Clinical trial NCT02936752
Conditions: Myelodysplastic Syndrome
Sponsor: National Cancer Institute (NCI)
Start date: 2017-06-23; Completion date: 2025-02-12
Enrollment: 28 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This phase Ib trial studies the side effects and best dose of entinostat when given together with pembrolizumab in treating patients with myelodysplastic syndrome after deoxyribonucleic acid (DNA) methyltransferase inhibitor (DNMTi) therapy failure. Entinostat may stop the growth of cancer cells by blocking some of the enzymes needed for cell growth. Immunotherapy with monoclonal antibodies, such as pembrolizumab, may help the body's immune system attack the cancer, and may interfere with the ability of tumor cells to grow and spread. Giving entinostat together with pembrolizumab may work better in treating patients with myelodysplastic syndrome after DNMTi therapy failure.

Detailed description:
PRIMARY OBJECTIVE:

I. To assess safety, tolerability, and identify the maximum tolerated dose (MTD) of entinostat given in combination with MK-3475 (pembrolizumab).

SECONDARY OBJECTIVE:

I. To obtain a preliminary estimate of efficacy of entinostat in combination with MK-3475 (pembrolizumab).

EXPLORATORY OBJECTIVE:

I. To assess the dynamic quantitative change in measurable immunological biomarkers (proportions of myeloid-derived suppressor cells \[MDSCs\], and programmed death protein-1 \[PD-1\] expression in bone marrow) with the combined epigenetic-immunotherapy and correlation with any observed clinical responses.

OUTLINE: This is a dose-escalation study of entinostat.

Patients receive lower dose entinostat orally (PO) on days 1 and 8 or higher dose entinostat PO on days 1, 8, and 15, and pembrolizumab intravenously (IV) over 30 minutes on day 1 of cycle 2 and cycles thereafter. Treatment repeats every 21 days for up to 4 cycles in the absence of disease progression or unacceptable toxicity. Patients who achieve an objective response or maintain a stable disease (SD) status after the first 4 cycles may continue to receive entinostat and pembrolizumab for up to 1 year.

After completion of study treatment, patients are followed up monthly for 6 months.

Arms: arm 1: Patients receive lower dose entinostat PO on days 1 and 8 or higher dose entinostat PO on days 1, 8, and 15, and pembrolizumab IV over 30 minutes on day 1 of cycle 2 and cycles thereafter. Treatment repeats every 21 days for up to 4 cycles in the absence of disease progression or unacceptable toxicity. Patients who achieve an objective response or maintain a SD status after the first 4 cycles may continue to receive entinostat and pembrolizumab for up to 1 year.
Interventions: intervention 1: Entinostat intervention 2: Pembrolizumab
Intervention descriptions: intervention 1: Given PO intervention 2: Given IV

Protocol documents:
Prot_SAP_000.pdf: 
i 
 
 
SUMMARY OF CHANGES – Protocol 
For Protocol Version 14 to 15, A11  
NCI Protocol #: 10009 
Local Protocol #: HIC 2000020860 
 
NCI Version Date: 09/04/2020 
Protocol Date: 09/04/2020 
 
 
I. 
CTEP Request for Amendment (RA) dated August 24, 2020: 
 
# 
Section 
Comments 
1. 
All 
Updated Version Date in Header 
2. 
Title 
Page 
Added “CATCHUP / Creating Access to Targeted Cancer Therapy for 
Underserved Populations” to the list of Participating Organizations 
3. 
Title 
Page 
Updated Protocol Type / Version # / Version Date 
 
II. 
Response to CTEP Amendment Review Recommendations dated August 3, 2020: 
 
# 
Section 
Comments 
1. 
Schema 
& 5.1 
Part 1 – remove the sentence “Entinostat will be stored at the pharmacy and it will 
not be given for the patient to take home.”   
PI Response: The procedure is indeed to give the entinostat in the clinic and 
not to give to the patient to take home, so this sentence was not removed.  
2. 
8.1.3.4 
 
At the time of the next amendment update the following: 
x PMB Online Agent Order Processing (OAOP) application:  
https://ctepcore.nci.nih.gov/OAOP   
PI Response: The PMB Online Agent Order Processing information has been 
updated as recommended.  
 
 
 
 
 


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NCI Protocol #: 
10009 
 
Local Protocol #: 
HIC 2000020860 
 
ClinicalTrials.gov Identifier: NCT02936752 
 
TITLE: A Phase 1b study of the anti-PD1 antibody Pembrolizumab in combination with the 
histone deacetylase inhibitor, Entinostat for treatment of patients with myelodysplastic 
syndromes after DNA methyltransferase inhibitor therapy failure 
 
Corresponding Organization: LAO-CT018 / Yale University Cancer Center LAO 
 
Principal Investigator: 
Amer M. Zeidan 
Yale University 
333 Cedar Street, PO Box 208028 
New Haven, CT, 06520, USA 
Telephone: 203-737-7103 
Fax (optional): 203-785-7232 
e-mail address: Amer.zeidan@yale.edu 
Participating Organizations 
 
LAO-11030 / University Health Network Princess Margaret Cancer Center LAO 
LAO-CA043 / City of Hope Comprehensive Cancer Center LAO 
LAO-CT018 / Yale University Cancer Center LAO 
LAO-MA036 / Dana-Farber - Harvard Cancer Center LAO 
LAO-MD017 / JHU Sidney Kimmel Comprehensive Cancer Center LAO 
LAO-MN026 / Mayo Clinic Cancer Center LAO 
LAO-NC010 / Duke University - Duke Cancer Institute LAO 
LAO-NJ066 / Rutgers University - Cancer Institute of New Jersey LAO 
LAO-OH007 / Ohio State University Comprehensive Cancer Center LAO 
LAO-PA015 / University of Pittsburgh Cancer Institute LAO 
LAO-TX035 / University of Texas MD Anderson Cancer Center LAO 
LAO-NCI / National Cancer Institute LAO 
EDDOP / Early Drug Development Opportunity Program 
CATCHUP / Creating Access to Targeted Cancer Therapy for Underserved Populations 


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Statistician: 
Name: Daniel Zelterman 
Address: 60 College street, LEPH 204 
New Haven, CT 06519 
Telephone: 203-785-5574 
Fax: (203) 737-4576 
e-mail address: daniel.zelterman@yale.edu 
 
Responsible Research Nurse: 
Name: Anne Caldwell, RN 
Address 100 Church St South, Suite F-220 
Address New Haven, CT 06519 
Telephone (203) 785-3465 
Fax (203)-785-6781 
e-mail address anne.caldwell@yale.edu 
Study Coordinator: 
Name: Grace Anne Valentin 
Address 37 College Street 
Address New Haven, CT 06510 
Telephone (203) 785-4699 
Fax (203)-737-3401 
e-mail address: 
graceanne.valentin@yale.edu 
 
Responsible Data Manager: 
Name: Madelynn Bonfiglio 
Address 37 College Street 
Address New Haven, CT 06510 
Telephone (203) 737-4616 
Fax (203)-737-3401 
e-m ail address 
madelynn.bonfiglio@yale.edu 
 
NCI-Supplied Agent(s): 
MS-275 (SNDX-275, entinostat, NSC 706995) 
MK-3475 (pembrolizumab, NSC 776864) 
Other Agent(s) : N/A 
IND #: 
IND Sponsor: DCTD, NCI 
Protocol Type / Version # / Version Date: Original/ Version#1/ September 9, 2016 
Revision/ Version#2/ November 30, 2016 
Revision/ Version#3/ February 12, 2017 
Revision/ Version#4/ February 24, 2017 
Revision/ Version#5/ April 25, 2017 
Revision/ Version#6/ February 01, 2018 
Revision/ Version#7/ April 02, 2018 
Revision/ Version#8/ May 04, 2018 
Revision/ Version#9/ June 06, 2018 
Revision/ Version#10/ October 18, 2018 
Revision/ Version#11/ September 10, 2019 
Revision/ Version#12/ November 25, 2019 
Revision/ Version#13/ March 02, 2020 
Revision/ Version#14/July 14, 2020 
Revision/ Version#15/ September 4, 2020 


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SCHEMA 
Title of Study: A Phase 1b study of the anti-PD1 antibody Pembrolizumab in combination with the 
histone deacetylase inhibitor entinostat for treatment of patients with myelodysplastic syndromes after 
DNA methyltransferase inhibitor therapy failure 
Investigators: Principal investigator: Amer Zeidan, MD, MHS 
Study Center(s): Yale Cancer Center 
Concept and Rationale: Besides allogeneic stem cell transplantation (allo-SCT), the DNA 
methyltransferase inhibitor (DMNTi) azacitidine (AZA) is the only treatment proven to prolong 
survival in patients with higher risk myelodysplastic syndrome (HR-MDS). Nonetheless, HR-MDS 
patients treated with AZA have a median survival advantage of 9.5 months (24.5 vs 15 months median 
OS)1 and the objective response rate is only 50% with most patients eventually relapsing within 2 years1. 
Failure of DNMTi therapy occurs in the majority of treated patients and carries a dismal prognosis with 
a median survival of less than 6 months 2. To date, no drug or combination of drugs have improved 
survival of MDS patients in the post-DNMTi failure setting in randomized trials, and there is currently 
no standard of care for those patients. Therefore, improving outcomes of MDS patients after failure of 
DNMTi remains a top clinical and research priority 3. 
Histone deacetylase inhibitors (HDACi) comprise a group of drugs that share the ability to inhibit the 
important epigenetic eraser enzymes HDACs. As single agents, several HDACi exhibited only modest 
clinical activities in MDS4,5. Therefore, the interest to develop these agents further has focused on 
combination-based strategies, usually with DNMTi. To date, no HDACi-DNMTi combination has 
improved survival of MDS patients in randomized studies when used in the frontline or the post-DNMTi 
failure setting. These observations suggest that while HDACi therapy has modest activity in MDS, 
DNMTi might not be the best choice for combination-based regimen with HDACi. Exploration of 
alternative agents to combine with HDACi in the post-DNMTi failure setting might be a more rational 
approach. 
Recent advances in tumor immunology have facilitated understanding the role of immune escape as 
important mechanism of cancer progression and treatment resistance. One important mechanism which 
cancers employ commonly to evade the immune system involve the exploitation of T-cell inhibitory 
pathways and molecules such as the programed death-1 (PD-1) and cytotoxic T-lymphocyte associated 
antigen-4 (CTLA-4) 6 7. Immune checkpoint blockade with antibodies directed against PD-1, itsligand 
(PD-L1), and CTLA-4 has emerged as promising approaches to reverse immune evasion, and have 
resulted in a significant and durable clinical activity in some patients with advanced solid malignancies 
especially metastatic melanoma, lung cancer, bladder cancer, and Hodgkin disease 8-11. Compared to 
expanding clinical applications of immune checkpoint inhibitors in solid tumors, the mechanisms of 
immune evasion in hematologic malignancies including MDS have not been fully elucidated. Several 
studies suggest that interaction between PD-1 and PD-L1 may induce immune evasion in MDS12 and 
moreover the upregulation of these molecules could be associated with resistance to DNMTi therapy in 
MDS13. 
Furthermore, recent studies suggest that myeloid-derived suppressor cells (MDSCs), which are 
significantly increased in bone marrows of MDS patients (~35% vs <5% in non-MDS bone marrow), 
play an important role in ineffective hematopoiesis in MDS14. MDSCs also induce resistance to immune 
checkpoint blockade (anti-CTLA4 and anti-PD1) in colon cancer bearing mice15. Importantly, in this 
mouse model MDSCs were significantly decreased by the use of the epigenetic modulator entinostat 
(ENT), leading to rejection of tumors when combined with immune checkpoint blockade15. 
Primary Objective(s): To identify the maximum tolerated total dose (MTD) of entinostat given in 
combination with pembrolizumab (MK-3475)  in post-HMA failure setting for patients with MDS. 
Secondary Objective(s): To obtain a preliminary estimate of efficacy of entinostat in combination 
with pembrolizumab (MK-3475). 


