel - Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), 
ii. Mineral Panel - Albumin, Calcium total, Magnesium total (Mg), 
Phosphorus
iii. Hepatic Panel - Alkaline Phosphatase, ALT/GPT, AST/GOT, Total 
Bilirubin, Direct Bilirubin
iv.  LD, Total Protein, Total CK, Uric Acid
c.
Fetal hemoglobin
d. CBC, differential, platelet count, PT, PTT
e.
T, B, NK cell subsets 
f.
Urinalysis and culture if indicated
g. Pulmonary Function Test
h. Arterial Blood Gas on room air (to be drawn if pulse oximetry <90% on room air).
i.
Research Labs (refer to Appendix 4 for details).
Within 48 Hours Prior to Initiation of DAC-THU/Pembrolizumab Therapy:
a.
Women of child-bearing potential will have a urine or serum hCG pregnancy test.


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3.4.2
Treatment Evaluation 
Twice Weekly During First Course of Therapy, and Weekly Thereafter Unless Clinically 
Indicated:
a.
CBC with differential
b. Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), Creatinine, 
Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), 
Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct 
Bilirubin, LD, Total Protein, Total CK, Uric Acid
c.
PT/PTT
Note: The patient may have lab work obtained through his or her local physician.
Every Three Weeks During Therapy: 
a.
T, B, NK cell subsets
Performed After Each Course:
a.
Physical examination including assessment of vital signs and ECOG status.
b. Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), Creatinine, 
Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), 
Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct 
Bilirubin, LD, Total Protein, Total CK, Uric Acid
c.
CBC, differential, platelet count, PT, PTT
d. Thyroid Function Tests
e.
T, B, NK cell subsets 
f.
Urinalysis and culture if indicated
g. Biopsy of index lesion (only on first treatment evaluation).
3.4.3
Research Evaluations (Refer to Appendix 4, Unless Otherwise Indicated Below)

Pharmacokinetic (PK) Studies 

Research Labs 

Radiological Assessments (refer to Appendix 3)

Tumor Biopsies (see Appendix 3)
3.4.4
Follow-Up Evaluations
Patients who are removed from treatment due to disease progression or unacceptable, dose-limiting 
toxicities or who voluntarily withdraw from treatment will be monitored weekly until treatment 
related toxicities have resolved to baseline at which time they will be removed from study.
Toxicity Evaluation:
o
Laboratory evaluation: CBC, Chemistries (Sodium, potassium, chloride, 
bicarbonate, creatinine, glucose, BUN, albumin, total calcium, ionized calcium, 
magnesium, phosphorus, alkaline phosphatase, ALT/SGPT, AST/SGOT, total 


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bilirubin, direct bilirubin, lactate dehydrogenase, total protein, creatine kinase, uric 
acid), PT/PTT
3.5
COSTS AND COMPENSATION
3.5.1
Costs
NIH does not bill health insurance companies or participants for any research or related clinical 
care that participants receive at the NIH Clinical Center. If some tests and procedures are 
performed outside the NIH Clinical Center, participants may have to pay for these costs if they are 
not covered by their insurance company. Medicines that are not part of the study treatment will 
not be provided or paid for by the NIH Clinical Center.
3.5.2
Compensation
Participants will not be compensated on this study.
3.5.3
Reimbursement
The NCI will cover the costs of some expenses associated with protocol participation. Some of 
these costs may be paid directly by the NIH and some may be reimbursed to the 
participant/guardian as appropriate. The amount and form of these payments are determined by 
the NCI Travel and Lodging Reimbursement Policy.
3.6
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA
Prior to removal from protocol, effort must be made to have all subjects complete a safety visit 
approximately 30 days after the last dose of study therapy.
3.6.1
Criteria for Removal from Protocol Therapy

Patient refusal of further treatments

It is deemed in the best interest of the patient

A patient who develops a concurrent serious medical condition that might preclude or 
contraindicate administration of DAC-THU/pembrolizumab therapy will be removed 
from treatment

A patient who becomes pregnant will be immediately taken off therapy

Progressive disease

Failure to meet criteria for additional treatment as defined in Sections 3.3.2 and 3.3.6

Delay of treatment for three weeks
Note: Patients will be followed until toxicities related to experimental therapy resolve to baseline.
3.6.2
Off-Study Criteria

Voluntary withdrawal for any reason, or noncompliance with protocol requirements

Completion of the follow up period (see Section 3.4.4)

Progressive disease unless the patient is being followed for a serious adverse event 
related to the research. The event must resolve to baseline prior to removing from the 
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
If the investigator determines that it is in the best interest of the patient to discontinue 
follow up

Lost to follow up

Death

PI decision to close the study
3.6.3
Lost to Follow-Up
A participant will be considered lost to follow-up if he or she fails to return for 3 scheduled visits 
and is unable to be contacted by the study site staff. 
The following actions must be taken if a participant fails to return to the clinic for a required study 
visit:

The site will attempt to contact the participant and reschedule the missed visit within 5 
business days and counsel the participant on the importance of maintaining the assigned 
visit schedule and ascertain if the participant wishes to and/or should continue in the 
study.

Before a participant is deemed lost to follow-up, the investigator or designee will make 
every effort to regain contact with the participant (where possible, 3 telephone 
calls/video calls and, if necessary, an IRB approved certified letter to the participant’s 
last known mailing address or local equivalent methods). These contact attempts should 
be documented in the participant’s medical record or study file. 

Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up.
4
CONCOMITANT MEDICATIONS/MEASURES
4.1
HEMATOLOGIC AND BLOOD PRODUCT SUPPORT
Blood product support should be provided to maintain platelets > 20,000 cells/mcl, Hgb > 8.0 
gm/dl and as clinically indicated. Growth factor support is not permitted with exception of 
administration of filgrastim. Filgrastim will not be administered prophylactically, but may be 
administered if a patient experiences neutropenic sepsis, or if clinically indicated.
4.2
ANTIDIARRHEALS
Antidiarrheal agents will be prescribed using standard clinical practice guidelines at the preference 
of the investigator.
4.3
ELECTROLYTE REPLACEMENT
Electrolyte replacement will be provided to maintain serum levels within normal limits.
4.4
PROTON PUMP INHIBITORS
Given the uncertainties regarding effects of proton pump inhibitors on absorption of DAC-THU, 
proton pump inhibitors will not be used except on days when DAC-THU is not administered. 


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5
CORRELATIVE STUDIES
5.1
BIOSPECIMEN COLLECTION AND PROCESSING FOR RESEARCH
5.1.1
Pharmacokinetic (PK) Sampling
Detailed plasma pharmacokinetic (PK) sampling of DAC and THU will be collected as outlined 
in Appendix 4. PK analysis will be performed at the NCI, Genitourinary Malignancies Branch 
(GMB). Samples will be stored in the Thoracic Epigenetics Lab (TEL) and batched and later 
transferred to the Figg Lab on dry ice for analysis. 
5.1.2
Studies on Tumor Tissue and Peripheral Blood
Portions of biopsy materials will be sent for frozen section or permanent section confirmation of 
malignancy and percent viable tumor cells. Tissue will be processed for focused gene, endogenous 
retroviral (ERV) and microRNA expressions, and DNA methylation signatures using quantitative 
RT-PCR, nanostring, pyrosequencing and digital droplet PCR techniques. Blood from EDTA and 
red top tubes (see Appendix 4) will be used to isolate plasma circulating tumor DNA and serum 
for focused methylation analysis and immune response to treatment. Remaining tissue samples 
will be snap frozen in liquid nitrogen for subsequent isolation of RNA and DNA for qRT-PCR and 
nanostring experiments. If sufficient tissue is available, another portion will be imbedded in 
paraffin for subsequent immunostaining experiments, focusing on expression of genes focusing on 
those proteins encoded by genes that have been identified to be clearly activated by epigenetic 
therapy. If sufficient materials are present, additional more comprehensive analyses including 
multiplex IHC analysis of tumor microenvironment may be performed with the focus of materials 
from patients treated at the MTD. All of the analyses, which are predicated on acquisition of 
sufficient materials, will be performed in the Thoracic Epigenetics Lab under direction of the PI 
or submitted to sequencing cores at the NCI.
5.1.3
Immune Subset Analyses
Peripheral blood mononuclear cells (PBMC) will be assessed using multiparameter flow cytometry 
for immune subsets including but not necessarily limited to Tregs, MDSC, effector and exhausted 
CD4+ or CD8+ T-cells, and CD14+ monocytes.  Assessment will include functional markers, i.e. 
PD-1, PD-L1, Tim-3, CTLA-4, HLA-DR and/or CD40. Samples will be drawn (as indicated in 
Appendix 4) and picked up by the Trepel Lab (see Section 5.2.4) for analysis. The Trepel Lab will 
prepare the samples for staining, stain and run the samples by multiparametric flow cytometry 
(MACSQuant, Miltenyi Biotec, Bergisch Gladbach, DE), and analyze the data using FlowJo 
(FlowJo LLC, Ashland, OR) software.  
5.1.4
Circulating Tumor Cells
Peripheral blood will be collected to correlate changes in circulating tumor cells with clinical 
response. CTCs will be assessed using ferrofluidic enrichment and multi-parameter flow 
cytometric detection.  Samples will be drawn (as indicated in Appendix 4) and picked up by the 
Trepel Lab (Section 5.2.4) for analysis. The Trepel Lab will process the samples, stain and run one 
tube immediately.  The other tube will be processed, 2D barcoded and viably stored for potential 
future analysis. The samples will be analyzed by multiparametric flow cytometry (MACSQuant, 
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5.2
SAMPLE STORAGE, TRACKING AND DISPOSITION
Samples will be ordered and tracked through Clinical Trial Data Management System. Should a 
CRIS screen not be available, the CRIS downtime procedures will be followed.  Samples will not 
be sent outside NIH without appropriate approvals and/or agreements, if required.
5.2.1
Samples Managed by Dr. Figg’s Blood Processing Core (BPC)
5.2.1.1
BPC Contact Information
Please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the 
Friday before is preferred).
For sample pickup, page 102-11964.
For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 
240-760-6190 (main clinical pharmacology lab number).
For questions regarding sample processing, send request to NCIBloodcore@mail.nih.gov.
5.2.1.2
Sample Data Collection
All samples sent to the Blood Processing Core (BPC) will be barcoded, with data entered and 
stored in the Labmatrix utilized by the BPC. This is a secure program, with access to Labmatrix 
limited to defined Figg lab personnel, who are issued individual user accounts. Installation of 
Labmatrix is limited to computers specified by Dr. Figg. These computers all have a password 
restricted login screen. 
Labmatrix creates a unique barcode ID for every sample and sample box, which cannot be traced 
back to patients without Labmatrix access. The data recorded for each sample includes the patient 
ID, name, trial name/protocol number, time drawn, cycle time point, dose, material type, as well 
as box and freezer location. Patient demographics associated with the clinical center patient 
number are provided in the system. For each sample, there are notes associated with the processing 
method (delay in sample processing, storage conditions on the ward, etc.).
5.2.1.3
Sample Storage and Destruction
Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -80C according 
to stability requirements. These freezers are located onsite in the BPC and offsite at NCI Frederick 
Central Repository Services in Frederick, MD. Visitors to the laboratory are required to be 
accompanied by laboratory staff at all times. 
Access to stored clinical samples is restricted. Samples will be stored until requested by a 
researcher named on the protocol. All requests are monitored and tracked in Labmatrix. All 
researchers are required to sign a form stating that the samples are only to be used for research 
purposes associated with this trial (as per the IRB approved protocol) and that any unused samples 
must be returned to the BPC. It is the responsibility of the NCI Principal Investigator to ensure that 
the samples requested are being used in a manner consistent with IRB approval.
Following completion of this study, samples will remain in storage as detailed above. Access to 
these samples will only be granted following IRB approval of an additional protocol, granting the 
rights to use the material.
If, at any time, a patient withdraws from the study and does not wish for their existing samples to 
be utilized, the individual must provide a written request. Following receipt of this request, the 
samples will be destroyed (or returned to the patient, if so requested). The PI will record any loss 


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or unanticipated destruction of samples as a deviation. Reporting will be per the requirements of 
Section 7.2.
Sample bar-codes are linked to patient demographics and limited clinical information. This 
information will only be provided to investigators listed on this protocol, via registered use of 
Labmatrix. It is critical that the sample remains linked to patient information such as race, age, 
dates of diagnosis and death, and histological information about the tumor, in order to correlate 
genotype with these variables.
5.2.2
Procedures for Storage of Tissue Specimens in the Laboratory of Pathology
Tissues designated for clinical diagnostics are transported to the Laboratory of Pathology (LP) 
where they are examined grossly and relevant portions fixed, embedded in paraffin and sectioned 
and stained for diagnostic interpretation. Unutilized excess tissue that is not embedded in paraffin 
is stored in formalin for up to three months, in accordance with College of American 
Pathologists/Joint Commission on Accreditation of Healthcare Organizations (CAP/JCAHO) 
guidelines, and then discarded. Following completion of the diagnostic workup, the slides and 
tissue blocks are stored indefinitely in the LP’s clinical archives. All specimens are catalogued and 
retrieved utilizing the clinical laboratory information systems, in accordance with CAP/JCAHO 
regulations. The use of any stored specimens for research purposes is only allowed when the 
appropriate IRB approval has been obtained. In some cases, this approval has been obtained via 
the original protocol on which the patient was enrolled.
5.2.3
Thoracic Epigenetics Laboratory
This study will be conducted within the Thoracic Surgery Branch, NCI. Sample collection and 
initial processing will be performed in the Thoracic Epigenetics Laboratory (TEL). Samples will 
be stored in designated monitored freezers (at least -20°C).  All samples obtained on this study 
will be tracked using Labmatrix. Pharmacokinetic samples will be analyzed in the Figg Lab, GMB.  
Samples will be identified and tracked using unique identifiers linked to each subject's unique 
patient number (study number). Codes linking personal identifiable information to the unique 
identifier will be stored in secure, computer servers with limited coded access or locked file 
cabinets in the Thoracic Surgery Branch, with access limited to the PI or study coordinator.  Gene 
expression, microRNA, and DNA methylation signatures, as well as methylation status of 
circulating tumor DNA and serologic responses to treatment will be analyzed in Dr. David 
Schrump’s laboratory.  
5.2.4
Trepel Laboratory
Contact the Trepel Lab by email (Dr. Jane Neckers (Trepel): trepel@helix.nih.gov; Min-Jung Lee: 
leemin@mail.nih.gov; Akira Yuno: akira.yuno@nih.gov and Sunmin Lee: lees@pop.nci.nih.gov) 
when the patient is scheduled and by phone as soon as the blood is drawn at 240-760-6330. 
A lab member will come to pick up the blood. Please keep blood at ambient temperature. Members 
of the lab will enter the samples into a secure password protected patient’s sample tracking 
database (Translational Pharmacodynamics Research Group Patient Sample Management System) 
and process the samples. 
Sample barcodes are linked to patient demographics and limited clinical information. This 
information will only be provided to investigators listed on this protocol.  It is critical that the 
sample remains coded and linked to patient information such as race, age, dates of diagnosis and 
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Blood samples will be stored initially in the Trepel Lab in the Magnuson Clinical Center. If, at any 
time, a subject withdraws from the study and does not wish for their existing samples to be utilized, 
the individual must provide a written request. Following receipt of this request, the samples will 
be destroyed (or returned to the patient, if so requested). When a patient withdraws consent the 
participant’s data will be excluded from future distributions, but data that have already been 
distributed for approved research use will not be able to be retrieved. The PI will record any loss 
or unanticipated destruction of samples as a deviation (refer to Section 5.2.5). Reporting will be 
per the requirements of Section 7.2.
5.2.5
Sample Destruction, End of Protocol Procedures
All specimens obtained in the protocol are used as defined in the protocol. Any specimens that are 
remaining at the completion of the protocol will be stored in the conditions described below. The 
study will remain open so long as sample or data analysis continues.  
Following completion of this study, if the subject has co-enrolled on 06C0014, samples will be 
transferred to the Tissue Procurement protocol and remain in storage in the Blood Processing Core 
(BPC) as detailed within 06C0014. Access to these samples will only be granted following IRB 
approval of an additional protocol, granting the rights to use the material.  If the subject has not 
co-enrolled on the Tissue Procurement protocol, the samples will be destroyed.
If the patient withdraws consent the participants data will be excluded from future distributions, 
but data that have already been distributed for approved research use will not be able to be 
retrieved.
The PI will record any loss or unanticipated destruction of samples as a deviation. Reporting will 
be per the requirements of Section 7.2.
6
DATA COLLECTION AND EVALUATION
6.1
DATA COLLECTION
For the purposes of the research sample analyses and correlation with clinical outcomes, 
demographic information, histology, operative and peri-operative interventions, pathologic 
findings, laboratory and imaging parameters (performed as part of routine or protocol specified 
patient care) may be collected on this study. The PI will be responsible for overseeing entry of data 
into a 21 CFR Part 11-compliant data capture system provided by the NCI CCR and ensuring data 
accuracy, consistency and timeliness. The principal investigator, associate investigators/research 
nurses and/or a contracted data manager will assist with the data management efforts.  Primary 
and final analyzed data will have identifiers so that research data can be attributed to an individual 
human subject participant.
End of Study Procedures: Data will be stored according to HHS, FDA regulations, and NIH 
Intramural Records Retention Schedule as applicable. 
Loss or Destruction of Data: Should we become aware that a major breach in our plan to protect 
subject confidentiality and trial data has occurred, this will be reported expeditiously per 
requirements in Section 7.2.1. 
6.1.1
Routine Data Collection
Following registration, all adverse events will be described in the source documents, reviewed by 
the designated research nurse, and captured.


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All adverse events, including clinically significant abnormal findings on laboratory evaluations, 
regardless of severity, will be followed until return to baseline or stabilization of event. Document 
AEs from the first study intervention, Study Day 1, through 30 days after the last administration 
of study therapy. Beyond 30 days after the last intervention, only adverse events which are serious 
and related to the study intervention need to be recorded. 
6.1.2
Exclusions to Routine Data Collection
6.1.2.1
Adverse Events
The following events will be captured only in the source documents and will not be recorded: 
• All Grade 1 events. 
• Grade 2 events that are not related to the treatment.
• Grade 1, 2, and 3 Lab values drawn outside of the protocol specified time points that are not 
assessed as clinically significant by the PI or his designee and are not associated with an 
adverse event. 
• Events related to vascular access devices (occlusion, thrombi, hospitalizations for insertion 
or removal). 
Note: Events that result in a hospitalization for convenience will not be reported.
6.1.2.2
Concomitant Medications/Measures
All concomitant medications and measures will be captured in the source documents. Only those 
medications that the patient is taking at baseline on a routine basis or medications that cause an 
AE will be captured (e.g., onetime medications, PRN medications, supportive medications, 
electrolyte replacement and medications given to treat adverse events will not be captured).
6.2
DATA SHARING PLANS
6.2.1
Human Data Sharing Plan
What data will be shared?
I will share human data generated in this research for future research as follows:

Coded, linked data in an NIH-funded or approved public repository.  

Coded, linked data in BTRIS (automatic for activities in the Clinical Center).

Identified or coded, linked data with approved outside collaborators under appropriate 
agreements.
How and where will the data be shared? 
Data will be shared through:

An NIH-funded or approved public repository, clinicaltrials.gov, dbGaP.

BTRIS (automatic for activities in the Clinical Center).

Publication and/or public presentations.
When will the data be shared? 

Before publication.

At the time of publication or shortly thereafter.


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6.2.2
Genomic Data Sharing Plan
Unlinked genomic data will be deposited in public genomic databases such as dbGaP in 
compliance with the NIH Genomic Data Sharing Policy. 
6.3
RESPONSE CRITERIA
For the purposes of this study, patients will be re-evaluated for response every 10 weeks (± 1 
week). In addition to a baseline scan, confirmatory scans should also be obtained not less than 4 
weeks following initial documentation of objective response. 
Objective response and progression will be evaluated in this study using the new international 
criteria proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) 
[(Version 1.1)(109)]. Changes in the largest diameter (unidimensional measurement) of the tumor 
lesions and the shortest diameter in the case of malignant lymph nodes are used in the RECIST 
criteria. 
Note: Lesions are either measurable or non-measurable using the criteria provided below. The term 
“evaluable” in reference to measurability will not be used because it does not provide additional 
meaning or accuracy.
6.3.1
Confirmatory Measurement/Duration of Response
•
Confirmation
To be assigned a status of PR or CR, changes in tumor measurements must be confirmed by 
repeat assessments that should be performed at least 4 weeks after the criteria for response 
are first met. 
•
Duration of Overall Response
The duration of overall response is measured from the time measurement criteria are met 
for CR or PR (whichever is first recorded) until the first date that recurrent or progressive 
disease is objectively documented (taking as reference for progressive disease the smallest 
measurements recorded since the treatment started).
The duration of overall CR is measured from the time measurement criteria are first met for 
CR until the first date that progressive disease is objectively documented.
6.3.2
Definitions
Evaluable for toxicity:  All patients will be evaluable for toxicity from the time of their first 
treatment with DAC/THU/pembrolizumab.  Dose limiting toxicities will be assessed during the 
first 3 weeks of DAC-THU/pembrolizumab therapy.
Evaluable for objective response: Patients who have measurable disease present at baseline, have 
received at least 9 weeks of DAC-THU/pembrolizumab  therapy (one full course), and have had 
their disease re-evaluated will be considered evaluable for response provided: (1) the patient 
demonstrates progressive disease or death while on protocol therapy; (2) the tumor is not removed 
surgically prior to the time complete response or partial response is confirmed; or (3) the patient 
demonstrates a complete or partial response as confirmed according to protocol criteria.  These 
patients will have their response classified according to the definitions stated below.  [Note:  
Patients who electively terminate therapy before completing 9 weeks of DAC-
THU/pembrolizumab therapy and do not expire within 14 days from start of treatment will be 
replaced. Patients who experience disease progression after 3 weeks of drug therapy but before 


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completing the minimum of 9 weeks of drug therapy will be taken off-treatment and will only be 
evaluable for toxicity (not evaluable for response)].
Evaluable Non-Target Disease Response:  Patients who have lesions present at baseline that are 
evaluable but do not meet the definitions of measurable disease, have received at least 8 weeks of 
DAC-THU/pembrolizumab therapy, and have had their disease re-evaluated will be considered 
evaluable for non-target disease.  The response assessment is based on the presence, absence, or 
unequivocal progression of the lesions. 
6.3.3
Disease Parameters
6.3.3.1
Measurable Disease
Measurable lesions are defined as those that can be accurately measured in at least one dimension 
(longest diameter to be recorded) as: 

By chest x-ray: > 20 mm; 

By CT scan: 
o
Scan slice thickness 5 mm or under as >10 mm 
o
Scan slice thickness > 5 mm: double the slice thickness 

With calipers on clinical exam: >10 mm.  
All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters).
6.3.3.2
Malignant Lymph Nodes
To be considered pathologically enlarged and measurable, a lymph node must be >15 mm in short 
axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 
mm). At baseline and in follow-up, only the short axis will be measured and followed.
6.3.3.3
Non-Measurable Disease
All other lesions (or sites of disease), including small lesions (longest diameter < 10 mm or 
pathological lymph nodes with ≥ 10 to < 15 mm short axis), are considered non-measurable 
disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis 
cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by CT or 
MRI), are considered as non-measurable.
Note: Cystic lesions that meet the criteria for radiographically defined simple cysts should not be 
considered as malignant lesions (neither measurable nor non-measurable) since they are, by 
definition, simple cysts.
‘Cystic lesions’ thought to represent cystic metastases can be considered as measurable lesions, if 
they meet the definition of measurability described above. However, if non-cystic lesions are 
present in the same patient, these are preferred for selection as target lesions.
6.3.3.4
Target Lesions
All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative 
of all involved organs, should be identified as target lesions and recorded and measured at 
baseline. Target lesions should be selected on the basis of their size (lesions with the longest 
diameter), be representative of all involved organs, but in addition should be those that lend 
themselves to reproducible repeated measurements. It may be the case that, on occasion, the largest 


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lesion does not lend itself to reproducible measurement in which circumstance the next largest 
lesion which can be measured reproducibly should be selected. A sum of the diameters (longest 
for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and 
reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then only 
the short axis is added into the sum. The baseline sum diameters will be used as reference to further 
characterize any objective tumor regression in the measurable dimension of the disease.
Progressive disease by RECIST criteria (109) noted after the first re-staging scan may represent 
disease that was not detected on the pre-study scan, and a confirmatory scan will be required at the 
next scheduled re-staging evaluation unless clinically not indicated. If confirmed, progression 
should be dated by the initial time when the lesions are first detected. If progressive disease by 
RECIST criteria is seen after Cycle 3, but not confirmed on subsequent restaging scan, the scans 
from after Cycle 3 would serve as the baseline scan to evaluate for disease progression (110).
6.3.3.5
Non-Target Lesions
All other lesions (or sites of disease) including any measurable lesions over and above the 5 target 
lesions should be identified as non-target lesions and should also be recorded at baseline. 
Measurements of these lesions are not required, but the presence, absence, or in rare cases 
unequivocal progression of each should be noted throughout follow-up.
6.3.4
Methods for Evaluation of Measurable Disease
6.3.4.1
Guidelines for Evaluation of Measurable Disease
All measurements should be taken and recorded in metric notation using a ruler or calipers. All 
baseline evaluations should be performed as closely as possible to the beginning of treatment and 
never more than 4 weeks before the beginning of the treatment.
The same method of assessment and the same technique should be used to characterize each 
identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is 
preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be 
imaged but are assessable by clinical exam.
6.3.4.2
Metastatic Bone Lesions
Disease progression is considered if a minimum of two new lesions is observed on bone scan. New 
lesions seen by the end of Cycle 3 (with the first re-staging bone scan) may represent disease that 
was not detected on the pre-study scan, and a confirmatory scan will be required at the next 
scheduled re-staging bone scan unless clinically not indicated. If confirmed, progression should 
be dated by the initial time when the lesions are first detected. If new lesions are seen after Cycle 
3, but no additional lesions are seen on confirmatory scans, the scans from post-Cycle 3 would 
serve as the baseline scan to evaluate for disease progression (109).
6.3.4.3
Clinical Lesions
Clinical lesions will only be considered measurable when they are superficial (e.g., skin nodules 
and palpable lymph nodes) and ≥ 10 mm diameter as assessed using calipers (e.g., skin nodules). 
In the case of skin lesions, documentation by color photography, including a ruler to estimate the 
size of the lesion, is recommended.
6.3.4.4
Methods of Measurement
Chest X-ray - Lesions on chest x-ray are acceptable as measurable lesions when they are clearly 
defined and surrounded by aerated lung. However, CT is preferable.