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Primary Endpoint(s): MTD as defined by the occurrence of dose-limiting toxicity (DLT) among 2 
dose schedules 
Secondary Endpoint(s): Overall response rate (ORR), with a target if at least 15% to be continued 
for a phase 2 study. 
Exploratory Endpoint(s): 
x Median response duration for responders, median time of progression to AML, median overall 
survival (OS) and 1- and 2-year OS rates. 
x Assessment of the dynamic quantitative change in proportion of MDSCs and PD-1 expression 
in BM with combined therapy and correlation with any observed clinical responses. 
Study Design: This will be a phase 1b study [see schema]. The proposed dosing of ENT has been 


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previously tested in MDS as single agent and shown to be well-tolerated. While the combination of 
ENT with the anti-PD1, pembrolizumab (MK-3475) has not been studied clinically in MDS patients, 
based on mechanisms of action it is unlikely that this combination would have overlapping toxicities. 
PART 1: ENT has been previously evaluated as a single agent therapy in patients with MDS at these 
doses and has been well tolerated. The ENT monotherapy run-in part (cycle 1) will allow to study the 
changes in MDSCs at pre- and post- ENT treatment (the end of cycle 1) to dissect the effects of ENT 
monotherapy from those of the combined ENT/anti-PD1 therapy. 
We will perform ‘3+3’ classical dose escalation design to determine DLT. The first 3 patients will 
receive ENT at dose level 1 (DL1: 8mg on D1, 8 of 21-day cycles). pembrolizumab (MK-3475) 
will be added starting day 1 cycle 2 at the approved dose (200mg intravenously [IV] over 30 
minutes on D1 of 21-day cycles) for 4 cycles. A safety committee that includes the study chair, 
the principal investigator from any study site that has enrolled a patient on the specific DL, as well 
as two sub-investigators from Yale will review safety data after enrollment of every 3 patients 
during the dose escalation phase. The committee will have the discretion to decide to enroll 3 
more patients in the specific dose level even if no DLT occurred in the first 3 treated patients in 
that dose level if there are toxicity/safety concerns that did not qualify as DLT (maximum of 6 
evaluable patients to be enrolled at any dose level). The committee can also decide to enroll 
patients at a lower dose level after the first 3 or after the first 6 patients enrolled in any dose level 
even if no or one DLT occurred at that dose level if there are toxicity/safety concerns that did not 
qualify as DLT. The following regarding enrollment in different dose levels will be considered 
guidance for the safety committee but can be modified in accordance of above: If no patient of the 
3 patients treated with combined therapy at DL1 develops DLT, then dose level 2 (DL2) will be 
tested. DL2 will be with ENT at 8mg on D1, 8 and 15 and pembrolizumab (MK-3475) at 200mg 
IV on D1 of 21-day cycles. If 1 patient out of the 3 patients has DLT at DL1, we will expand 
cohort to 3 more patients and if 1 patient of the 6 patients has DLT (i.e. only 0/3 or 1/6), we will 
test next dose (DL2). If no patient of the 3 patients treated with combined therapy at DL2 develop 
DLT, then DL2 will be used for part 2 (expansion cohort). If 1 patient out of the 3 patients has 
DLT at DL2, we will expand cohort to 3 more patients and if 1 patient of the 6 patients has DLT 
(i.e. only 0/3 or 1/6), dose level 2 (DL2) will be used for part 2 (expansion cohort). If 2 patients or 
more of the 6 patients at DL2 experience DLT, DL1 will be used for expansion cohort. The cycle 
duration will be 21 days, the maximum number of treatment cycles of entinostat will be 18 cycles 
and for pembrolizumab (MK-3475) the maximum number of cycles will be 17. Cycle 2 to cycle 
18, will have concurrent administration. In all the phases of the study, entinostat will be 
administered in clinic within 2 hours before administration of pembrolizumab (MK-3475). 
Entinostat will be stored at the pharmacy and it will not be given for the patient to take home. 
 
An additional dose level (DL -1) in which day 8 of ENT will be omitted (i.e. ENT at 8mg on D1, and 
pembrolizumab (MK-3475) at 200mg IV on D1 of 21-day cycles) will be used if 2 or more patients 
develop DLT during the first 2 cycles of combined therapy at DL1. If 0 or 1 patients of the 6 patients 
treated with combined therapy at DL-1 develop DLT then DL-1 will be used in the second part of the 
study. If 2 or more 6 patients enrolled in DL -1 develop DLT, then the study will be terminated. If the 
AE drug causality is clear (e.g. immune related event) and one of the drugs is discontinued, the patient 
can remain on the study and receive the non-causative agent. 
Toxicities will be tabulated and graded according to the Common Terminology Criteria for Adverse 
Events Version 5 (CTCAE-5). DLT will be assessed after the first 2 cycles of combined therapy. 
Responses will be as defined by International Working Group (IWG)-2006 criteria (CR, PR, or HI) and 


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assessed at the end of combined first 2 cycles, and every 2 cycles afterwards. Patients who achieve an 
objective response or maintain a stable disease (SD) status after the first 4 combination cycles will 
continue receiving both drugs at the same schedule till progression or up to 1 year of combined therapy. 
 
PART 2: After the safe dose of the combination is established, 15 patients will be treated with ENT+ 
pembrolizumab (MK-3475) combination at that same dose and schedule. Participants who go off-study 
during the 1st cycle of entinostat will be replaced in the escalation phase. Participants who do not 
complete the first 2 cycles of combined therapy without experiencing a DLT (as DLT period is defined 
in the first 2 combined therapy cycles) during dose escalation will also be replaced. To be DLT- 
evaluable, the patient must complete the first two cycles of combined therapy without experiencing a 
DLT. Any patient who is dose reduced during the entinostat run-in first cycle due to entinostat-related 
toxicity will be analyzed on the dose level that corresponds to the reduced dose level delivered during 
the first cycle of combined therapy. 
Assessment of response will occur after 4 combination cycles and similar to part 1, those who achieve 
an objective response or SD will continue to receive the therapy till progression. Patients who progress 
or respond and subsequently lose response will be withdrawn from study. BM biopsy/aspirate and PB 
samples will be done at baseline, after first cycle of ENT monotherapy, every 2 cycles of combined 
therapy, and at time of progression or study discontinuation. Formal safety assessment will be 
performed at end of DLT-observing period (i.e. 1 ENT monotherapy cycle and 2 cycles of combined 
therapy) for each patient) for each DL to assess for any unexpected autoimmune or other toxicity. The 
cycle duration will be 21 days, the maximum number of treatment cycles of entinostat will be 18 cycles 
and for pembrolizumab (MK-3475) the maximum number of cycles will be 17. Cycle 2 to cycle 18, 
will have concurrent administration. 
The following early stopping rule for excess toxicity in the expansion cohort will be used: We will cease 
enrollment in the expansion phase at the dose determined from dose escalation part of the study if we 
observe two or more DLTs among the first six evaluable patients enrolled in the expansion cohort. An 
evaluable patient is any patient who received a study drug. If the true toxicity rate is 50% or greater at 
the MTD then this rule will stop the trial with probability at least .89. Similarly, if the true toxicity rate 
is 33% at the MTD then this stopping rule will end the trial with probability .65. In that event, a new 
expansion cohort will start at the dose level immediately below the dose level that was expanded. If the 
dose level being expanded is dose level -1, then the study will be terminated. 