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CT and MRI - CT and MRI are the best currently available and reproducible methods to measure 
target lesions selected for response assessment. For this study helical Multi-detector CT will be 
performed with cuts of 5 mm in slice thickness for chest, abdomen and pelvis lesions and 2-3 mm 
thickness for head and neck lesions.
6.3.4.5
Additional Response Evaluation Using Volumetric Analysis
In addition, the utility of volumetric tumor measurement in patients with measurable disease will 
be prospectively evaluated and compared to 1D and 2D measurements.
6.3.5
Response Criteria for Radiographic Studies
6.3.5.1
Measuring of Soft Tissue Disease
a. Evaluation of Target Lesions
Complete Response (CR)
Disappearance of all target lesions. Any pathological lymph nodes (whether target or non- 
target) must have reduction in short axis to < 10 mm.
Partial Response (PR)
At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the 
baseline sum diameters.
Progressive Disease (PD)
At least a 20% increase in the sum of the diameters of target lesions, taking as reference the 
smallest sum on study (this includes the baseline sum if that is the smallest on study). In 
addition to the relative increase of 20%, the sum must also demonstrate an absolute increase 
of at least 5 mm. (Note: the appearance of one or more new lesions is also considered 
progressions).
Note: For this study, determination of PD will not be made prior to the Day 60 evaluation.
Stable Disease (SD)
Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, 
taking as reference the smallest sum diameters while on study.
b. Evaluation of Non-Target Lesions
Complete Response (CR)
Disappearance of all non-target lesions and normalization of tumor marker level. All lymph 
nodes must be non-pathological in size (< 10 mm short axis).
Note: Tumor markers alone cannot be used to assess response. If tumor markers are initially 
above the upper normal limit, they must normalize for a patient to be considered in complete 
clinical response.
Non-CR/Non-PD (Stable Disease, SD)
Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level 
above the normal limits.


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Progressive Disease (PD)
Appearance of one or more new lesions and/or unequivocal progression of existing non- 
target lesions. Unequivocal progression should not normally trump target lesion status. It 
must be representative of overall disease status change, not a single lesion increase.
Although a clear progression of “non-target” lesions only is exceptional, the opinion of the 
treating physician should prevail in such circumstances, and the progression status should 
be confirmed at a later time by the review panel (or Principal Investigator).
c. Evaluation of Best Overall Response
The best overall response is the best response recorded from the start of the treatment until 
disease progression/recurrence (taking as reference for progressive disease the smallest 
measurements recorded since the treatment started). The patient's best response assignment 
will depend on the achievement of both measurement and confirmation criteria.
For Patients with Measurable Disease (i.e., Target Disease)
Target 
Lesions
Non-
Target 
Lesions
New Lesions
Overall 
Response
Best Overall Response 
when Confirmation is 
Required*
CR
CR
No
CR
> 4 wks. Confirmation**
CR
Non-CR
Non-PD
No
PR
CR
Not evaluated
No
PR
PR
Non-
CR
Non-PD Not 
evaluated
No
PR
> 4 wks. Confirmation**
SD
Non-
CR
Non-PD Not 
evaluated
No
SD
Documented at least once 
> 4 wks. from baseline**
PD
Any
Yes or No
PD
Any
PD***
Yes or No
PD
Any
Any
Yes
PD
No prior SD, PR or CR
*
See RECIST 1.1 manuscript for further details on what is evidence of a new lesion.
**
Only for non-randomized trials with response as primary endpoint.
***
In exceptional circumstances, unequivocal progression in non-target lesions may be 
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d. Patients with a global deterioration of health status requiring discontinuation of 
treatment without objective evidence of disease progression at that time should be 
reported as “symptomatic deterioration.” Every effort should be made to document 
the objective progression even after discontinuation of treatment.
e.
In some circumstances, it may be difficult to distinguish residual disease from normal 
tissue. When the evaluation of complete response depends on this determination, it is 
recommended that the residual lesions be investigated (fine needle aspirate/biopsy) 
before confirming the complete response status.
6.4
TOXICITY CRITERIA
The following adverse event management guidelines are intended to ensure the safety of each 
patient while on the study. 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 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 web site (http://ctep.cancer.gov).
7
NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN
7.1
DEFINITIONS
Please refer to definitions provided in Policy 801: Reporting Research Events found here.
7.2
OHSRP OFFICE OF COMPLIANCE AND TRAINING/IRB REPORTING
7.2.1
Expedited Reporting
Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 
802 Non-Compliance Human Subjects Research found here. Note: Only IND Safety Reports that 
meet the definition of an unanticipated problem will need to be reported per these policies. 
7.2.2
IRB Requirements for PI Reporting Continuing Review
Please refer to the reporting requirements in Policy 801: Reporting Research Events 
found here. 
7.3
NCI CLINICAL DIRECTOR REPORTING 
Problems expeditiously reported to the OHSRP in iRIS will also be reported to the NCI Clinical 
Director.  A separate submission is not necessary as reports in iRIS will be available to the Clinical 
Director.
In addition to those reports, all deaths that occur within 30 days after receiving a research 
intervention should be reported via email to the Clinical Director unless they are due to progressive 
disease.  
To report these deaths, please send an email describing the circumstances of the death to  the 
Clinical Director/designee at NCICCRQA@mail.nih.gov within one business day of learning of 
the death.


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7.4
NIH REQUIRED DATA AND SAFETY MONITORING PLAN
7.4.1
Principal Investigator/Research Team 
The clinical research team will meet weekly when patients are being actively treated on the trial 
to discuss each patient in detail. Decisions about dose level enrollment and dose escalation if 
applicable will be made based on the toxicity data from prior patients.
All data will be collected in a timely manner and reviewed by the principal investigator or a clinical 
associate investigator in a timely manner. Events meeting requirements for expedited reporting as 
described in Section 7.2.1 will be submitted within the appropriate timelines.
The principal investigator will review adverse event and response data on each patient to ensure 
safety and data accuracy. The principal investigator will personally conduct or supervise the 
investigation and provide appropriate delegation of responsibilities to other members of the 
research staff.
8
SPONSOR SAFETY REPORTING
8.1
DEFINITIONS
8.1.1
Adverse Event 
Any untoward medical occurrence in a patient or clinical investigation subject administered a 
pharmaceutical product and which does not necessarily have a causal relationship with this 
treatment. An adverse event (AE) can therefore be any unfavorable and unintended sign (including 
an abnormal laboratory finding), symptom, or disease temporally associated with the use of a 
medicinal (investigational) product, whether or not related to the medicinal (investigational) 
product (ICH E6 (R2)).
8.1.2
Serious Adverse Event (SAE)
An adverse event or suspected adverse reaction is considered serious if in the view of the 
investigator or the sponsor, it results in any of the following:

Death.

A life-threatening adverse event (see Section 8.1.3).

Inpatient hospitalization or prolongation of existing hospitalization.
o
A hospitalization/admission that is pre-planned (i.e., elective or scheduled surgery 
arranged prior to the start of the study), a planned hospitalization for pre-existing 
condition, or a procedure required by the protocol, without a serious deterioration in 
health, is not considered a serious adverse event.
o
A hospitalization/admission that is solely driven by non-medical reasons (e.g., 
hospitalization for patient or subject convenience) is not considered a serious adverse 
event. 
o
Emergency room visits or stays in observation units that do not result in admission to 
the hospital would not be considered a serious adverse event. The reason for seeking 
medical care should be evaluated for meeting one of the other serious criteria.


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
Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions.

A congenital anomaly/birth defect.

Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate 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.
8.1.3
Life-threatening 
An adverse event or suspected adverse reaction is considered "life-threatening" if, in the view of 
either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of 
death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a 
more severe form, might have caused death. (21CFR312.32)
8.1.4
Severity
The severity of each Adverse Event will be assessed utilizing the CTCAE version 5.0.
8.1.5
Relationship to Study Product
All AEs will have their relationship to study product assessed using the terms:  related or 
not related.  

Related – There is a reasonable possibility that the study product caused the adverse 
event. Reasonable possibility means that there is evidence to suggest a causal relationship 
between the study product and the adverse event.

Not Related – There is not a reasonable possibility that the administration of the study 
product caused the event.
8.2
ASSESSMENT OF SAFETY EVENTS
AE information collected will include event description, date of onset, assessment of severity and 
relationship to study product and alternate etiology (if not related to study product), date of 
resolution of the event, seriousness and outcome. The assessment of severity and relationship to 
the study product will be done only by those with the training and authority to make a diagnosis 
and listed on the Form FDA 1572 as the site principal investigator or sub-investigator.  AEs 
occurring during the collection and reporting period will be documented appropriately regardless 
of relationship.  AEs will be followed through resolution.
SAEs will be:

Assessed for severity and relationship to study product and alternate etiology (if not 
related to study product) by a licensed study physician listed on the Form FDA 1572 as 
the site principal investigator or sub-investigator.

Recorded on the appropriate SAE report form, the medical record and captured in the 
clinical database.


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
Followed through resolution by a licensed study physician listed on the Form FDA 1572 
as the site principal investigator or sub-investigator.
For timeframe of recording adverse events, please refer to Section 6.1. All serious adverse events 
recorded from the time of first investigational product administration must be reported to the 
Sponsor with the exception of any listed in Section 8.4.
8.3
REPORTING OF SERIOUS ADVERSE EVENTS
Any AE that meets protocol-defined serious criteria or meets the definition of Adverse Event of 
Special Interest that require expedited reporting must be submitted immediately (within 24 hours 
of awareness) to OSRO Safety using the CCR SAE report form. Any exceptions to the expedited 
reporting requirements are found in Section 8.4.
All SAE reporting must include the elements described in Section 8.2.
SAE reports will be submitted to the Center for Cancer Research (CCR) at: 
OSROSafety@mail.nih.gov and to the CCR PI and study coordinator.  CCR SAE report form 
and instructions can be found at: 
https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions 
Following the assessment of the SAE by OSRO, other supporting documentation of the event may 
be requested by the OSRO Safety and should be provided as soon as possible.
8.4
WAIVER OF EXPEDITED REPORTING TO CCR 
Death or Hospitalization that is deemed to be due to disease progression, and not attributable to 
the intervention will not be reported as an SAE. The event, and the assessment that it was caused 
by disease progression will be documented in the medical records. The causality assessment of 
Hospitalization will be re‐evaluated any time when new information is received. If the causality 
assessment changes from disease progression to related to the study intervention, SAE report will 
be sent to the Sponsor immediately in an expedited manner according to Section 8.3. If there is 
any uncertainty whether the intervention is a contributing factor to the event, the event should be 
reported as AE or SAE as appropriate.
8.5
REPORTING PREGNANCY 
All 
required 
pregnancy 
reports/follow-up 
to 
OSRO 
will 
be 
submitted 
to: 
OSROSafety@mail.nih.gov and to the CCR PI and study coordinator.  Forms and instructions can 
be found here: https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions. 
8.5.1
Maternal Exposure
If a patient becomes pregnant during the course of the study, the study treatment should be 
discontinued immediately, and the pregnancy reported to the Sponsor no later than 24 hours of 
when the Investigator becomes aware of it. The Investigator should notify the Sponsor no later 
than 24 hours of when the outcome of the Pregnancy becomes known.
Pregnancy itself is not regarded as an SAE. However, congenital abnormalities or birth defects 
and spontaneous miscarriages that meet serious criteria (Section 8.1.2) should be reported as SAEs. 
The outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, 
normal birth, or congenital abnormality) should be followed up and documented.


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8.5.2
Paternal Exposure
Male patients should refrain from fathering a child or donating sperm during the study and for 60 
days after the last dose of DAC-THU and pembrolizumab.
Pregnancy of the patient’s partner is not considered to be an AE. However, the outcome of all 
pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or 
congenital abnormality) occurring from the date of the first dose until 60 days after the last dose 
should, if possible, be followed up and documented.
8.6
REGULATORY REPORTING FOR STUDIES CONDUCTED UNDER CCR-SPONSORED IND
Following notification from the investigator, CCR, the IND sponsor, will report any suspected 
adverse reaction that is both serious and unexpected in expedited manner to the FDA in accordance 
to 21 CFR 31.2.32.  CCR will report an AE as a suspected adverse reaction only if there is evidence 
to suggest a causal relationship between the study product and the adverse event.  CCR will notify 
FDA and all participating investigators (i.e., all investigators to whom the Sponsor is providing 
drug under its INDs or under any investigator’s IND) in an IND safety report of potential serious 
risks from clinical trials or any other source, as soon as possible, in accordance to 21 CFR Part 
312.32. 
All serious events will be reported to the FDA at least annually in a summary format.
8.7
SPONSOR PROTOCOL DEVIATION REPORTING
A Protocol Deviation is defined as any non-compliance with the clinical trial Protocol, Manual of 
Operational Procedures (MOP) and other Sponsor approved study related documents, GCP, or 
protocol-specific procedural requirements on the part of the participant, the Investigator, or the 
study site staff inclusive of site personnel performing procedures or providing services in support 
of the clinical trial.
It is the responsibility of the study Staff to document any protocol deviation identified by the Staff 
or the site Monitor in the CCR Protocol Deviation Tracking System (PDTS) online application. 
The entries into the PDTS online application should be timely, complete, and maintained per CCR 
PDTS user requirements. 
In addition, any deviation to the protocol should be documented in the participant’s source records 
and reported to the reviewing IRB per their guidelines. OSRO required protocol deviation 
reporting is consistent with E6(R2) GCP: Integrated Addendum to ICH E6(R1): 4.5 Compliance 
with Protocol; 5.18.3 (a), and 5.20 Noncompliance; and ICH E3 16.2.2 Protocol deviations.
9
CLINICAL MONITORING
Clinical site monitoring is conducted to ensure:

that the rights of the participants are protected; 

that the study is implemented per the approved protocol, Good Clinical Practice and 
standard operating procedures; and 

the quality and integrity of study data and data collection methods are maintained.
Monitoring for this study will be performed by NCI CCR Office of Sponsor and Regulatory 
Oversight (OSRO) Sponsor and Regulatory Oversight Support (SROS) Services contractor. 
Clinical site monitoring activities will be based on OSRO standards, FDA Guidance E6(R2) Good 


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Clinical Practice: Integrated Addendum to ICH E6(R1) March 2018, and applicable regulatory 
requirements.
Details of clinical site monitoring will be documented in a Clinical Monitoring Plan (CMP) 
developed by OSRO. CMPs will be protocol-specific, risk-based and tailored to address human 
subject protections and integrity of the study data. OSRO will determine the intensity and 
frequency of monitoring based on several factors, including study type, phase, risk, complexity, 
expected enrollment rate, and any unique attributes of the study and the site. The Sponsor will 
conduct a periodic review of the CMP to confirm the plan's continued appropriateness. A change 
to the protocol, significant or pervasive non-compliance with GCP, or the protocol may trigger 
CMP updates.
OSRO SROS Monitoring visits and related activities will be conducted throughout the life cycle 
of each protocol. The first activity is before the study starts to conduct a Site Assessment Visit 
(SAV) (as warranted), followed by a Site Initiation Visit (SIV), Interim Monitoring Visit(s) 
(IMVs), and a study Close-Out Visit (COV).
Some monitoring activities may be performed remotely, while others will occur at the study site(s). 
Monitoring visit reports will describe visit activities, observations, and associated action items or 
follow-up required for resolution of any issues, discrepancies, or deviations. Monitoring reports 
will be distributed to the study PI, NCI CCR QA, CCR Protocol Support Office, coordinating 
center (if applicable), and the Sponsor regulatory file.
The site Monitor will inform the study team of any deviations observed during monitoring visits. 
If unresolved, the Monitor will request that the site Staff enter the deviations in the CCR Protocol 
Deviation Tracking System (PDTS) for deviation reporting to the Sponsor and as applicable per 
institutional and IRB guidance. 
10 STATISTICAL CONSIDERATIONS
The primary objectives of this trial are to determine the objective response rates of DAC/THU plus 
pembrolizumab in patients with NSCLC, EsC, and MPM who have intra-tumoral PD-L1 
expression of at least 50% and those who do not. Approximately 30% of NSCLC exhibit  PD-L1 
expression in 50% or more cancers cells, another 30-50% have PD-L1 expression in 1-49% of 
cancer cells, and the remaining NSCLC have no PD-L1 expression by IHC (25, 111-113). In 
contrast, 20-40% of gastroesophageal cancers exhibit PD-L1 expression (25, 111, 112); in the 
majority of these cancers, PD-L1 is expressed at low levels, and tends not to be expressed on the 
cancer cells (112). Despite these differences regarding PD-L1 expression, present data indicate 
that responses to immune checkpoint inhibitors in EsC are comparable to NSCLC with low PD-
L1 expression (114). Approximately 25%-30% of MPM exhibit some level of PD-L1 expression, 
with the expression being primarily on tumor-associated immune cells (115). PD-L1 expression 
tends to be higher on sarcomatoid MPM and lower on the more common epithelioid subtype. 
Response rates in MPM following PD-1 blockade are comparable to NSCLC with similar levels 
of PD-L1 expression (99, 100).
Patients will be initially enrolled onto a multi-dose level dose escalation portion of the trial 
following a 3+3 design. Patients will not be stratified for the Phase I component, which will 
accelerate our ability to define MTD. Thus, for the dose escalation phase, up to 6 patients per dose 
level may be enrolled, with a total of 12 patients required with a maximum of 2 dose levels. At the 
MTD, an additional 4 patients will be enrolled to further estimate the safety of the combination.  


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Since published data have demonstrated a response rate of 45% for first-line pembrolizumab alone 
in NSCLC patients with PD-L1 expression of 50% or greater (95), (98), this trial will try to 
demonstrate if a response rate in excess of 45% may be realized in NSCLC patients with ≥ 50% 
PD-L1 expression by the addition of DAC/THU. Briefly, NSCLC patients with high intratumoral 
(50% or greater) PD-L1 expression will be evaluated for response as part of a Simon optimal two-
stage Phase II trial design (116) in order to rule out an unacceptably low PR+CR rate of 35% 
(p0=0.35) in favor of an improved response rate of 60% (p1=0.60). With alpha=0.10 (probability 
of accepting a poor treatment=0.10) and beta=0.20 (probability of rejecting a good 
treatment=0.20), the first stage will determine the response rate in 10 evaluable patients, including 
those NSCLC patients among the 10 who are treated at the MTD from the Phase I cohort who have 
high intra-tumoral PD-L1 expression. If 0 to 4 of the 10 patients have a clinical response, then no 
further patients will be accrued. If 5 or more of these 10 patients have a response, then accrual 
would continue until a total of 23 evaluable patients have been treated at the MTD. As it may take 
up to several months to determine if a patient has experienced a response, a temporary pause in 
the accrual may be necessary to ensure that enrollment to the second stage is warranted. If there 
are 5 to 10 patients with a response out of 23 patients, this would be an uninterestingly low 
response rate. If there were 11 or more of 23 (47.8%) who experienced a response, this would be 
sufficiently interesting to warrant further study in later trials. Under the null hypothesis (35% 
response rate), the probability of early termination is 75.2%. 
NSCLC patients with low (0-49%) intratumoral PD-L1 expression will be evaluated for response 
using a Simon optimal two-stage Phase II trial design (116) in order to rule out an unacceptably 
low PR+CR rate of 10% (p0=0.10) in favor of an improved response rate of 30% (p1=0.30). 
Variable mutational burdens among NSCLC with low PD-L1 expression could affect responses to 
immune checkpoint inhibitors, potentially making this cohort more heterogeneous than the 
NSCLC high PD-L1 expresser cohort. For example, NSCLC patients without activating mutations 
in EGFR, HER2 or ALK with PD-L1 tumor proportion score (TPS) < 1% have an expected 
response rate of less than 10%, whereas those with activating mutations involving these genes have 
response rates of 5% or less; patients with PD-L1 TPS of 1-49% have a 17% response rate (25, 
103, 104, 113). Therefore, with alpha=0.05 (probability of accepting a poor treatment=0.05) and 
beta = 0.20 (probability of rejecting a good treatment=0.20), the first stage will determine the 
response rate in 10 evaluable patients, including those among the 10 who are treated at the MTD 
from the Phase I cohort who have low intra-tumoral PD-L1 expression, and if 0 to 1 of the 10 have 
a clinical response, then no further patients will be accrued. If 2 or more of these 10 patients have 
a response, then accrual would continue until a total of 29 evaluable patients have been treated in 
this cohort. As it may take up to several months to determine if a patient has experienced a 
response, a temporary pause in the accrual may be necessary to ensure that enrollment to the 
second stage is warranted. If there are 2 to 5 patients with a response out of 29 patients, this would 
be an uninterestingly low response rate. If there were 6 or more of 29 (20.7%) who experienced a 
response, this would be sufficiently interesting to warrant further study in later trials. Under the 
null hypothesis (10% response rate), the probability of early termination is 73.6%.
While accrual of NSCLC patients is underway, up to 10 EsC patients including any from the Phase 
I component after the MTD has been identified, will be accrued to examine responses in EsC 
patients treated with DAC-THU at the MTD and pembrolizumab. Since it is anticipated that most 
if not all of these 10 patients will have low or no PD-L1 expression in their tumor tissues, and 
because response rates to immune checkpoint inhibitors in EsC appear to be comparable to NSCLC 
with low PD-L1 expression, criteria similar to those reported for the NSCLC low PD-L1 cohort 


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will be used to identify a potential signal of activity. Specifically, if there are 2 or more responses 
in these 10 patients, this might warrant amendment of the current protocol to enable expansion of 
this cohort with appropriate statistical criteria, or to justify a larger, focused Phase II trial.
Additionally, up to 10 MPM patients including any from the Phase I component after the MTD 
has been identified, will be accrued to examine responses in MPM patients treated with DAC-THU 
at the MTD and pembrolizumab. Since it is anticipated that most if not all of these 10 patients will 
have low PD-L1 expression in their tumor tissues, and because response rates to immune 
checkpoint inhibitors in MPM patients appear to be comparable to NSCLC with low PD-L1 
expression, criteria similar to those reported for the NSCLC low PD-L1 cohort will be used to 
identify a potential signal of activity. Specifically, if there are 2 or more responses in these 10 
patients, this might warrant amendment of the current protocol to enable expansion of this cohort 
with appropriate statistical criteria, or to justify a larger, focused Phase II trial.
It is expected that approximately 2-4 patients per month may enroll onto this trial. Thus, it is 
expected that 24-30 months may be required in order to enroll up to 78 evaluable patients. 
Assuming two dose levels will be used to define MTD, the Phase I component of the trial will 
require 16 patients. The Phase II portion of the trial may require 29 NSCLC patients for the PD-
L1 high, and 23 NSCLC patients for the PD-L1 low cohorts, plus 10 EsC patients, plus 10 MPM 
patients, less 10 patients treated at the MTD in the Phase I portion of the protocol; hence 
16+29+23+10+10-10=78 patients In order to allow for a small number of inevaluable patients, the 
accrual ceiling will be set at 85 patients, acknowledging that the trial can be completed with 60 or 
fewer patients if response goals for each cohort are not met.
Twelve months after Amendment E has been implemented, accrual will be assessed. If the trial 
is not on track for accrual targets, e.g., if fewer than 12 patients have been accrued within 12 
months, the protocol will either be terminated, or the trial will be opened at an additional site to 
accelerate patient accrual, and ensure timely completion of this trial.
Pharmacokinetic 
analysis 
will 
be 
conducted 
using 
non-compartmental 
methods. 
Pharmacodynamic analyses, as well as analyses of molecular endpoints in tumor tissues and 
PBMC before and after treatment will be performed, with the primary focus being with respect to 
results at the MTD. For pharmacodynamic endpoints, descriptive statistics will be used for each 
endpoint.  Briefly, pharmacodynamic and molecular endpoints in target tissues will be assessed 
before and after DAC-THU/pembrolizumab treatment, with the primary focus being comparison 
of microarray, qRT-PCR and IHC results relative to profiles identified in our preclinical studies. 
It is anticipated that non-parametric statistical methods will be used to compare paired results 
before and after treatment (but not between groups), as well as to perform comparisons or 
correlations among parameters.  As the exact set of comparisons and analyses to be performed will 
be determined following completion of the trial and will be based on limited numbers of patients, 
the analyses will be considered exploratory and hypothesis generating rather than definitive. 
10.1 METHODS OF ANALYSIS
Response rates will be calculated as the percent of patients whose best response is a CR or PR. 
Toxicity information recorded will include the type, severity, time of onset, time of resolution, and 
the probable association with the study regimen.  Tables will be constructed to summarize the 
observed incidence by severity and type of toxicity.