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Study Schema 
 
Study design 
 
 
 
 
 
 
 


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Dose Escalation Schedule 
Dose Level 
Dose* 
Entinostat 
Pembrolizumab 
 
 
Level -1(Backup dose 
level) 
8mg po D1 
200mg 
Level 1 (starting dose 
level) 
8mg po D1,8 
200mg 
Level 2 
8mg po D1,8,15 
200mg 
Number of Patients: 6-27 
 
Main Criteria for Inclusion/Exclusion: 
 
Pathologically confirmed MDS diagnosis (regardless of initial IPSS risk category) or oligoblastic 
AML with 21-30% BM blasts in whom DNMTi have failed. Patients who have developed AML after 
DNMTi therapy can be enrolled as long as they have initiated DNMTi therapy while they were in the 
MDS or oligoblastic AML (20-30% BM blasts) phase and the study chair agrees. Failure of DNMTis 
is defined as: Failure to achieve a CR, PR or HI after at least 4 cycles of DNMTi or progressed after 
such therapy. 
The patients should also have 
x ECOG performance status of 0, 1, or 2 at study entry 
x Laboratory test results within these ranges: 
Calculated creatinine clearance by MDRD (CrCl) =>60 ml/min/1.73 squared meter 
x 7RWDOELOLUXELQPJG/XQOHVVGXHWR*LOEHUW¶VV\QGURPHKHPRO\VLVRULQHIIHFWLYH
KHPDWRSRLHVLVDQG$676*27DQG$/76*37[ULN Females of childbearing 
potential must have a negative serum or urine pregnancy test within 72 hours prior to start of 
first cycle of therapy. 
x Patients must have no clinical evidence of CNS or pulmonary leukostasis, disseminated 
intravascular coagulation, or CNS leukemia. 
x Patients must have no serious or uncontrolled medical conditions 
• 
Patients, who relapsed 6 months after bone marrow transplant and have no evidence 
of active graft versus host disease and are off systemic immunosuppressant 
medications for at least 2 months and have received HMA therapy before or after 
transplant and meet other eligibility criteria of progression after at least 4 months of 
DNMTi therapy, are eligible to be enrolled in this clinical trial. 
 
 
 
Exclusion criteria: 
x Any patients eligible for allo-SCT and willing to undergo allo-SCT as determined at time of 
screening for trial. Patients who are ineligible or not interested in undergoing allo-SCT will be 
eligible for the trial. 
x Any serious medical condition, uncontrolled intercurrent illness (e.g., active infection, 
symptomatic congestive heart failure (CHF), unstable angina, cardiac arrhythmias, laboratory 
abnormalities, or psychiatric illness and/or biopsychosocial conditions that may limit 


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compliancePatients with known active cancers whop are receiving active therapy 
x Patients with a known positive test for hepatitis B virus surface antigen (HBV sAg) or hepatitis 
C virus ribonucleic acid (HCV antibody) indicating acute or chronic infection might be 
enrolled if the viral load by PCR is undetectable with/without active treatment. 
x Pregnant or breast-feeding females (lactating females must agree not to breast feed while taking 
the study drugs) 
x Use of any other experimental drug or therapy within 21 days of baseline - patients who have 
had chemotherapy or radiotherapy within 4 weeks of entering the study or those who have not 
recovered from adverse events due to agents administered more than 4 weeks earlier 
x Known hypersensitivity to pembrolizumab (MK-3475) or entinostat or history of allergic 
reactions to compounds of similar chemical or biologic composition to anti-PD1 antibodies or 
entinostat. 
x Prior treatment with any anti-PD-1 blocking therapies or HDACi, or anti-CTLA-4 antibody, 
CD137 agonist or other immune activating therapy such as anti-CD 40 antibody within the last 
3 months of enrollment in the study. 
x Any history of active or severe autoimmune disease including: 
• 
Inflammatory bowel disease, including ulcerative colitis and Crohn’s Disease 
Rheumatoid arthritis, systemic progressive scleroderma, systemic lupus 
erythematosis, autoimmune vasculitis (e.g., Wegener’s Granulomatosis). 
• 
Note: patients with hypothyroidism who are on stable doses of hormone replacement 
therapy are allowed 
x CNS or motor neuropathy considered of autoimmune origin (e.g. Guillain-Barre Syndrome, 
myasthenia gravis, multiple sclerosis) 
Has a history of (non-infectious) pneumonitis that required steroids or has current pneumonitisPatients 
who are Human Immunodeficiency Virus (HIV) positive may participate IF theymeet the following 
eligibility requirements: They must be stable on their anti-retroviral regimen, and they must be healthy 
from an HIV perspective. 
They must have a CD4 count of greater than 250 cells/mcL. 
They must not be receiving prophylactic therapy for an opportunistic infection. 


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Intervention and Mode of Delivery: Entinostat, PO administration, pembrolizumab (MK-3475), IV 
infusion 
Duration of Intervention and Evaluation: 1 year or until disease is progressed 
 
Statistical Methods: 
(a) Definition of primary outcome/endpoint: The primary endpoint is the maximum tolerated dose 
(MTD), which is the highest dose level where 1 or fewer of 6 participants experience a dose 
limiting toxicity (DLT) during the first two cycles of treatment. The RP2D is the same as the 
MTD. The maximally administered dose is the dose at which two or more of 6 participants 
experience a DLT. 
 
Definition of DLT: DLT will be defined as the occurrence of any of the following during the 
first 2 cycles of combined therapy unless the event can clearly be determined to be unrelated to 
the study drugs. 
 
x Any grade 3 or 4 non-hematologic toxicity with the following exceptions: A) Transient 
laboratory abnormalities that can be treated or resolve to grade 2 or less within 72 hours. 
x Grade 4 hematologic toxicity, including treatment-associated aplasia lasting >42 days from the 
last dose of study drug. Given that severe anemia, neutropenia and thrombocytopenia are 
features of advanced MDS and commonly encountered in this patient population, they will not 
be used to define DLT except if associated with prolonged treatment-associated aplasia as 
described above. 
x Patients with grade 3 immune related adverse events will be removed from the treatment and 
this will be considered to be a DLT as well as any other immune-related adverse event that 
results in pembrolizumab interruption, dose modification, or discontinuation of therapy. 
x
x Definition of secondary outcomes/endpoints: Overall response rate (ORR: The sum of rates of 
complete response [CR], partial response [PR], and hematologic improvement [HI]) as defined 
by the modified IWG-2006 criteria (Appendix A) with a goal of at least 20% to be considered 
clinically meaningful for further evaluation in larger studies. 
x Analytic plan for primary objective: Toxicities will be tabulated and graded according to the 
Common Terminology Criteria for Adverse Events Version 5 (CTCAE-5). DLT will be 
assessed after the first 2 cycles of combined therapy. 
x Analytic plan for secondary objectives/ Sample size justification: Currently there is no therapy 
that has been shown to prolong survival in MDS in the post-DNMTi setting and that the 
expected best response in patients receiving supportive care only in post-DNMTi setting is 
QHJOLJLEOHRUOHVV,WLVJHQHUDOO\DFFHSWHGWKDWD255LQWKHSRVW-DNMTi is 
clinically meaningful and would be worth further evaluation in larger studies. We therefore 
calculated that treating a total of 21 patients in the combination arm (6 in the first part of the 
study and 15 in the second part of the study) to test the difference of ORR between 1% (null 
hypothesis-supportive care only) and 15% (alternative hypothesis, combination therapy). The 
null hypothesis response rate of 1% will be rejected if we observe 2 or more favorable 
responses. This criterion has significance level between .02 and .03 using a one-sided binomial 
test. The criterion has power between .84 and .93 for testing an alternative response rate of 15%. 
The total number of patients enrolled in the study will range from 6 to 27. 
x Analytic plan for exploratory objectives: The quantitative change in MDSCs (% MDSCs by 


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flow cytometry, QIF score by AQUA) during treatment with the ENT/anti-PD1 combined 
therapy will be estimated using mixed effects models to take into account the within-patient 
correlation. Likelihood ratio tests will be performed to confirm if random intercepts and slopes 
are necessary in the model. The fixed effect for change in MDSCs over time will be evaluated 
for significance. The variability in the rate of change in MDSCs across patients will also be 
examined. The association between the clinical outcome and a meaningful reduction in MDSCs, 
which will be defined after a review of the data, will be assessed with the chi- square test. The 
quantity of MDSCs at baseline and during treatment as continuous variables can also be 
compared between responding and non-responding patients using a t-test or Mann- Whitney U- 
Test, if more appropriate. 


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TABLE OF CONTENTS 
SCHEMA .........................................................................................................................................2 
TABLE OF CONTENTS ...............................................................................................................10 
1. 
OBJECTIVES ....................................................................................................................13 
1.1 
Primary Objectives .................................................................................................13 
1.2 
Secondary Objectives .............................................................................................13 
1.3 
Exploratory objectives ...........................................................................................13 
2. 
BACKGROUND ...............................................................................................................13 
2.1 
Myelodysplastic syndrome (MDS) ........................................................................13 
2.2 
CTEP IND Agents..................................................................................................16 
2.3 
Rationale for combining anti-PD1 antibody with entinostat in MDS after 
DNMTi failure .......................................................................................................21 
2.4 
Correlative Studies Background (full correlative proposal attached in 
Appendix J) ............................................................................................................21 
3. 
PATIENT SELECTION ....................................................................................................23 
3.1 
Eligibility Criteria ..................................................................................................23 
3.2 
Exclusion Criteria ..................................................................................................24 
3.3 
Inclusion of Women and Minorities ......................................................................25 
4. 
REGISTRATION PROCEDURES (Rostered Protocol Model)........................................25 
4.1 
Investigator and Research Associate Registration with CTEP ..............................25 
4.2 
Site Registration .....................................................................................................26 
4.3 
Patient Registration ................................................................................................28 
4.4 
General Guidelines.................................................................................................29 
5. 
TREATMENT PLAN ........................................................................................................29 
5.1 
Agent Administration.............................................................................................29 
5.2 
Definition of Dose-Limiting Toxicity ....................................................................34 
5.3 
Dose Expansion Cohorts ........................................................................................36 
5.4 
General Concomitant Medication and Supportive Care Guidelines ......................36 
5.5 
Duration of Therapy ...............................................................................................38 
5.6 
Duration of Follow Up ...........................................................................................40 
5.7 
Criteria for Removal from Study ...........................................................................41 
5.8 
Criteria to Resume Treatment ................................................................................41 
5.9 
Treatment Beyond Progression ..............................................................................41 
5.10 
Discontinuation of Treatment Following Complete Response ..............................42 
5.11 
Treatment Up to 1 Years ........................................................................................43 
6. 
DOSING DELAYS/DOSE MODIFICATIONS................................................................43 
6.1 
Entinostat Dose Modifications and Supportive Care Guidelines for Drug- 
Related Adverse Events .........................................................................................43 
6.2 
MK-3475 Dose Modifications and Supportive Care Guidelines for Drug- 


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Related Adverse Events .........................................................................................45 
7. 
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS .............................63 
7.1 
Comprehensive Adverse Events and Potential Risks List(s) (CAEPRs) ...............63 
7.2 
Adverse Event Characteristics ...............................................................................71 
7.3 
Expedited Adverse Event Reporting ......................................................................71 
7.4 
Routine Adverse Event Reporting .........................................................................74 
7.5 
Secondary Malignancy ...........................................................................................74 
7.6 
Second Malignancy ................................................................................................74 
7.7 
Reporting of Pregnancy and Lactation to the Sponsor ...........................................75 
8. 
PHARMACEUTICAL INFORMATION ..........................................................................75 
8.1 
CTEP IND Agent(s) ...............................................................................................75 
9. 
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES .........................................80 
9.1 
Exploratory/Ancillary Correlative Studies .............................................................80 
9.2 
Special Studies: N/A ..............................................................................................82 
10. 
STUDY CALENDAR........................................................................................................83 
11. 
MEASUREMENT OF EFFECT ........................................................................................85 
11.1 
Definitions..............................................................................................................85 
11.2 
Response criteria ....................................................................................................85 
11.3 
Other response parameters .....................................................................................86 
12. 
STUDY OVERSIGHT AND DATA REPORTING / REGULATORY 
REQUIREMENTS .............................................................................................................86 
12.1 
Study Oversight ......................................................................................................86 
12.2 
Data Reporting .......................................................................................................87 
12.3 
Collaborative Agreements Language .....................................................................89 
13. 
STATISTICAL CONSIDERATIONS ...............................................................................91 
13.1 
Study Design/Endpoints .........................................................................................91 
13.2 
Sample Size/Accrual Rate ......................................................................................93 
13.3 
Stratification Factors ..............................................................................................93 
13.4 
Analysis of Secondary Endpoints ..........................................................................93 
13.5 
Reporting and Exclusions ......................................................................................94 
14. 
Study Status Updates and Study Closure ...........................................................................95 
14.1 
Definitions of Study Status Changes......................................................................95 
14.2 
Responsibility for Filing Protocol Status Update Forms........................................96 
REFERENCES ..............................................................................................................................98 
APPENDIX A1 
Modified International Working Group (IWG)-2006 response criteria for 
altering natural history of MDS .......................................................................................104 
APPENDIX A2 
Modified International Working Group (IWG)-2006 response criteria for 


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hematologic improvement................................................................................................106 
 
APPENDIX A3 
International Working Group Acute Myeloid Leukemia Response Criteria 
107 
 
APPENDIX B ....International Prognostic Scoring System (IPSS) for Myelodysplastic Syndrome 
108 
 
APPENDIX C 
Performance Status by ECOG Grading Criteria.......................................109 
 
APPENDIX D ..................................... Protocol for mononuclear cells isolation (Ficoll Hypaque): 
110 
 
APPENDIX E 
PATIENT DRUG INFORMATION HANDOUT AND WALLET CARD 
111 
 
APPENDIX F 
TRANSPORTER MDR1/PgP TRANSPORTER MDR1/PGP ................114 
 
APPENDIX G 
examples of sensitive in vivo cyp substrates and cyp substrates with 
narrow therapeutic range1 .................................................................................................................................................116 
APPENDIX H 
Gastric acid reducing drugs......................................................................118 
 
APPENDIX I 
BIOASSAY TEMPLATES......................................................................119 
 
APPENDIX J 
Correlative study proposal .......................................................................120 


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1. OBJECTIVES 
 
1.1 
Primary Objectives 
 
To assess safety, tolerability, and identify the maximum tolerated dose (MTD) of 
entinostat given in combination with pembrolizumab (MK-3475). 
 
1.2 
Secondary Objectives 
 
To obtain a preliminary estimate of efficacy of entinostat in combination with 
pembrolizumab (MK-3475). 
 
1.3 
Exploratory objectives 
 
To assess the dynamic quantitative change in measurable immunological biomarkers 
(proportions of myeloid-derived suppressor cells [MDSCs], and programmed death 
protein-1 [PD-1] expression in bone marrow) with the combined epigenetic- 
immunotherapy and correlation with any observed clinical responses. 
 
 
2. BACKGROUND 
 
2.1 
Myelodysplastic syndrome (MDS) 
 
Novel treatment options after azacitidine failure represent a major unmet clinical need in 
MDS 
Myelodysplastic syndromes (MDS) are among the most common hematologic malignancies and 
constitute the most common chronic myeloid malignancy in USA16,17. The age-adjusted incidence 
in USA has been estimated at 3.3-4.6 per 100,000 persons (exceeding that of acute myeloid 
leukemia [AML]) which translates into more 20,000 cases per year16,17 with some estimates putting 
the number at 45,000 new cases in the year 200318. Patients with MDS have widely variable 
prognosis with expected survival that ranges from few months to many years reflecting the 
complex pathophysiology of the disease19. Approximately one third of MDS patients have higher- 
risk (HR)-disease, typically defined with International Prognostic Scoring System (IPSS) of 
intermediate-2 or high risk groups [Appendix B], and have an expected median survival of less 
than one year if treated only with supportive measures20. The only curative treatment modality for 
HR-MDS is allogeneic bone marrow transplantation (allo-SCT), but due to the typically advanced 
age and multiple comorbidities that characterize these patients, less than 5% of patients undergo 
allo-SCT21. Aside of allo-SCT, the DNA methyltransferase inhibitor (DNMTi) azacitidine is the 
only treatment proven to prolong survival in HR-MDS1. Nonetheless, the survival advantage with 
azacitidine has a median duration of only 9.5 months (24.5 vs 15 months in median overall survival 
[OS])1. Additionally, only 50% of patients receiving azacitidine exhibit an objective response 
(complete response rate [CR] only 17%) with most patients eventually relapsing within 2 years1. 
 
Failure of DNMTi therapy is associated with dismal prognosis with a survival of less than 6 
months2. To date and despite extensive research, no drug or combination of drugs have improved 


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survival of MDS patients in the post-DNMTi failure setting in randomized trials, and there is 
currently no standard of care. Therefore, improving outcomes of MDS patients with failure of 
DNMTi remains a top clinical priority 3. For those patients with DNMTi failure who are not 
candidates or do not want to undergo allo-SCT, clinical trials or best supportive therapy are 
considered reasonable treatment approaches as intensive chemotherapy is associated with 
significant toxicity and low response rates, and is usually only used in a minority of patients as a 
bridge to allo-SCT for eligible patients who are willing to undergo the procedure and have a graft 
source. As the mechanisms of primary and secondary resistance to DNMTi therapy are poorly 
understood, it has been difficult to rationally design targeted therapies that are effective to salvage 
patients after DNMTi therapy failure occurs. 
 
Histone deacetylase inhibitors (HDACi) demonstrated modest activity in MDS 
Histone deacetylase inhibitors (HDACi) comprise a group of drugs that share the ability to inhibit 
the important epigenetic eraser enzymes histone deacetylases (HDAC) but vary significantly in 
terms of their structure, target specificity, and pharmacokinetic profiles. There are 18 different 
HDACs in human that belong to 4 different classes, and the HDACi vary significantly in their class 
and enzyme specificity. In addition, HDACi have different pharmacokinetic profiles with 
significant difference in their half-lives, administration modes and metabolism. As single agents, 
multiple HDACi have been well tolerated in MDS but exhibited only a modest clinical activity 4,5. 
Therefore, interest to develop these agents further has focused on combination-based strategies, 
usually with HMAs. To date, no HDACi-HMA combination has improved survival of MDS 
patients in randomized studies when used in the frontline or post-HMA failure setting. Several 
studies of HDACi are ongoing in the post-HMA failure setting, usually by adding the HDACi to 
azacitidine with the primary goal of achieving (in case of primary resistance) or re-acquiring (in 
case of secondary resistance) a clinical response. 
 
One of the HDACs studied in MDS in entinostat: an orally bioavailable, isotype-selective class 1- 
specific HDACi with long half-life (100 hours). A phase I clinical trial published in abstract form 
at Johns Hopkins University combined entinostat with a lower dose, prolonged administration 
schedules of azacitidine22. In this trial, patients were treated with SQ azacitidine for 10 days at 
different dose levels (30, 40, or 50mg/m2/day) and given oral entinostat (2, 4, 6, 8 mg/m2/dose) on 
days 3 and 10. Out of 31 enrolled patients, 13 had MDS, 4 had chronic myelomonocytic leukemia 
(CMML), and 14 had acute myeloid leukemia (AML). Patients were evaluable for response if they 
received 4 or more cycles of treatment. Of the 27 evaluable patients, 12 had responses (2 CR, 4 
had partial responses [PR], and 6 bilineage hematologic improvement [HI]) according to the 
International Working Group [IWG] 2000 criteria. The 50mg/m2/day dose of azacitidine D1-10 
and the entinostat 4 mg/m2/dose on D3 and D10 were subsequently studied in the randomized 
phase 2 ECOG1905 study versus azacitidine monotherapy at the same 50mg/m2/day dose D1- 
D1023. Both arms achieved rates of trilineage hematologic normalization (32% for azacitidine 
monotherapy and 27% for combination) that were exceeded that of the historical control in which 
the standard regimen of azacitidine at 75mg/m2/day D1-7 was used (16% in CALBG9221 trial). 
While there was no survival advantage with the combination compared to azacitidine, this could 
be potentially related to pharmacologic antagonism caused by early administration of entinostat, a 
potent cell-cycle inhibitor with a long half-life of 4.5 days, in each cycle (day 3) which could have 
inhibited incorporation of azacitidine into DNA and subsequent cell cycling23. These findings 
suggest that while entinostat has a modest activity in MDS, HMAs might not be the best choice 


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for combination-based regimen with entinostat. Exploration of alternative agents to combine with 
entinostat in the post-DNMTi failure setting might be a more rational approach. 
 