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11 HUMAN SUBJECTS PROTECTIONS
11.1 RATIONALE FOR SUBJECT SELECTION
The patients to be entered on this protocol have advanced NSCLC, EsC, or MPM and have limited 
life expectancies. This population was selected because of the unknown outcome of this treatment 
in terms of its effectiveness. The experimental treatment has a chance to provide clinical benefit 
although this is unknown. Subjects of both genders and all racial/ethnic groups are eligible. Efforts 
will be made to extend accrual to a representative population, but in this preliminary study, a 
balance must be struck between patient safety considerations and limitations on the number of 
individuals exposed to potentially toxic and/or ineffective treatments on the one hand and the need 
to explore gender and ethnic aspects of clinical research on the other hand. If differences in 
outcome that correlate with gender or ethnic identity are noted, accrual may be expanded, or a 
follow-up study may be written to investigate these differences more fully. One strategy for 
recruitment may be to distribute an IRB approved protocol recruitment letter to General and 
Oncology physicians and nurses.
11.2 PARTICIPATION OF CHILDREN
It is anticipated that most children with cancer will not have disease that is appropriate for study. 
Because the effects of DAC-THU have not been evaluated in children, individuals <18 will be 
excluded from this study until more data are available.
11.3 PARTICIPATION OF SUBJECTS UNABLE TO GIVE CONSENT
Adults unable to give consent are excluded from enrolling in the protocol.  However, re-consent 
may be necessary and there is a possibility, though unlikely, that subjects could become 
decisionally impaired. For this reason and because there is a prospect of direct benefit from 
research participation (Section 11.4), all subjects will be offered the opportunity to fill in their 
wishes for research and care, and assign a substitute decision maker on the “NIH Advance 
Directive for Health Care and Medical Research Participation” form so that another person can 
make decisions about their medical care in the event that they become incapacitated or cognitively 
impaired during the course of the study. 
Note: The PI or AI will contact the NIH Ability to Consent Assessment Team (ACAT) for 
evaluation to assess ongoing capacity of the subjects and to identify an LAR, as needed.
Please see Section 11.5.1 for consent procedure. 
11.4 EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS
11.4.1 Risks
11.4.1.1 Study Drug Risks
The risks to the patient participating in this trial are anticipated to be small and are primarily the 
risks associated with administering the DAC-THU with pembrolizumab. These potential risks are 
addressed in Sections 13.1.9, 13.2.8 and 13.3.6.  There is the potential risk of not responding to 
the investigational treatment, and being unfit to receive and potentially benefit from standard of 
care cytotoxic chemotherapy. Risks and benefits will be carefully discussed with the patient at the 


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time consent is obtained.  Patients will be monitored throughout the study in order to limit the 
consequences of any adverse events.
11.4.1.2 Blood Sampling
Side effects of blood draws include pain and bruising, lightheadedness, and rarely, fainting.
11.4.1.3 Urine Collection
There is no physical risk involved with urine collection.
11.4.1.4 Biopsies
The risks associated with biopsies are pain and small amounts of bleeding at the biopsy site, and 
rarely, a small infection at the site. Biopsy sites usually heal very well and with very little scarring. 
In order to minimize pain, local anesthesia will be used; side effects of the local anesthesia may 
include a mild burning sensation when the numbing medicine is injected into the skin. 
11.4.1.5 Conscious Sedation
The common side effects of conscious sedation include drowsiness, delayed reflexes, hypotension, 
headache, and nausea. These are generally mild and last no more than a few hours.
11.4.1.6 Pulmonary Function Tests
These tests are safe and side effects are unlikely, but may include brief light headedness or slight 
soreness of the chest.
11.4.1.7 Scans and Contrast 
The most common discomfort is the length of time a patient must lay still during a scan. Patients 
may also become uncomfortable with the closed space of the machines.
There is a small risk of reaction in scans involving contrast. Common reactions include pain in the 
vein where the contrast was given, a metallic or bitter taste in the mouth, headache, nausea and a 
warm or flushing feeling that lasts from 1-3 minutes. In very rare cases, severe reactions that affect 
breathing, heart rhythm or blood pressure have occurred. 
An IV catheter may need to be inserted for administration of the contrast agent or anesthetic, which 
may cause pain at the site where the IV is placed and there is a small risk of bruising or infection, 
or inflammation of the skin and vein with pain and swelling. 
11.4.1.8 Risks of Exposure to Ionizing Radiation 
This research study involves the potential for 5 CT CAP scans, 5 PET/CT scans, 5 brain CT scans 
as well as 2 CT-guided biopsies over the course of the first year on study. This radiation exposure 
is not required for medical care and is for research purposes only. Subjects will be exposed to 
approximately 13.6 rem. This amount of radiation is above the guideline of 5 rem per year and 
will expose the subject to the roughly the same amount of radiation as 45.3 years of background 
radiation.
11.4.2 Benefits
There may be some direct benefit to patients who participate in this trial since it is anticipated that 
DAC-THU with pembrolizumab may cause tumor stabilization or some tumor regression. The 
greatest benefit will be the information regarding the feasibility, toxicity and dosing of DAC-THU 
with pembrolizumab administration, as well as the information on changes in the epigenome of 
lung and gastroesophageal cancers and systemic immunity to up-regulated CTAs. 


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11.5 CONSENT PROCESS AND DOCUMENTATION
The informed consent document will be provided as a physical or electronic document to the 
participant or consent designee(s) as applicable for review prior to consenting.  A designated study 
investigator will carefully explain the procedures and tests involved in this study, and the 
associated risks, discomforts and benefits. In order to minimize potential coercion, as much time 
as is needed to review the document will be given, including an opportunity to discuss it with 
friends, family members and/or other advisors, and to ask questions of any designated study 
investigator. A signed informed consent document will be obtained prior to entry onto the study.
The initial consent process as well as re-consent, when required, may take place in person or 
remotely (e.g., via telephone or other NIH approved remote platforms used in compliance with 
policy, including HRPP Policy 303) per discretion of the designated study investigator and with 
the agreement of the participant/consent designee(s).  Whether in person or remote, the privacy of 
the subject will be maintained. Consenting investigators (and participant/consent designee, when 
in person) will be located in a private area (e.g., clinic consult room). When consent is conducted 
remotely, the participant/consent designee will be informed of the private nature of the discussion 
and will be encouraged to relocate to a more private setting if needed.
Consent will be documented with required signatures on the physical document (which includes 
the printout of an electronic document sent to participant) or as described below, with a manual 
(non-electronic) signature on the electronic document.   When required, witness signature will be 
obtained similarly as described for the investigator and participant.
Manual (Non-Electronic) Signature on Electronic Document:
When a manual signature on an electronic document is used for the documentation of consent 
at the NIH Clinical Center, this study will use the following to obtain the required signatures:

Adobe platform (which is not 21 CFR Part 11 compliant); or,  

iMedConsent platform (which is 21 CFR Part 11 compliant)
During the consent process, participants and investigators will view individual copies of the 
approved consent document on screens at their respective locations (if remote consent); the 
same screen may be used when in the same location, but is not required.  
Both the investigator and the participant will sign the document using a finger, stylus or mouse. 
Note: Refer to the CCR SOP PM-2, Obtaining and Documenting the Informed Consent Process 
for additional information (e.g., verification of participant identity when obtaining consent 
remotely) found here.
11.5.1 Consent Process for Adults Who Lack Capacity to Consent to Research 
Participation
For participants addressed in Section 11.3, an LAR will be identified consistent with Policy 403 
and informed consent obtained from the LAR, as described in Section 11.5.
12 REGULATORY AND OPERATIONAL CONSIDERATIONS
12.1 STUDY DISCONTINUATION AND CLOSURE
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause. Written notification, documenting the reason for study suspension or 


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termination, will be provided by the suspending or terminating party to investigators, funding 
agencies, the Investigational New Drug (IND) sponsor and regulatory authorities, as applicable.  
If the study is prematurely terminated or suspended, the Principal Investigator (PI) will promptly 
inform study participants, the Institutional Review Board (IRB), and sponsor and will provide the 
reason(s) for the termination or suspension. Study participants will be contacted, as applicable, 
and be informed of changes to study visit schedule.
 Circumstances that may warrant termination or suspension include, but are not limited to:

Determination of unexpected, significant, or unacceptable risk to participants

Demonstration of efficacy that would warrant stopping   

Insufficient compliance to protocol requirements

Data that are not sufficiently complete and/or evaluable

Determination that the primary endpoint has been met

Determination of futility
Study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the sponsor, IRB and as applicable, Food and Drug Administration (FDA).
12.2 QUALITY ASSURANCE AND QUALITY CONTROL
The clinical site will perform internal quality management of study conduct, data and biological 
specimen collection, documentation and completion.  An individualized quality management plan 
will be developed to describe a site’s quality management.
Quality control (QC) procedures will be implemented beginning with the data entry system and 
data QC checks that will be run on the database will be generated. Any missing data or data 
anomalies will be communicated to the site(s) for clarification/resolution.
Following written Standard Operating Procedures (SOPs), the monitors will verify that the clinical 
trial is conducted and data are generated and biological specimens are collected, documented 
(recorded), and reported in compliance with the protocol, International Conference on 
Harmonization Good Clinical Practice (ICH GCP), and applicable regulatory requirements (e.g., 
Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)). 
The investigational site will provide direct access to all trial related sites, source data/documents, 
and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local and 
regulatory authorities.
12.3 CONFLICT OF INTEREST POLICY
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical.  Therefore, any actual conflict of interest of persons who have 
a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and 
managed. Furthermore, persons who have a perceived conflict of interest will be required to have 
such conflicts managed in a way that is appropriate to their participation in the design and conduct 
of this trial.  The study leadership in conjunction with the National Cancer Institute has established 
policies and procedures for all study group members to disclose all conflicts of interest and will 
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12.4 CONFIDENTIALITY AND PRIVACY
Participant confidentiality and privacy is strictly held in trust by the participating investigators, 
their staff, and the sponsor(s). This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participants. Therefore, 
the study protocol, documentation, data, and all other information generated will be held in strict 
confidence. No information concerning the study or the data will be released to any unauthorized 
third party without prior written approval of the sponsor. 
All research activities will be conducted in as private a setting as possible.
The study monitor, other authorized representatives of the sponsor, representatives of the 
Institutional Review Board (IRB), and/or regulatory agencies may inspect all documents and 
records required to be maintained by the investigator, including but not limited to, medical records 
(office, clinic, or hospital) and pharmacy records for the participants in this study. The clinical 
study site will permit access to such records.
The study participant’s contact information will be securely stored at the clinical site for internal 
use during the study. At the end of the study, all records will continue to be kept in a secure location 
for as long a period as dictated by the reviewing IRB, Institutional policies, or sponsor 
requirements.
Study participant research data, which is for purposes of statistical analysis and scientific reporting, 
will be stored at the NCI CCR. This will not include the participant’s contact or identifying 
information. Rather, individual participants and their research data will be identified by a unique 
study identification number. The study data entry and study management systems used by the 
clinical site and by NCI CCR research staff will be secured and password protected. At the end of 
the study, all study databases will be archived at the NIH.
To further protect the privacy of study participants, a Certificate of Confidentiality has been issued 
by the National Institutes of Health (NIH). This certificate protects identifiable research 
information from forced disclosure. It allows the investigator and others who have access to 
research records to refuse to disclose identifying information on research participation in any civil, 
criminal, administrative, legislative, or other proceeding, whether at the federal, state, or local 
level. By protecting researchers and institutions from being compelled to disclose information that 
would identify research participants, Certificates of Confidentiality help achieve the research 
objectives and promote participation in studies by helping assure confidentiality and privacy to 
participants.  
13 PHARMACEUTICAL INFORMATION 
13.1 DECITABINE 
13.1.1 Synonyms
DAC
deoxyazacytidine
NSC 127716
Dacogen®