Rationale for use of immune-check point inhibitors in MDS after DNMTi failure 
Programmed death receptor-1 (PD-1) is a molecule expressed on antigen-stimulated T-cells and 
induces downstream signaling that cause inhibition of T-cell proliferation, cytokine release, and 
cytotoxicity24. Recent data showed that multiple types of tumor are capable of evading the immune 
system by expressing PD-1 ligands (e.g. PD-L1) which engages the PD-1 receptor on the surface 
of cytotoxic T-cell leading to suppression of their activity6. Immune checkpoint blockade with 
anti-PD-1 and anti-PDL-1 antibodies (and anti-cytotoxic T-Lymphocyte-associated antigen 4 
[CTLA-4] antibodies) has emerged as a novel promising approach to reverse this phenomenon and 
has resulted in a significant and durable clinical activity in some patients with advanced solid 
malignancies especially metastatic melanoma9. Pembrolizumab (MK-3475, KEYTRUDA®) is a 
humanized monoclonal IgG4-kappa antibody that inhibits PD-1 with highly selectivity which has 
recently been granted an accelerated approval in 2014 by the FDA for treatment of refractory 
metastatic 
melanoma 
(Keytruda 
product 
information. 
http://www.merck.com/product/usa/pi_circulars/k/ keytruda/keytrudapi. pdf.). In a large open- 
label, multicenter phase 1b trial, 173 adult patients with refractory metastatic melanoma were 
randomized to IV pembrolizumab at 2 or 10 mg/kg every 3 weeks9. With a median follow-up of 8 
months, the ORR was 21-26% at both doses. Survival data has not yet been reported. Most 
common drug-related adverse events of any grade in the 2 and 10 mg/kg doses were fatigue (33% 
vs 37%), pruritus (26% vs 19%), and rash (18% vs 18%). Grade 3 fatigue (in 3%) was the only 
drug-related grade 3 to 4 adverse event reported in more than one patient9, suggesting that the drug 
is well tolerated and is potentially associated with less severe side effects than the CTLA-4 
inhibitor ipilimumab. Advanced non-small cell lung cancer showed 19.4% of ORR and 12.5 
months of median duration of response25. Metastatic or refractory squamous cell carcinoma of the 
head and neck showed 18% of ORR to pembrolizumab in a phase Ib trial26. Pembrolizumab is now 
approved for advanced melanoma, non-small cell lung cancer, and advanced head and neck cancer. 
 
Several lines of evidence suggest that the immune system dysregulation is important in the 
pathogenesis and progression of MDS and that targeting key immune effector and regulatory steps 
can be a novel therapeutic approach for disease control in MDS27. We are in fact evaluating the 
use of the CTLA-4 inhibitor ipilimumab in a multi-center CTEP-sponsored phase 1 study in 
DNMTi-refractory MDS and there are other active phase 1 studies of anti-PD1 and anti-PD-L1 
antibodies in MDS. 
 
Data suggests that during disease progression in MDS the clonal blasts become more aggressive 
while the non-clonal immune cells become less efficient via unknown mechanisms12. In a Chinese 
study of 38 MDS patients, the percentages of CTLA-4, PD-1 and CD25 were significantly higher 
in patients with MDS compared to normal and the differences of CTLA-4, PD-1 and the ratio of 
CTLA-4/CD28 between patients with HR-MDS and lower-risk (LR)-MDS were more significant 
with progression of MDS further suggesting involvement of the PD-1 axis in MDS progression28. 
Using MDS cell lines and patient samples, Kondo and colleagues12 showed that the expression of 
an immune-inhibitory molecule PD-L1 (B7-H1, CD274) was induced by interferon-ܵ (IFN-ܵ and 
tumor necrosis factor-Į 71)-Į RQ 0'6 EODVWV DQG WKDW WKHVH EODVWV H[KLELWHG PRUH SRWHQW
intrinsic proliferative capacity than PD-L1-negative MDS blasts. Additionally, these PD-L1- 


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positive blasts suppressed T-cell proliferation and induced T-cell apoptosis in allogeneic co- 
cultures. In BM samples, blasts from HR-MDS patients expressed PD-L1 molecules more often 
compared with those from LR-MDS patients while the BM T-cells over-expressed PD-1 
molecules12. These findings suggest that PD-L1-positive MDS blasts have an intrinsic proliferative 
advantage and induce T-cell suppression through interactions with PD-1 receptors leading 
potentially to disease progression and suggesting a role for therapeutic intervention12. The MD 
Anderson group studied mRNA in CD34+ cells from 124 patients with MDS, AML and chronic 
myelomonocytic leukemia (CMML) and found that 34%, 15%, and 8% had aberrant up- regulation 
(စ2-fold) in PD-L1, PD-1 and CTLA4, respectively13. PD-L1 protein expression was observed in 
MDS CD34+ cells, whereas stromal and non-blast cellular elements were positive for PD-1. More 
importantly, in subgroup of patients treated with DNMTi therapy, PD-L1, PD- 1 and CTLA4 
expression was increased with the patients who exhibited resistance to DNMTi therapy showing 
higher relative up-regulation in gene expression than responding patients. Furthermore, treatment 
of leukemia cells with decitabine resulted in a dose-dependent up- regulation of these genes and 
partial hypomethylation of PD-1 in leukemia cell lines and human samples13. In addition to 
providing further evidence to the role of PD-1/PD-L1 axis in MDS progression, these results 
suggest this axis might mediate resistance to DNMTi therapy and provide rationale for combining 
epigenetic therapy with immune checkpoint blockade using an anti-PD1 antibody as a novel 
therapeutic approach to improve outcomes of HR-MDS patients. 
 
2.2 
CTEP IND Agents 
 
 
2.2.1 Pembrolizumab (MK-3475) 
 
MK-3475 (SCH 900475, pembrolizumab) is a humanized immunoglobulin (Ig) G4 
monoclonal antibody (mAb) which binds the programmed death 1 (PD-1) receptor, thus 
inhibiting the interaction with its ligands, PD-L1 or PD-L2 (Investigator’s Brochure, 
2014). PD-1 is an immune-checkpoint receptor expressed by T cells. When bound to 
either PD-L1 or PD-L2, the PD-1 pathway negatively regulates T-cell effector functions. 
The PD-1 pathway functions to limit unwanted or excessive immune responses, including 
autoimmune reactions. PD-L1 is typically expressed at low levels on various non- 
hematopoietic tissues, and PD-L2 is only detectably expressed on antigen-presenting 
cells in the lymphoid tissue or chronic inflammatory environments. 
 
PD-L1 is also expressed in the tumor microenvironment of various cancers29. Activation 
of the PD-1 pathway may be a critical mechanism to evade T-cell mediated tumor 
rejection30,31. High levels of PD-L1 expression are correlated with poor prognosis and 
survival in renal cell carcinoma (RCC)32, pancreatic carcinoma33, hepatocellular 
carcinoma (HCC)34, and ovarian carcinoma35. 
 
Immune-checkpoint inhibition of another inhibitory T-cell receptor, cytotoxic T- 
lymphocyte-associated antigen 4 (CTLA-4), with the mAb ipilimumab demonstrated 
significant prolongation of overall survival (OS) in patients with melanoma in two phase 
3 trials 36-38. As an immunotherapy target, PD-1 is distinct from CTLA-4 because it can 
be activated directly by the cancer and it regulates the effector phase of T-cell response, 


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whereas CTLA-4 regulates the initial stage of T-cell activation6,39. Antibodies targeting 
the PD-1 pathway have demonstrated durable objective responses in phase 1 and 2 trials. 
Nivolumab showed an overall response rate (ORR) of approximately 28% in subjects 
with advanced melanoma, 27% in subjects with RCC, and 18% in subjects with non- 
small cell lung cancer (NSCLC) who had failed prior therapy40. Pembrolizumab (MK-
3475) has shown an ORR of approximately 38% in patients with melanoma41 and ~20% 
in patients with NSCLC (Investigator’s Brochure, 2014). 
 
2.1.2.1 Clinical Development of pembrolizumab (MK-3475) 
 
Pharmacokinetics 
The half-life (t1/2) of pembrolizumab (MK-3475) is approximately 4 weeks and there 
is no indication of dose dependency of half-life in the three dose groups (1, 3, and 10 
mg/kg) (Investigator’s Brochure, 2014). The long t1/2 supports a dosing interval of 
every 2 or 3 weeks. 
 
There was a dose-related increase in exposure from 1 to 10 mg/kg (Investigator’s 
Brochure, 2014). Serum concentrations of MK-3475 were lower by a factor of 
approximately 5 in patients receiving 2 mg/kg Q3W than in those receiving 10 mg/kg 
Q3W 41 (Investigator’s Brochure, 2014). Steady-state trough concentrations were 
20% greater in the patients receiving 10 mg/kg Q2W than in those receiving the same 
dose Q3W. 
 
Anti-Drug Antibodies (ADA) Data 
The occurrence of ADA has been observed in less than 1% of the patients screened, 
indicating a low potential of pembrolizumab (MK-3475) to elicit the formation of 
ADA (Investigator’s Brochure, 2014). No impact of ADA on MK-3475 exposure has 
been observed. 
 
Efficacy 
When treated with pembrolizumab (MK-3475) monotherapy, the ORR for IPI-
treated patients with melanoma (Part B) was 25%/27% according to the Response 
Evaluation Criteria in Solid Tumors (RECIST)/investigator-assessed immune-related 
response criteria (irRC), respectively (Investigator’s Brochure, 2014). The ORR for 
IPI-naïve patients with melanoma (Parts B and D) was 39%/43% by 
RECIST/investigator-assessed irRC, respectively. The majority of responses were 
seen in patients with melanoma by 16 weeks of therapy with pembrolizumab (MK-
3475); however, some responses have been reported after 24 weeks or more of 
therapy with MK-3475. Responses can be delayed, and in some patients, a RECIST-
defined progression followed by a response has been observed. 
 
The preliminary objective response rate for 38 patients with NSCLC (Part C) was 
21%/24% by RECIST/investigator-assessed irRC, respectively (Investigator’s 
Brochure, 2014). 
 