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13.1.2 Description
Decitabine is an analog of 2'-deoxycytidine in which the carbon at position 5 in the pyridine ring 
has been replaced by nitrogen. In acid media, decitabine undergoes cleavage at the glycosidic bond 
to yield 5-azacytosine and deoxyribose. In acid media, a rapid, reversible opening of the 
5-azacytosine 
ring 
occurs 
between 
positions 
1 
and 
6, 
producing 
N-(formylamidino)-N'-D-2'-deoxyribofuranosylurea which decomposes with the loss of a formyl 
group.
Decitabine is enzymatically phosphorylated in sequence by deoxycytidine kinase, deoxycytidylic 
kinase, and nucleoside diphosphonucleoside kinase to the active forms, 5-Aza-dCMP, 
5-Aza-dCDP, 5-Aza-dCTP, respectively. The latter form is incorporated into DNA. Decitabine is 
enzymatically deaminated by cytidine deaminase; phosphorylated decitabine nucleoside 
(5-Aza-dCMP) is catabolized by deoxycytidylic deaminase.
13.1.2.1 Mode of Action
Inhibition of DNA methylation, and induction of cellular differentiation.
13.1.3 Chemical Names
(1)
1-(2'-deoxy-D-ribofuranosyl)-5-azacytosine
(2)
3,5-Triazin-2(1H)-one,4-amino-l-(2-deoxy-ß-D-erythro-pentofuranosyl)- 
(3)
4-Amino-l-(2-deoxy-ß-D-erythro-entofuranosyl)-1,3,5-triazin-2(1H)-one
(4)
5-aza-2'-deoxycytidine
13.1.4 Chemical Identification
Empiric formula C8Hl2N404
Molecular weight 228.21 daltons
CAS registry No. 2353-33-5 pertains to free base.
13.1.5 Formulation
Decitabine is manufactured by IriSys LLC (San Diego, CA) and supplied by DCTD as a 
freeze-dried powder in 20 mL-capacity vials containing DAC 50 mg with 68 mg potassium 
phosphate, and NaOH. Decitabine will be encapsulated by IriSys LLC under contract with the NIH 
CC Pharmacy per federal Good Manufacturing Practice guidelines. In this formulation decitabine 
is supplied as capsules containing 5 mg of decitabine per capsule, in bottles containing 30 capsules, 
together with a drying agent. The label on the bottles includes the following information:  the (i) 
name of the drug; (ii) quantity of drug per capsule; (iii) date of packaging; (iv) recommended 
storage temperature; (v) lot number of bulk drug used to generate the capsules
13.1.6 Stability
Powder: The glass bottles containing decitabine should be stored at -20°C. The drug substance has 
been tested after 18 months of storage at -20°C and no change in appearance, assay or impurities 
was observed. Decitabine is stable as a dry powder, however, degrades in a pH dependent manner 
in aqueous solution (optimal pH for stability is 7.0). Decitabine is hydrolyzed in aqueous solution 
at pH 7.4 at a rate of approximately 2.5% every 7 hours at 4°C.
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13.1.7 Storage
Powder: Containers should be tightly closed and stored in the freezer at -20°C.
Capsules: Bottles should be tightly closed and stored at refrigerated temperatures (2-8°C).
13.1.8 Administration
Please see Section 3.2.1.
13.1.9 Toxicities
There is substantial information regarding the toxicity of decitabine in humans from clinical trials 
in patients with MDS and AML, including relapsed or poor prognosis cases. Some of this 
information is summarized in the package insert for this FDA approved drug. In almost all studies, 
the patients received doses much higher than the decitabine doses planned in this study. 
Leukopenia was a major toxicity and nausea, or vomiting were common non-hematologic 
toxicities. A better guide to anticipated side-effects in this clinical trial are observations from 
clinical studies of decitabine repositioned for non-cytotoxic DNMT1-depletion for sickle cell 
disease (SCD), -thalassemia intermedia and MDS. In these clinical trials there were no episodes 
of non-hematologic CTCAE Grade 2 or higher toxicity from decitabine. In the SCD and -
thalassemia trials, consistent with a non-cytotoxic mechanism of action, the main side-effect was 
an increase in the platelet count (as opposed to the usual side-effect of thrombocytopenia seen with 
cytotoxic treatments). Cytotoxicity/DNA damage assays based on bone marrow morphological 
examination, bone marrow DNA content analysis, VDJ recombination assay, erythrocyte 
micronucleus assay and gamma-H2AX assay were also negative. These assays did not reveal 
evidence of DNA damage or cytotoxicity. The increase in the platelet count was not associated 
with any clinical adverse events.  Other common side effects are fatigue, fever, nausea, cough, 
petechiae, constipation, diarrhea, and hyperglycemia.
Expected side effects in current trial:
1. Thrombocytosis: Decitabine-induced shifts in differentiation include increased erythropoiesis 
and megakaryopoiesis (60, 61). Therefore, increases in platelet counts are expected, especially in 
patients who are hyposplenic (e.g., patients with SCD) or asplenic (many patients with -
thalassemia). Although it is intuitive to expect an association between platelet count and 
thrombosis risk, this has not been evident in clinical observations and studies, whereas qualitative 
RBC defects from underlying disease can be pro-thrombotic (60, 117-124). Hence, in a SCD study, 
SC decitabine-induced improvement in multiple indices of RBC pathology was accompanied by 
improvement in multiple markers of coagulation pathway activity, despite concurrent platelet 
count increases to > 800x109/L (60). Thus, it is possible that decitabine-induced improvements in 
RBC phenotype could reduce thrombophilia despite concurrent platelet count increases. 
Coagulation pathway evaluation (for example by measurement of D-dimer levels) should probably 
be a component of studies with decitabine to treat hemoglobinopathies. 
2. Neutropenia: Decitabine induced shifts in differentiation include favoring erythroid-
megakaryocyte progenitor production over granulocyte-monocyte progenitor production by 
hematopoietic stem cells (60, 61). Hence, even at low, non-cytotoxic concentrations, a downward 
shift in neutrophil counts is expected. Of note, the current standard of care for symptomatic SCD 
is DNA damaging, cytotoxic doses of HU.  Consequently, in SCD subjects treated with HU, 


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hematologic toxicities are pancellular, with neutrophil recovery taking up to 2 weeks (AHFS Drug 
Information, 2000).  
3.  Lymphopenia: Decitabine induced shifts in hematopoietic differentiation may also reduce 
lymphocyte production.
3.  Testicular toxicity: Pre-clinical studies suggest an effect on sperm-counts can occur even 
with the intended low, non-cytotoxic concentrations of decitabine. This toxicity appears 
reversible. Of note, the current standard of care for symptomatic SCD with HU also causes 
testicular toxicity (AHFS Drug Information, 2000).  
4.  Teratogenicity: Pre-clinical data indicates that there is a teratogenic risk even at low doses of 
decitabine.  Therefore, all clinical studies with decitabine must take into account, and incorporate 
precautions, to address a risk for teratogenicity. Current standard therapy for SCD with either HU 
or stem cell transplantation also has teratogenic risks (pregnancy category D). 
13.2 TETRAHYDROURIDINE
13.2.1 Names
Chemical Name: 2(1H)-pyrimidinone, tetrahydro-4-hydroxy-1.beta.-D-ribofuranosyl-
Other Names: THU, H4U
CAS Registry Number:  18771-50-1
Molecular Formula: C9H16N2O6
M.W.: 248
Approximate Solubility: The drug is highly soluble in water, up to 200 mg/mL.
13.2.2 Mode of Action: 
Cytidine deaminase inhibitor
13.2.3 How Supplied: 
Tetrahydrouridine is manufactured by KP Pharmaceutical Technology Inc (Bloomington, IN, 
USA) and will be purchased from the Cleveland Clinic. THU is supplied as capsules containing 
250 mg of THU per capsule, in bottles containing 8 capsules per bottle together with a drying agent. 
The label on the bottles includes the following information: the (i) name of the drug; (ii) quantity 
of drug per bottle; (iii) date of manufacture; (iv) recommended storage temperature; (v) 
manufacturer’s lot number.
13.2.4 Storage: 
Capsules: Bottles should be tightly closed and stored at refrigerated temperatures 2°-8°C (35-46°F)
13.2.5 Stability: 
Capsules: Shelf life stability testing of the intact bottles is on-going.  
13.2.6 Route of Administration: 
Oral
13.2.7 Method of Administration: 
Please see Section 3.2.1.


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13.2.8 Toxicities:
Most reports discuss the use of THU co-administered with a cytidine analogue in order to inhibit 
the metabolism of that cytidine-based therapeutic, and the side-effects are that of the active agent 
cytidine analogue therapeutic. The single dose toxicology of THU was evaluated in dogs and 
Rhesus monkeys at intravenous doses of up to 1000 mg/kg: with the exception of local 
inflammation at the injection site, no notable toxic effects were attributed to the administration of 
THU (125).  Although there is only limited non-clinical toxicology data with THU alone, the data 
that exists suggests that THU by itself does not pose a risk for toxicity, even at doses considerably 
higher than the dose in the planned study.
13.3 PEMBROLIZUMAB 
(Refer to FDA-approved package insert for complete product information)
13.3.1 Description: 
Pembrolizumab is a humanized monoclonal IgG4 antibody directed against the human cell surface 
receptor, programmed death-1, (PD-1).  Following administration, pembrolizumab binds to PD-1 
and blocks the interaction between PD-1 and its ligands.  
13.3.2 How Supplied: 
Keytruda (pembrolizumab) will be purchased by the NIH Clinical Center Pharmacy Department 
from commercial sources.  Pembrolizumab is supplied in a single-use 50 mg vial as 
pembrolizumab lyophilized powder and as a single use 25 mg/mL ready-to-use solution containing 
4 mL (100 mg/vial).  
13.3.3 Reconstitution/Dose Preparation: 
The lyophilized powder is reconstituted with 2.3 mL Sterile Water for injection, USP, and the 
resultant concentration is 25 mg/mL. Diluent should be directed against the side of the vial to avoid 
excess foaming.  Swirl contents gently until completely dissolved.  Let contents sit for up to 5 
minutes to allow all bubbles to clear.  Do not shake.  
The ready-to-use solution or reconstituted lyophilized powder must be further diluted prior to 
administration. It may be diluted in 0.9% Sodium Chloride injection, USP or 5% Dextrose 
Injection, USP. The final concentration of the dilution solution should be between 1 mg/ml to 10 
mg/ml.
13.3.4 Stability/Storage: 
The reconstituted lyophilized power is stable for up to 6 hours at room temperature or up to 24 
hours at when stored under refrigeration (2-8°C or 36-46°F).
The diluted solution is stable for up to 6 hours at room temperature or up to 24 hours at when 
stored under refrigeration (2-8°C or 36-46°F).
13.3.5 Administration: 
Pembrolizumab will be given at a fixed dose of 200 mg as an intravenous infusion over 30 minutes 
through an intravenous line containing a sterile, non-pyrogenic, low-protein binding 0.2 micron to 
5 micron in-line or add-on filter.


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13.3.6 Toxicities:  
This protocol uses commercial supply of pembrolizumab; please refer to current FDA-approved 
package insert. 


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15 APPENDICES AND SUPPLEMENTARY TABLES
15.1 SUPPLEMENTARY TABLES
15.1.1 Supplementary Table 1: Cancer/Testis Genes and Their Chromosomal Locations 
(Partial List)
Gene family
Family members
CT 
identifier
Chromosomal 
localization
MAGEA
MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA5, MAGEA6, 
MAGEA8, MAGEA9, MAGEA10, MAGEA11, MAGEA12
CT1
Xq28
BAGE
BAGE
CT2
21p11.1
MAGEB
MAGEB1, MAGEB2, MAGEB3, MAGEB4, MAGEB5, MAGEB6
CT3
Xp21.3
GAGE
GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, 
GAGE8
CT4
Xp11.23
SSX
SSX1, SSX2, SSX3, SSX4
CT5
Xp11.23-p11.22.
NY-ESO-1
CTAG1B, CTAG2
CT6
Xq28
MAGEC1
MAGEC1
CT7
Xq26
SYCP1
SYCP1
CT8
1p12-p13
BRDT
BRDT
CT9
1p22.1
MAGEC2
MAGEC2
CT10
Xq27
SPANX
SPANXA1, SPANXB1, SPANXC, SPANXD
CT11
Xq27.1
XAGE
XAGE1
CT12
Xp11.22
HAGE
DDX43
CT13
6q12-q13.
SAGE
SAGE1
CT14
Xq26.
ADAM2
ADAM2
CT15
8p11.2.
PAGE-5
PAGE5
CT16.1
Xp11.23
LIPI
LIPI
CT17
21q11.2
NA88A pseudogene
VENTXP1
CT18
Xp21.3.
IL13RA
IL13RA2
CT19
Xq13.1-q28
TSP50
TSP50
CT20
3p12–14
CTAGE-1
CTAGE1
CT21.1
18p11.2
SPA17
SPA17
CT22
11q24.2
ACRBP
ACRBP
CT23
12p12-p13.
CSAG
CSAG1, CSAG2
CT24
Xq28.
MMA1
DSCR8
CT25
21q22.2.
CAGE/DDX53
DDX53
CT26
Xp22.11
BORIS
CTCFL
CT27
20q13.31
HOM-TES-85
LUZP4
CT28
Xq23
AF15q14
CASC5
CT29
15q14
HCA661
TFDP3
CT30
Xq26.2
JARID1B
JARID1B
CT31
1q32.1.
LDHC
LDHC
CT32
11p15.3-p15.5


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
114
Gene family
Family members
CT 
identifier
Chromosomal 
localization
MORC
MORC1
CT33
3q13
SGY-1
DKKL1
CT34
19q13.33
SPO11
SPO11
CT35
20q13.2-q13.3
TPX1
CRISP2
CT36
6p21-qter
NY-SAR-35
FMR1NB
CT37
Xq27.3-q28.
FTHL17
FTHL17
CT38
Xp21
NXF2
NXF2
CT39
Xq22.1
TAF7L
TAF7L
CT40
Xq22.1
TDRD1
TDRD1
CT41
10q25.3.
TEX15
TEX15
CT42
8p12
FATE
FATE1
CT43
Xq28
TPTE
TPTE
CT44
21p11
CT45
CT45-1, CT45-2, CT45-3, CT45-4, CT45-5, CT45-6
CT45
Xq26.3
HORMAD1
HORMAD1
CT46
1q21.2.
CT47
CT47-1, CT47-2, CT47-3, CT47-4, CT47-5, CT47-6, CT47-7, CT47-
8, CT47-9, CT47-10, CT47-11, CT47-12
CT47
Xq24
SLCO6A1
SLCO6A1
CT48
5q21.1
TAG
TAG
CT49
Na
LEMD1
LEMD1
CT50
1q32.1
HSPB9
HSPB9
CT51
1p36.2-p35
CCDC110
CCDC110
CT52
4q35.1
ZNF165
ZNF165
CT53
6p21.3
SPACA3
SPACA3
CT54
17q11.2
CXorf48
CXorf48
CT55
Xq26.3
THEG
THEG
CT56
19pter-p13
ACTL8
ACTL8
CT57
1p36.2-p35
NLRP4
NLRP4
CT58
1p36.2-p35
COX6B2
COX6B2
CT59
19q13.42
LOC348120
LOC348120
CT60
15q11.2
CCDC33
CCDC33
CT61
15q24.1
LOC196993
LOC196993
CT62
15q23
PASD1
PASD1
CT63
Xq28
CT64
CT64/BX103208
CT64
3q26.1.
TULP2
TULP2
CT65
19q13.1.
CT66
CT66/AA884595
CT66
7q11.22.
KLKBL4
KLKBL4
CT67
16q21
RBM46
RBM46
CT68
4q32.1
CT69
CT69/BC040308
CT69
6q23.2
CT70
CT70/BI818097
CT70
Na


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
115
Gene family
Family members
CT 
identifier
Chromosomal 
localization
SPINLW1
SPINLW1
CT71
20q12-q13.2
TSSK6
TSSK6
CT72
19p13.11
ADAM29
ADAM29
CT73
4q34.
CCDC36
CCDC36
CT74
3p21.31
LOC440934
LOC440934
CT75
2q36.1
SYCE1
SYCE1
CT76
10q26.3
CPXCR1
CPXCR1
CT77
Xq21.3
TSPY1
TSPY1
CT78
Yp11.2
TSGA10
TSGA10
CT79
2q11.2.
PIWIL2
PIWIL2
CT80
8p21.3
ARMC3
ARMC3
CT81
10p12.31
AKAP3
AKAP3
CT82
12p13.3.
CXorf61
Cxorf61
CT83
Xq23
PBK
PBK
CT84
8p21.2
C21orf99
C21orf99
CT85
21q11.2
OIP5
OIP5
CT86
15q15.1
CEP290
CEP290
CT87
12q21.32
CABYR
CABYR
CT88
18q11.2
SPAG9
SPAG9
CT89
17q21.33.
MPHOSPH1
MPHOSPH1
CT90
10q23.31
ROPN1
ROPN1
CT91
3q21.1.
PLAC1
PLAC1
CT92
Xq26
CALR3
CALR3
CT93
19p13.1
PRM
PRM1/PRM2
CT94.1
16p13.2
CAGE1
CAGE1
CT95
6p24.3
CT96
TTK
CT96
6q13-q21
LY6K
LY6K
CT97
8q24.3
IMP-3
IMP-3
CT98
7p11.
AKAP4
AKAP4
CT99
Xp11.2
DPPA2
DPPA2
CT100
3q13.13
KIAA0100/MLAA-22
KIAA0100
CT 101
7q11.2
TCC52
TCC52
CT102
9p13.3
SEMG1
SEMG1
CT103
20q12-q13.2
POTE
POTE21, POTE2, POTE8, POTE14, POTE15, POTE18, POTE22
CT104
2q21.1
FLJ36144/MAD-CT2
MAD-CT2
CT105
15q11.2.
NUF2/CDCA1
CDCA1
CT106
1q23
RHOXF2/PEPP2
PEPP2
CT107
Xq24


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
116
Gene family
Family members
CT 
identifier
Chromosomal 
localization
OTOA
OTOA
CT108
16p12.2
CCDC62
CCDC62
CT109
12q24.31
GPATCH2
GPATCH2
CT110
1q41
Na: not available


Abbreviated Title: DAC-THU/Pembro in NSCLC & EsC
Version Date:  8/20/2019
117
15.1.2 Supplementary Table 2: Frequency of CT Gene Expression in Various Human 
Cancers as Determined by Semi-Quantitative RT-PCR
Frequency (%) of Expression in Tumor Type
CT 
Family
(Member)
Bla
d
Brn
Brst
Col
Eso
Gas
H/N
Live
r
Leu
k/
Ly
mp
h
Lun
g
(NS
CL
C)
Mel
Ov
Pan
cr
Pro
s
Ren
al
Sarc
MAGEA1/
CT1.1
22
-
18
2
53
29
28
80
0
49
48
28
-
15
0
14
BAGE1/C
T2.1
15
-
10
0
-
-
8
-
0
4
26
15
-
0
0
6
MAGEB1/
CT3.1
0
0
17
0
-
0
0
-
0
14
22
-
-
0
0
9
GAGE/CT
4.1
12
-
9
0
-
-
19
38b
1
19
28
31
-
10
0
25
SSX2/CT5
.2
44
6
7
12
-
-
35
9b
36
16
35
-
-
40
5
50
NY-ESO-
1/CT6.1
80
0
30
0
-
0
-
29
0
17
34
25
0
25
9
0
MAGEC1/
CT7.1
44
-
30
10
-
-
36
-
-
33
70
-
-
-
-
60
SYCP1/C
T8
-
47
20
0
-
7
-
28b
0
7
14
0
-
0
8
0
BRDT/CT
9
0
-
0
0
8
-
8
-
-
25
0
-
-
-
0
-
MAGEE1/
CT10
44
-
38
0
-
-
36
-
-
24
50
-
-
-
-
0
SPANXC/
CT11.3
9
-
25
22
0
-
-
-
-
33
70
-
0
-
-
-
XAGE-
1a/CT12.1
a
-
-
-
-
-
-
-
-
-
-
8
-
-
-
-
22
HAGE/CT
13
24
37
5
31
27
-
-
20
9
32
17
-
-
22
6
20
SAGE/CT
14
12
0
5
0
20
-
17
-
4
22
4
-
-
0
5
5
ADAM2/
CT15
-
-
0
0
-
-
-
-
-
0
0
0
-
-
12
-
PAGE-
5/CT16
-
-
5
11
-
-
-
-
-
39
22
0
-
-
44
-
LIP1/CT1
7
-
-
5
0
-
-
-
-
-
0
0
0
-
-
25
-


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
118
Frequency (%) of Expression in Tumor Type
CT 
Family
(Member)
Bla
d
Brn
Brst
Col
Eso
Gas
H/N
Live
r
Leu
k/
Ly
mp
h
Lun
g
(NS
CL
C)
Mel
Ov
Pan
cr
Pro
s
Ren
al
Sarc
NA88/CT
18
-
-
-
-
-
-
-
-
-
-
11
-
-
-
-
-
TSP50/CT
20
-
-
28
-
-
-
-
-
-
-
-
-
-
-
-
-
CTAGE-
1/CT21.1
-
-
-
-
-
-
-
-
35
-
-
-
-
-
-
-
SPA17/CT
22
-
-
-
-
-
-
-
-
26
-
-
-
-
-
-
-
OYTES1/
CT23
28
-
40
15
-
0
-
40
-
20
-
-
-
-
0
-
MMA1a/C
T25.1a
-
-
0
0
0
-
-
-
-
40
26
-
0
-
-
18
CAGE/CT
26
-
-
-
-
-
89
-
-
-
100
-
-
-
-
-
-
HOMTES
85/CT28
-
35
0
10
-
-
-
19
-
28
36
32
-
0
-
-
D40/CT29
-
20
-
13
-
0
-
-
-
41
-
36
27
-
-
-
HCA661/
CT30
0
-
-
-
-
0
0
29
-
-
20
-
-
-
-
-
PLU-
1/CT31
-
-
86
-
-
-
-
-
-
-
-
-
-
-
-
-
LDHC/CT
32
-
-
35
15
-
-
-
-
-
47
44
42
-
37
57
-
MORC/C
T33
-
-
0
0
-
-
-
-
-
18
18
14
-
0
0
-
SGY-
1/CT34
-
-
20
0
-
-
-
-
-
12
25
57
-
12
0
-
SPO11/CT
35
-
-
0
0
-
-
-
-
-
0
6
0
-
0
0
-
TPX1/CT3
6
-
-
15
0
-
-
-
-
-
-
6
14
-
37
14
-
NYSAR35
/CT37
42
-
23
0
8
-
-
-
-
17
6
8
-
-
0
8
FTHL17/C
T38
22
-
14
0
0
-
10
-
0
25
0
-
-
0
0
0
NXF2/CT
39
19
-
0
11
12
-
5
-
0
15
55
-
-
14
0
27


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
119
Frequency (%) of Expression in Tumor Type
CT 
Family
(Member)
Bla
d
Brn
Brst
Col
Eso
Gas
H/N
Live
r
Leu
k/
Ly
mp
h
Lun
g
(NS
CL
C)
Mel
Ov
Pan
cr
Pro
s
Ren
al
Sarc
TAF7L/C
T40
10
-
0
0
0
-
10
-
0
9
21
-
-
0
0
12
TDRD1/C
T41.1
28
-
37
0
10
-
22
-
5
5
0
-
-
38
0
0
TEX15/C
T42
21
-
0
0
20
-
11
-
0
21
27
-
-
12
33
28
FATE/CT
43
-
-
-
21
-
7
-
66
-
0
-
-
-
-
-
-
TPTE/CT4
4
-
-
-
0
-
0
-
39
-
36
-
-
-
-
-
-
aAbbreviations: Blad, bladder; Brn, brain; Brst, breast; Col, colon; Gas, gastric; H/N, head and neck; Leuk, leukemia; 
Lymph, lymphoma, NSCLC, non-small cell lung carcinoma; Mel, melanoma; Ov, ovarian; Pancr, pancreatic; Pros, 
prostate; Sarc, sarcoma.
Reprinted from ref (126) 


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
120
15.2 APPENDICES
15.2.1 Appendix 1: Performance Status Criteria
ECOG Performance Status Scale
Karnofsky Performance Scale
Grade
Descriptions
Percent
Description
100
Normal, 
no 
complaints, 
no 
evidence of disease.
0
Normal activity.  Fully active, able 
to 
carry 
on 
all 
pre-disease 
performance without restriction.
90
Able to carry on normal activity; 
minor signs or symptoms of 
disease.
80
Normal activity with effort; some 
signs or symptoms of disease.
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).
70
Cares for self, unable to carry on 
normal activity or to do active 
work.
60
Requires occasional assistance, 
but is able to care for most of 
his/her needs.
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.
50
Requires considerable assistance 
and frequent medical care.
40
Disabled, requires special care 
and assistance.
3
In bed >50% of the time.  Capable 
of only limited self-care, confined 
to bed or chair more than 50% of 
waking hours.
30
Severely disabled, hospitalization 
indicated.  Death not imminent.
20
Very 
sick, 
hospitalization 
indicated. Death not imminent.
4
100% bedridden.  Completely 
disabled.  Cannot carry on any 
self-care.  Totally confined to bed 
or chair.
10
Moribund, 
fatal 
processes 
progressing rapidly.
5
Dead.
0
Dead.