Pharmacodynamics/Biomarkers 
PD-L1 is being investigated as a predictive biomarker for pembrolizumab (MK-3475) 
treatment. At the 


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15th World Conference on Lung Cancer, Garon et al. presented preliminary data on a 
subset of patients suggesting that higher levels of tumor PD-L1 expression are 
associated with increased clinical activity. Preliminary clinical safety and activity of MK- 
3475 monotherapy for the treatment of previously treated patients with non-small cell lung 
cancer (NSCLC). J Thor Oncol. 8 Suppl 2:S364-365). Objective responses by RECIST 
1.1 occurred in 4 out of 7 patients with higher levels of PD-L1 expression (57%, 95% 
confidence interval [CI] 18-90%) vs. 2 out of 22 patients with lower levels of PD-L1 
expression (9%, 95% CI 1-29%).  These data are extremely preliminary, and PD-L1 
is not being used for patient selection. 
 
Biomarkers to evaluate immune modulation and markers in the tumor 
microenvironment, such as T-cell infiltration, the baseline expression of markers of 
T-cell suppression FoxP3 or the immunoregulatory enzyme indoleamine 2,3- 
dioxygenase (IDO) in tumor biopsies, were associated with a high response rate42. 
 
2.1.2.2 Safety data 
 
The most frequent treatment-related adverse events (AEs) were fatigue, nausea, 
cough, pruritus, diarrhea, and rash (Investigator’s Brochure, 2014). Most AEs were 
not considered serious. The most commonly reported immune-related AEs were rash, 
pruritus, vitiligo, hypothyroidism, arthralgia, diarrhea, and pneumonitis. 
 
Important identified risks include: pneumonitis, thyroid disorders (hypothyroidism 
and hyperthyroidism), colitis, diarrhea, hepatitis, nephritis, uveitis, rash/pruritus and 
neuropathy. 
 
2.2.2 Entinostat (NSC 706995) 
 
Entinostat, SNDX-275, an orally available synthetic pyridylcarbamate licensed from 
Bayer Schering AG by Syndax Pharmaceuticals and previously named MS-275, inhibits 
HDACs. SNDX-275 promotes hyperacetylation of nucleosomal histones, allowing 
transcriptional activation of a distinct set of genes. This ultimately leads to the inhibition 
of cell proliferation, induction of terminal differentiation, and/or apoptosis43. DNA within 
the cell nucleus combines with a class of proteins called histones to form chromatin. 
Histones have amino terminal groups that are positively charged and are hypo-acetylated 
by HDACs. The positive charge tightly binds the histones to the negatively charged DNA 
phosphodiester backbone. Gene transcription and expression are inhibited by such a 
condensed conformation of the DNA. Histone acetyltransferases acetylate the amino 
terminal ends and neutralize their positive charges, thus leading to a more open chromatin 
conformation, facilitating DNA transcription. Altered activity of HDACs and inactivation 
of histone acetyltransferases within transformed cells are key events that affect chromatin 
remodeling. There is evidence that HDACs are associated with a wide range of tumors 
including melanomas, neuroblastomas, lymphomas, and lung, breast, prostate, ovarian, 
bladder, and colon cancers. In a number of in vitro models, HDAC inhibitors triggered 
growth arrest and induced cell differentiation or apoptosis. In acute promyelocytic 
leukemia, recruitment of HDACs by aberrant fusion proteins repressed constitutive gene 
transcription and thus prevented promyelocytic differentiation. 


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Entinostat inhibited HDAC in various tumor cell lines. In particular, entinostat induced 
accumulation of acetylated histones adjacent to the promoter of the transforming growth 
factor (TGF)-ȕW\SH,,UHFHSWRUJHQHZLWKUHVXOWLQJJHQHH[SUHVVLRQ0XWDWLRQV affecting 
the TGF-ȕVLJQDOLQJSDWKZD\KDYHEHHQDVVRFLDWHGZLWKGHYHORSPHQWDQGSURJUHVVLRQRI
human malignancies, including carcinomas of the lung, breast, prostate, and colon. 
Entinostat also induced histone hyperacetylation and induced expression of various tumor 
suppressor genes. 
Various in vitro studies in a range of human cancer cell lines have demonstrated the 
antiproliferative activity of entinostat. In vivo, entinostat inhibited the growth of a range 
of human tumor xenografts, including models of lung, prostate, breast, pancreatic, renal 
cell, and glioblastoma. More recently, entinostat has been shown to modify the phenotype 
of cancer cells from a mesenchymal to an epithelial one, with impact on reducing the 
metastatic potential of the cancer cells44. In addition, there is a suggestion that entinostat 
may have longer term effects on cancer phenotypes, cancer stem cells (CSCs) or 
progenitor cell pool and potential sensitization to subsequent post-study treatments45. 
 
2.2.1.1 Entinostat in Patients with Solid Tumors, including Lung Cancer and 
Melanoma 
To date, entinostat has been investigated alone or in combination in >900 patients 
with cancer in clinical studies, including >600 patients with solid tumors, including 
NSCLC and melanoma. Entinostat has been investigated specifically in patients with 
NSCLC in combination with erlotinib in clinical studies sponsored by Syndax and in 
combination with 5-azacitadine (AZA) in clinical studies sponsored by the NCI. Of 
the studies of entinostat in combination with AZA, evidence of the anti-tumor activity 
of this combination was demonstrated in an initial study45, in which adults with 
metastatic NSCLC who experienced disease progression after at least 1 anticancer 
regimen received 7 mg of entinostat by mouth (PO) on Days 3 and 10 and 30 or 40 
mg/m2/day of AZA subcutaneously (SC) on Days 1-6 and 8-10 of a 28-day cycle. 
Of 31 evaluable patients, 1 experienced a CR, with a duration of 14 months; a second 
patient experienced a PR, with a duration of 8 months; and 10 patients experienced 
stable disease for at least 12 weeks. Furthermore, 4 patients had major objective 
responses to the immediate subsequent therapy. Based on these encouraging findings, 
the NCI has initiated a follow-up study utilizing this combination regimen to 
investigate the premise that epigenetic therapy can augment the clinical utility of 
cytotoxic therapy in patients with advanced disease. 
Evidence of the efficacy of entinostat also was seen in patients with Stage IV 
nonresectable melanoma, with 25% of patients experiencing disease stabilization46. 
Based on these encouraging findings, further investigation of entinostat in 
combination was considered warranted in patients with melanoma. 
Overall, among all patients treated, entinostat has been well tolerated at the doses and 
schedules investigated. Regardless of indication and regimen, the most frequently 
reported AEs with entinostat included gastrointestinal (GI) disturbances, primarily 
nausea with or without vomiting and diarrhea; fatigue; and hematologic 
abnormalities, primarily anemia, thrombocytopenia, neutropenia, and leukopenia. 
Most occurrences of these events are Grade 1 or 2 in severity and non-serious. Grade 
3 and 4 hematologic abnormalities are commonly seen in patients with hematologic 


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malignancies, but are much less prevalent in patients with solid tumors. 
As would be expected, the AE profile of entinostat when given in combination varies 
somewhat based on the agent with which it is given and the corresponding patient 
population. Entinostat in combination with AZA was generally associated with an 
increased number and rate of AEs relative to its use in combination with an aromatase 
inhibitor, erlotinib, or other agents. Consistent with the overall AE profile of 
entinostat, nausea with or without vomiting, fatigue, and anemia were the most 
prevalent AEs regardless of the patient population or the agent given in combination. 
 
2.2.1.2 Entinostat in Patients with MDS and AML 
One of the HDACs studied in MDS in entinostat: an orally bioavailable, isotype- 
selective class 1-specific HDACi with long half-life (100 hours). A phase I clinical 
trial published in abstract form at Johns Hopkins University combined entinostat with 
a lower dose, prolonged administration schedules of azacitidine22. In this trial, 
patients were treated with SQ azacitidine for 10 days at different dose levels (30, 40, 
or 50mg/m2/day) and given oral entinostat (2, 4, 6, 8 mg/m2/dose) on days 3 and 10. 
Out of 31 enrolled patients, 13 had MDS, 4 had chronic myelomonocytic leukemia 
(CMML), and 14 had acute myeloid leukemia (AML). Patients were evaluable for 
response if they received 4 or more cycles of treatment. Of the 27 evaluable patients, 
12 had responses (2 CR, 4 had partial responses [PR], and 6 bilineage hematologic 
improvement [HI]) according to the International Working Group [IWG] 2000 
criteria. The 50mg/m2/day dose of azacitidine D1-10 and the entinostat 4 mg/m2/dose 
on D3 and D10 were subsequently studied in the randomized phase 2 ECOG1905 
study versus azacitidine monotherapy at the same 50mg/m2/day dose D1-D1023. Both 
arms achieved rates of trilineage hematologic normalization (32% for azacitidine 
monotherapy and 27% for combination) that were exceeded that of the historical 
control in which the standard regimen of azacitidine at 75mg/m2/day D1-7 was used 
(16% in CALBG9221 trial). While there was no survival advantage with the 
combination compared to azacitidine, this could potentially have been related to 
pharmacologic antagonism caused by early administration of entinostat, a potent cell- 
cycle inhibitor with a long half-life of 4.5 days, in each cycle (day 3) which could 
have inhibited incorporation of azacitidine into DNA and subsequent cell cycling23. 
2.2.1.3. Clinical Development of Entinostat 
Potential Drug Interactions: Metabolism: Data from in vitro metabolism experiments 
in human tissues demonstrated that entinostat is not metabolized by CYP enzymes47, 
but UGT 1A4 did metabolize entinostat to its M2 glucuronide metabolite. No 
metabolites could be detected after incubation of entinostat in human liver 
microsomes47. While inhibition of CYP enzymes 2B6 and 3A4 was seen, the data 
show that the degree of the inhibition makes it unlikely that any in vivo systemic 
interactions would occur. Intestinal CYP 3A4 may be inhibited by entinostat. 
However, entinostat did not inhibit any UGT enzymes tested. Entinostat was found to 
induce CYP 1A2, CYP 2C6, and CYP 2B8 as well as UGT 1A4. Finally, entinostat 
was found to be a substrate for P-gp and BCRP transporters, but did not inhibit either 
of these transport proteins. 


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Patient Care Implications: 
Entinostat may cause fatigue or malaise; advise patient to exercise caution while 
driving a vehicle or operating machinery. 
 