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
121
15.2.2 Appendix 2: Sample Medication Self-Administration Record and Patient Diary
NAME
 
DATE
April 2015 
Sun
Mon
Tue
Wed
Thu
Fri
Sat
 
 
 
1
2
3
4
 
___________
_________
___________
_________
___________
_________
___________
_________
___________
_________
___________
_________
5
6
7
8
9
10
11
___________
_________
___________
_________
___________
_________
___________
_________
___________
_________
___________
_________
___________
_________
12
13
14
15
16
17
18
___________
_________
___________
_________
___________
_________
___________
_________
___________
_________
___________
_________
___________
_________
19
20
21
22
23
24
25
___________
_________
___________
_________
___________
_________
___________
_________
___________
_________
___________
_________
___________
_________
26
27
28
 29
30
 
 
___________
_________
___________
_________
___________
_________
 
 
 
 
 
 
 
 
 
 
 
Take Medications: DAC and THU once per day on assigned days. THU should be taken approximately 2 hours before 
or 2 hours after a meal. DAC should be taken 1 hour after THU.
GI
 
Dates
Treated at 
Home
Treated in 
doctor's 
office 
Hospitalized
Comments
diarrhea
 
 
 
 
 
 
nausea
 
 
 
 
 
 
vomiting
 
 
 
 
 
 
mouth sores
 
 
 
 
 
 
neuropathy
 
 
 
 
 
 
tingling hands and feet
 
 
 
 
 
sensitivity to cold
 
 
 
 
 
general
 
 
 
 
 
 
Fever
 
 
 
 
 
 
Infection
 
 
 
 
 
 
swelling arms/legs
 
 
 
 
 
fatigue
 
 
 
 
 
 
Injection site
 
 
 
 
 
 
redness/swelling
 
 
 
 
 
pain
 
 
 
 
 
 
shortness of breath
 
 
 
 
 
bruising/bumps
 
 
 
 
 
 


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
122
swollen glands 
near injection 
site
 
 
 
 
 
 
rash
 
 
 
 
 
 
Respiratory
 
 
 
 
 
 
sore throat
 
 
 
 
 
 
runny nose
 
 
 
 
 
 
cough
 
 
 
 
 
 
Other:
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
123
15.2.3 Appendix 3: Study Calendar
Screening to Completion of DAC-THU/Pembrolizumab
Courses 1 and Course N+1 [Course 2 Onwards]*
* Note: Couse N+1 administered only for patients who exhibit stable disease 
or disease regression 
During Treatment
Procedure
Screening
Within 4 weeks 
prior to enrollment
Baseline 
Within 10 
days prior to 
start of DAC-
THU
Day 1 
of 
DAC-
THU
Day 2 
of 
DAC-
THU
Within 3 
days prior 
to 
completion 
of DAC-
THU
At the end of course 
completion
End of 
Treatment 
Visit7
History and PE
X
X
X
X
Vital signs
X
X
X
X
Performance Score
X
X
X
NCI LP Confirmation of Dx1
X
CBC with differential; Acute 
Care Panel, Hepatic Panel 
(Sections 2.2 and 3.4.1)
X
X
Twice weekly during Course 1, 
and then weekly unless clinically 
indicated
X
X
PT, PTT
X
X
Twice weekly during Course 1, 
and then weekly unless clinically 
indicated
X
Thyroid Function Tests
X
Albumin, Ca2+ total, Mg2+ 
total, Phosphorus, LD, Total 
Protein, Total CK, Uric Acid
X
Twice weekly during Course 1, 
and then weekly unless clinically 
indicated
X
X
Fetal Hemoglobin
X
Urinalysis 
X4
X4
Anti HIV, anti HCV, 
HBsAg, anti-HAV IgM, and 
if needed, HCV RNA
X
Urine or serum HCG in 
women of childbearing 
potential
X3
X3
T, B, NK cell subsets
X9
X


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
124
Courses 1 and Course N+1 [Course 2 Onwards]*
* Note: Couse N+1 administered only for patients who exhibit stable disease 
or disease regression 
During Treatment
Procedure
Screening
Within 4 weeks 
prior to enrollment
Baseline 
Within 10 
days prior to 
start of DAC-
THU
Day 1 
of 
DAC-
THU
Day 2 
of 
DAC-
THU
Within 3 
days prior 
to 
completion 
of DAC-
THU
At the end of course 
completion
End of 
Treatment 
Visit7
HLA class I and class II 
analysis of PBMC
X1
EKG
X
Echocardiogram
X
PFTs
X
X
ABGs (to be drawn if pulse 
oximetry <90% on room 
air)
X
X
Lupus Anticoagulant and 
ANA
X
Radiological Assessments
X2
X2
DAC-THU
Once a day on days according to 
dose level
Tumor sample for research8
X
X
Correlative Research Studies 
X5
X5
X5
PK/PD 
X6
X6
Adverse Events10
X
Concomitant Medications10
X
1.
May be obtained any time prior to initiation of study therapy. Does not need to be repeated if already done at NCI.
2.
Radiological Assessments: Pre-enrollment screening assessment: X-rays, contrast enhanced CT scans of chest, abdomen, pelvis, as 
well as brain MR (no gadolinium contrast), and fused PET-CT scans to evaluate the status of disease. (All imaging must be obtained 
within 4 weeks prior to treatment.) At the end of each course completion assessment: CT scan (C/A/P) (with standard oral/IV 
contrast), fused PET/CT scan and brain MR without gadolinium contrast (or CT scan if MR contraindicated).
3.
Must be completed within 3 days prior to enrollment or 48 hours prior to start of study drugs.
4.
Urinalysis and culture if indicated.


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
125
5.
See Appendix 4 for details of Research Labs timepoints and collection details. Note: Tubes/media may be adjusted at the time of 
collection based upon materials available if approved by the PI/laboratory investigator.
6.
See Appendix 4 for PK sampling timepoints and collection details. Note: PK samples will only be drawn on Day 1 and Day 2 of 
Cycle 1.
7.
End of treatment visit will occur approximately 30 days after the last dose of study drug. If the patient cannot return to the Clinical 
Center for this visit, a request will be made to collect required clinical labs (specify as needed) from a local physician or laboratory.  
If this is not possible, patients may be assessed by telephone/or other NIH approved remote platforms for symptoms.
8.
Tumor biopsy will be performed at baseline if screening biopsy was not performed and after one course of therapy (Week 10 +/- 
one week).
9.
Every three weeks during therapy.
10. Adverse events and concomitant medications will be assessed weekly during the dose finding portion of the trial and every three 
weeks prior to the administration of pembrolizumab during the expansion portion of the trial.


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
126
15.2.4 Appendix 4: Pharmacokinetic Samples and Research Bloods
15.2.4.1 Course 1 Cycle 1
TEL Research 
Sample #
(2 red top tubes, 
2 lavender top 
tubes)
Immune 
Subset & CTC 
Samples #
(2 CPT tubes, 
and 2 lavender
top tubes)
PK Sample 
#
(1 6 mL 
green top 
tube)
Hour
(pre dose = within 1 
hour prior to THU)
Target Time Actual Time Initials
Baseline (post -
enrollment)
R1
T1
n/a
n/a
n/a
n/a
n/a
Day 1 DAC-THU
R2 (within 1 hour 
prior to THU)
T2 (within 1
hour prior to
THU)
PK 1
pre dose
n/a
n/a
PK 2
0.5h 
post 
DAC dose
n/a
n/a
PK 3
1h post DAC 
dose
n/a
n/a
PK 4
1.5h 
post 
DAC dose
n/a
n/a
PK 5
2h post DAC 
dose
n/a
n/a
PK 6
3h post DAC 
dose
n/a
n/a
PK 7
4h post DAC 
dose
n/a
n/a
PK 8
8h post DAC 
dose
Day 2 DAC-THU
n/a
n/a
PK 9
pre dose
n/a
n/a
PK 10
0.5h 
post 
DAC dose
n/a
n/a
PK 11
1h post DAC 
dose
n/a
n/a
PK 12
1.5h 
post 
DAC dose
n/a
n/a
PK 13
2h post DAC 
dose
n/a
n/a
PK 14
3h post DAC 
dose
n/a
n/a
PK 15
4h post DAC 
dose
n/a
n/a
PK 16
8h post DAC 
dose


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
127
After completion of 
each course of DAC-
THU
R3
T3
n/a
n/a
n/a
n/a
n/a
15.2.4.2 Course N+1 (Course 2 Onwards)*
TEL Research 
Sample #
(2 red top tubes, 
2 lavender top 
tubes)
Immune 
Subset & CTC 
Samples #
(2 CPT tubes, 
and 2 lavender
top tubes)
PK Sample 
#
(1 6 mL 
green top 
tube)
Hour
(pre dose = within 1 
hour prior to THU)
Target Time Actual Time Initials
Day 1 DAC-THU
R4* (within 1 hour 
prior to THU)
T4* (within 1 
hour prior to 
THU)
n/a
pre dose
After completion of 
each course of DAC-
THU
R5*
T5*
n/a
n/a
n/a
n/a
n/a
*Note: Labels should list the specific collection number as appropriate; indicated collection numbers are 
for Course # (e.g., Course 2) draws. 
15.2.4.3 Instructions
PK Studies (PK)
Collect 6 mL blood in 1 green top tube. Tubes should then be placed on crushed ice, and samples 
should be centrifuged for 15 minutes at approximately 1000 x g at 0-5 °C within 2 hours after 
collection. The plasma should be transferred to separate pre-labeled screw-capped polypropylene 
transfer tubes and stored at -80 °C in the Thoracic Epigenetics Lab (TEL) until batched and later 
sent to the Figg Lab on dry ice for analysis.
Note: All samples are to be placed on wet ice and transported to the Thoracic Epigenetics Lab 
(TEL) within one hour of draw.
Research Labs (R)
Samples will be collected in 2 red top (10 mL each) tubes, and 2 lavender top tubes (EDTA; 10 
mL each) for focused methylation analysis and immune response to treatment (see Section 5.1.2). 
Note: All samples are to be placed on wet ice and transported to the Thoracic Epigenetics Lab 
(TEL) within one hour of draw.
Research Labs will be collected at the following timepoints (see also Appendix 3: Study 
Calendar):

Baseline (once), see Section 3.4.1;

Each Course
o
Prior to DAC-THU initiation, 
and at


Abbreviated Title: DAC-THU/Pembro: NSCLC, EsC, PM
Version Date:  08/01/2022
128
o
DAC-THU completion. 
Immune Subset and Circulating Tumor Cell Analysis (T)
Samples will be collected in 2 CPT citrate tubes (BD; 8 mL) for immune subset analysis (see 
Section 5.1.3) and 2 lavender top tubes (10 mL) for CTCs analysis (Section 5.1.4). The Trepel Lab 
will be notified to pick up the samples (as indicated in Section 5.2.4) as soon as the blood is drawn 
for sample processing and analysis. 
Samples will be drawn at the following timepoints (see also Appendix 3):

Each course
o
Prior to DAC-THU initiation, 
and at
o
DAC-THU completion.
Labeling (PK and Research Labs)
Prior to the infusion, the PK /research lab sheet and labels will be placed in the patient's room and 
a separate set of labels will be given to the Thoracic Epigenetics Lab staff. The labels will list the 
patient identifiers as well as the Cycle and draw number as appropriate. As the RN (or designee) 
draws the bloods, the RN (or designee) will place a label on the tube and write the sample reference 
# on the label on the tube. When the Thoracic Epigenetics Lab personnel or fellow processes the 
sample, the TEL personnel will label the storage tube with the label and will transcribe the 
corresponding sample reference # on the label on the tube.
Tracking (PK and Research Labs)
Upon completion of the PK/Research sample draws, the research nurse will make 3 copies of the 
PK sheet. The original and one copy should be placed in the research binder. 2 copies should be 
given to the TEL personnel. TEL will retain one copy for their records and will send the other copy 
with the PK samples to the Figg Lab. Note: Prior to sending the PK sheets to the Figg Lab, the 
patient name should be crossed out.

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