Administration of entinostat is contraindicated in patients with a history of allergy 
to entinostat or other medications that have a benzamide structure (eg, tiapride, 
remoxipride, clebropride). 
Careful monitoring of patients for signs of infection or reactivation of past 
infections is recommended, as reactivation of infection has been reported in 
patients treated with entinostat, in some cases without evidence of neutropenia. 
The clinical significance of this finding and the potential association with 
entinostat is unknown. 
 
Entinostat must not be used during pregnancy or while breast-feeding. Women 
and men participating in entinostat clinical studies must agree to use acceptable 
contraceptive methods, as indicated in the clinical study protocol, during 
treatment and for 4 months thereafter. 
 
2.3 
Rationale for combining anti-PD1 antibody with entinostat in MDS after DNMTi 
failure 
More recently, myeloid-derived suppressor cells (MDSCs) were found to be significantly 
expanded in Bone marrows of patients with MDS and to play an important role in the pathogenesis 
of the disease especially in driving ineffective hematopoiesis14. In another very provoking study 
by Kim and colleagues, the authors demonstrated in murine models with metastatic tumors that 
MDSCs can mediate resistance to immune checkpoint blockade therapy. In this study, treatment 
with anti-PD-1 and anti-CTLA-4 inhibitor antibodies could not eliminate CT26 tumors or 
metastatic 4T1 tumors 15. Interestingly, when the animals were co-treated with epigenetic 
modifiers in addition to the antibodies, the outcomes improved significantly with cure rates in 
excess of 80% of the tumor-bearing mice. Specifically, the use of anti-PD1 + anti-CTLA-4 with 
azacitidine plus entinostat or the combination of the antibodies with entinostat alone eradicated the 
tumors while when the antibody combination was used with azacitidine monotherapy the primary 
tumors were not eradicated. Azacitidine and entinostat (alone or together) in absence of the 
immune-checkpoint inhibitors also failed to eradicate the tumors. These findings suggested that 
the combination of the class 1 HDACi entinostat with immune-checkpoint inhibition was sufficient 
to eradicate the primary tumors and their metastasis. Elegant functional studies showed that 
epigenetic modulators exerted their effect primarily by down-regulating MDSCs suggesting that 
cancers which are resistant to immune checkpoint blockade can be potentially controlled or cured 
by targeting MDSCs. 
 
2.4 
Correlative Studies Background (full correlative proposal attached in Appendix J) 
 
The role of MDSCs in MDS and the preclinical data of HDACi to suppress MDSCs: 
Myelopoiesis is altered in cancer, which induces expansion of relatively immature and activated 
myeloid cells, now called MDSCs48. MDSCs have been reported to facilitate tumor metastasis 
and angiogenesis49-51. Recently, MDSCs were found to be significantly expanded in bone 
marrows of patients with MDS and to play an important role in the pathogenesis of the disease 


24 
NCI Protocol #:10009  
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especially in driving ineffective hematopoiesis14. The role of MDSCs in pathogenesis of MDS 
and the strategy to target MDSCs remain under investigation. Kim and colleagues recently 
demonstrated that MDSCs can mediate resistance to immune checkpoint blockade therapy in a 
murine tumor model15. In this study, treatment with anti-PD-1 and anti-CTLA-4 inhibitor 
antibodies could not completely eliminate murine colon cancers or metastatic murine breast 
cancers15. Interestingly, when the animals were co-treated with epigenetic modifiers (i.e. 
ENT+AZA or ENT alone) in addition to the antibodies, the outcomes improved significantly 
with cure rates in excess of 80% of the tumor-bearing mice15. Importantly, AZA and ENT (alone 
or together) in absence of the immune-checkpoint inhibitors also failed to eradicate the tumors. 
These findings suggested that the combination of the class 1 HDACi ENT with immune- 
checkpoint inhibition was sufficient to eradicate the primary tumors and their metastasis. 
Mechanistic studies showed that epigenetic modulators exerted their effect primarily by down- 
regulating MDSCs, suggesting that cancers which are resistant to immune checkpoint blockade 
can be potentially cured by targeting MDSCs. Another study showed that epigenetic 
modification by HDAC-2 regulates the differentiation of MDSCs in a murine cancer model and 
HDACi induces differentiation of MDSCs to normal macrophages and dendritic cells rather 
thanexpanding MDSCs52. Based on the significant role of MDSCs in pathogenesis and 
progression of MDS, the potentially important role of MDSCs in mediating resistance to immune 
checkpoint blockade therapy, and the preclinical data of the efficacy of the HDACi ENT in 
suppressing MDSCs, we will assess the quantitative and qualitative change of MDSCs in BM of 
MDS patients pre- and post- ENT/anti-PD1 therapy to achieve our primary goal of this study via 
Specific Aim 1. 
 
PD-L1 expression in MDS and its correlation with the response to anti-PD1 therapy: Kondo 
and et al. demonstrated that PD-L1-positive MDS blasts have an intrinsic proliferative advantage 
and induce T-cell suppression through interactions with PD-1 receptors potentially contributing 
to disease progression12. They also showed in BM samples, blasts from HR-MDS patients 
expressed PD-L1 molecules more often compared with those from LR-MDS patients while the 
BM T-cells over-expressed PD-1 molecules12. The MD Anderson group studied mRNA in 
CD34+ cells from 124 patients with MDS, AML and chronic myelomonocytic leukemia 
(CMML) and found that 34%, 15%, and 8% had aberrant up-regulation (စ2-fold) in PD-L1, PD- 
1 and CTLA4, respectively13. PD-L1 protein expression was observed in MDS CD34+ cells, 
whereas stromal and non-blast cellular elements were positive for PD-1. More importantly, in a 
subgroup of patients treated with DNMTi therapy, PD-L1, PD-1 and CTLA4 expression was 
increased. Furthermore, the patients who exhibited resistance to DNMTi therapy showed higher 
relative up-regulation in gene expression than responding patients. Treatment of leukemia cells 
with decitabine resulted in a dose-dependent up-regulation of these genes and partial 
hypomethylation of PD-1 in leukemia cell lines and human samples13. These results suggest the 
PD-1/PD-L1 axis might mediate resistance to DNMTi therapy and provide rationale for using 
immune checkpoint blockade with an anti-PD1 antibody as a novel therapeutic approach to 
improve outcomes of HR-MDS patients. However, preliminary results from ongoing early phase 
clinical trials of ant-CTLA-4 and anti-PD1/PD-L1 agents in MDS patients suggest that immune 
checkpoint therapy as a monotherapy only leads to disease stabilization in some patients but very 
few responses. This observation provides rationale for combining immune checkpoint therapy 
with other novel agents with distinct mechanisms of action to improve patient outcomes. 
Additionally, there is no single biomarker to predict the response to anti-PD1 therapy in cancers. 


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Previous data indicate that the response rate of anti-PD-1 therapy is higher in PD-L1 expressing 
tumors40. On the other hand, recent two landmark studies suggest that the predicted response of 
anti-PD-L1 is associated with PD-L1 expression in tumor-infiltrating immune cells8,10. These 
studies have led us to test the question about the expression of PD-L1 in CD34+ blasts from 
human MDS BM and its correlation with the response to combined anti-PD1/ENT therapy in 
MDS in specific aim 2. 
 
 
3. PATIENT SELECTION 
 
3.1 
Eligibility Criteria 
 
3.1.1 Pathologically confirmed MDS diagnosis (regardless of initial IPSS risk 
category) or oligoblastic AML with 21-30% BM blasts in whom DNMTi have 
failed. Patients who have developed AML after DNMTi therapy can be enrolled 
as long as they have initiated DNMTi therapy while they were in the MDS or 
oligoblastic AML (20-30% BM blasts) phase and the study chair agrees. Failure 
of DNMTis is defined as: Failure to achieve a CR, PR or HI after at least 4 cycles 
of DNMTi or progressed after such therapy. 
 
3.1.2 
$JH years 
 
 Because no dosing or adverse event data are currently available on the use of 
entinostat in combination with pembrolizumab (MK-3475) in patients <18 years 
of age, children are excluded from this study, but will be eligible for future 
pediatric trials. 
 
3.1.3 
(&2*SHUIRUPDQFHVWDWXVAppendix C). 
 
3.1.4 
Patients must have laboratory test results within these ranges: 
x Calculated creatinine clearance by MDRD (CrCl) =>60 ml/min/1.73 squared meter 
x 7RWDOELOLUXELQPJG/XQOHVVGXHWR*LOEHUW¶VV\QGURPH
hemolysis, or ineffective hematopoiesis and AST (SGOT) and 
$/76*37[ ULN 
 
3.1.5 Females of childbearing potential must have a negative serum or urine 
pregnancy test within 72 hours prior to start of first cycle of therapy. 
• 
Patients must have no clinical evidence of CNS or pulmonary 
leukostasis, disseminated intravascular coagulation, or CNS 
leukemia. 
• 
Patients must have no serious or uncontrolled medical conditions 


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3.1.6 
The effects of entinostat and pembrolizumab (MK-3475) on the developing 
human fetus are unknown. For this reason, women of child-bearing potential and 
men who are sexually active with women of childbearing potential must agree to 
use adequate contraception (hormonal or barrier method of birth control; 
abstinence) prior to study entry and for the duration of study participation. 
Should a woman become pregnant or suspect she is pregnant while she or her 
partner is participating in this study, she should inform her treating physician 
immediately. Men who are sexually active with women of childbearing 
potential, treated or enrolled on this protocol must also agree to use adequate 
contraception prior to the study, for the duration of study participation, and 4 
months after completion of entinostat and MK3475 (pembrolizumab) 
administration. 
 
3.1.7 Ability to understand and the willingness to sign a written informed 
consent document.   
 
3.1.8 Patients, who relapsed 6 months after bone marrow transplant and 
have no evidence of active graft versus host disease and are off 
systemic immunosuppressant medications for at least 2 months and 
have received HMA therapy before or after transplant and meet 
other eligibility criteria of progression after at least 4 months of 
DNMTi therapy, are eligible to be enrolled in this clinical trial. 
 
3.1.9 Patients who are Human Immunodeficiency Virus (HIV) positive 
may participate IF they meet the following eligibility requirements:  
x Must be on an effective anti-retroviral therapy with undetectable viral load 
within 6 months are eligible for this trial 
x They must have a CD4 count of greater than 250 cells/mcL. 
x They must not be receiving prophylactic therapy for an opportunistic infection. 
 
 
3.2 
Exclusion Criteria 
 
3.2.1. Any patients eligible for allo-SCT and willing to undergo allo-SCT as determined 
at time of screening for trial. Patients who are ineligible or not interested in undergoing 
allo-SCT will be eligible for the trial. 
3.2.2. Any serious medical condition, uncontrolled intercurrent illness (e.g., active 
infection, symptomatic CHF, unstable angina, cardiac arrhythmias, laboratory 
abnormalities, or psychiatric illness and/or biopsychosocial conditions that may limit 
compliance 
3.2.3. Patients with known active cancers who are on therapy for those cancers at time of 
screening. 
3.2.4. Patients with a known positive test for hepatitis B virus surface antigen (HBV sAg) 
or hepatitis C virus ribonucleic acid (HCV antibody) indicating acute or chronic infection 
might be enrolled if the viral load by PCR is undetectable with/without active treatment. 
3.2.5. Pregnant or breast feeding females (lactating females must agree not to breast feed 
while taking the study drugs) 


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3.2.6. Use of any other experimental drug or therapy within 21 days of baseline - patients 
who have had chemotherapy or radiotherapy within 4 weeks of entering the study or  
                   those who have not recovered from adverse events due to agents administered more than 4 weeks earlier 
3.2.7. Known hypersensitivity to pembrolizumab (MK-3475) or history of allergic 
reactions to compounds of similar chemical or biologic composition to anti-PD1 or PD- 
L1 antibodies or entinostat. 
3.2.8. Prior treatment with any anti-PD-1 blocking therapies or HDACi, or anti-CTLA-4 
antibody, CD137 agonist or other immune activating therapy such as anti-CD 40 antibody 
within the last 3 months of enrollment in the study. 
3.2.9. Any history of active or severe autoimmune disease: Inflammatory bowel disease, 
including ulcerative colitis and Crohn’s Disease, rheumatoid arthritis, systemic 
progressive scleroderma, systemic lupus erythematosis, autoimmune vasculitis (e.g., 
Wegener’s Granulomatosis), CNS or motor neuropathy considered of autoimmune origin 
(e.g. Guillain-Barre Syndrome, myasthenia gravis, multiple sclerosis). Patients with 
hypothyroidism with stable hormone replacement therapy dosing are allowed on study. 
3.2.10. Has a history of (non-infectious) pneumonitis that required steroids or has current 
pneumonitis 
 
3.3 
Inclusion of Women and Minorities 
 
NIH policy requires that women and members of minority groups and their subpopulations be 
included in all NIH-supported biomedical and behavioral research projects involving NIH- 
defined clinical research unless a clear and compelling rationale and justification establishes to 
the satisfaction of the funding Institute & Center (IC) Director that inclusion is inappropriate 
with respect to the health of the subjects or the purpose of the research. Exclusion under other 
circumstances must be designated by the Director, NIH, upon the recommendation of an IC 
Director based on a compelling rationale and justification. Cost is not an acceptable reason for 
exclusion except when the study would duplicate data from other sources. Women of 
childbearing potential should not be routinely excluded from participation in clinical research. 
Please see http://grants.nih.gov/grants/funding/phs398/phs398.pdf. 
 
4. REGISTRATION PROCEDURES (ROSTERED PROTOCOL MODEL) 
 
4.1 
Investigator and Research Associate Registration with CTEP 
 
Food and Drug Administration (FDA) regulations and National Cancer Institute (NCI) policy 
require all individuals contributing to NCI-sponsored trials to register and to renew their 
registration annually. To register, all individuals must obtain a Cancer Therapy Evaluation 
Program (CTEP) Identity and Access Management (IAM) account 
(https://ctepcore.nci.nih.gov/iam). In addition, persons with a registration type of Investigator 
(IVR), Non-Physician Investigator (NPIVR), or Associate Plus (AP) (i.e., clinical site staff 
requiring write access to OPEN or RAVE or acting as a primary site contact) must complete their 
annual registration using CTEP’s web-based Registration and Credential Repository (RCR) 
(https://ctepcore.nci.nih.gov/rcr). Documentation requirements per registration type are outlined 
in the table below. 


28 
NCI Protocol #:10009  
Version Date: 09/04/2020 
 
 
 
Documentation Required 
IVR 
NPIVR AP 
A 
FDA Form 1572 
 
D
 
D
 
 
Financial Disclosure Form 
 
D
 
D
 
D
 
NCI Biosketch (education, training, employment, 
license, and certification) 
 
 
D
 
 
D
 
 
D
 
HSP/GCP training 
 
D
 
D
 
D
 
Agent Shipment Form (if applicable) 
 
D
 
 
 
CV (optional) 
 
D
 
D
 
D
 
An active CTEP-IAM user account and appropriate RCR registration is required to access all 
CTEP and CTSU (Cancer Trials Support Unit) websites and applications. In addition, IVRs and 
NPIVRs must list all clinical practice sites and IRBs covering their practice sites on the FDA 
Form 1572 in RCR to allow the following: 
x Added to a site roster 
x Assigned the treating, credit, consenting, or drug shipment (IVR only) tasks in OPEN 
x Act as the site-protocol PI on the IRB approval. 
Additional information can be found on the CTEP website at 
https://ctep.cancer.gov/investigatorResources/default.htm. For questions, please contact the RCR 
Help Desk by email at < RCRHelpDesk@nih.gov >. 
 
 
4.2 
Site Registration 
 
This study is supported by the NCI Cancer Trials Support Unit (CTSU). 
 
Each investigator or group of investigators at a clinical site must obtain IRB approval for this 
protocol and submit IRB approval and supporting documentation to the CTSU Regulatory Office 
before they can be approved to enroll patients.  Assignment of site registration status in the 
CTSU Regulatory Support System (RSS) uses extensive data to make a determination of whether 
a site has fulfilled all regulatory criteria including but not limited to the following: 
x An active Federal Wide Assurance (FWA) number, 
x An active roster affiliation with the Lead Network or a participating organization, 
x A valid IRB approval 
x Compliance with all protocol specific requirements. 


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In addition, the site-protocol Principal Investigator (PI) must meet the following criteria: 
x Active registration status 
x The IRB number of the site IRB of record listed on their Form FDA 1572 
x An active status on a participating roster at the registering site 
Sites participating on the NCI CIRB initiative that are approved by the CIRB for this study are 
not required to submit IRB approval documentation to the CTSU Regulatory Office. For sites 
using the CIRB, IRB approval information is received from the CIRB and applied to the RSS in 
an automated process. Signatory Institutions must submit a Study Specific Worksheet for Local 
Context (SSW) to the CIRB via IRBManager to indicate their intent to open the study locally. 
The CIRB’s approval of the SSW is then communicated to the CTSU Regulatory Office. In 
order for the SSW approval to be processed, the Signatory Institution must inform the CTSU 
which CIRB-approved institutions aligned with the Signatory Institution are participating in the 
study. 
 
4.2.1 Downloading Regulatory Documents 
 
Site registration forms may be downloaded from the NCI protocol # 10009 protocol 
page located on the CTSU Web site. Permission to view and download this protocol 
is restricted and is based on person and site roster data housed in the CTSU RSS. To 
participate, Investigators and Associates must be associated with the Corresponding 
or Participating protocol organization in the RSS. 
 
x Go to https://www.ctsu.org and log in using your CTEP-IAM username and 
password. 
x Click on the Protocols tab in the upper left of your screen. 
x Either enter the protocol # in the search field at the top of the protocol tree, or 
x Click on the By Lead Organization folder to expand, and then select LAO-CT018 
and protocol #10009 
x Click on LPO Documents, select the Site Registration documents link, and 
download and complete the forms provided. (Note: For sites under the CIRB 
initiative, IRB data will load to RSS as described above.) 
 
4.2.2 Requirements For NCI protocol #10009 Site Registration: 
 
x IRB approval (For sites not participating via the NCI CIRB; local IRB 
documentation, an IRB-signed CTSU IRB Certification Form, Protocol of Human 
Subjects Assurance Identification/IRB Certification/Declaration of Exemption 
Form, or combination is accepted). 
 
4.2.3 Submitting Regulatory Documents 
 
Submit required forms and documents to the CTSU Regulatory Office, where they will 
be entered and tracked in the CTSU RSS. 
Regulatory Submission Portal: www.ctsu.org (members’ area) t Regulatory 


30 
NCI Protocol #:10009  
Version Date: 09/04/2020 
 
 
Tab tRegulatory Submission 
When applicable, original documents should be mailed to: 
CTSU Regulatory Office 
1818 Market Street, Suite 3000 
Philadelphia, PA 19103 
Institutions with patients waiting that are unable to use the Portal should alert the CTSU 
Regulatory Office immediately at 1-866-651-2878 in order to receive further instruction 
and support. 
 
4.2.4 Checking Site Registration Status 
 
You can verify your site registration status on the members’ section of the CTSU 
website. 
x Go to https://www.ctsu.org and log in to the members’ area using your CTEP-IAM 
username and password 
x Click on the Regulatory tab at the top of your screen 
x Click on the Site Registration tab 
x Enter your 5-character CTEP Institution Code and click on Go 
 
Note: The status given only reflects compliance with IRB documentation and institutional 
compliance with protocol-specific requirements as outlined by the Lead Network. It does 
not reflect compliance with protocol requirements for individuals participating on the 
protocol or the enrolling investigator’s status with the NCI or their affiliated networks. 
 
4.3 
Patient Registration 
 
4.3.1 OPEN / IWRS 
 
Patient enrollment will be facilitated using the Oncology Patient Enrollment Network 
(OPEN). OPEN is a web-based registration system available to users on a 24/7 basis. It 
is integrated with the CTSU Enterprise System for regulatory and roster data interchange 
and with the Theradex Interactive Web Response System (IWRS) for retrieval of patient 
registration/randomization assignment. Patient enrollment data entered by Registrars in 
OPEN / IWRS will automatically transfer to the NCI’s clinical data management system, 
Medidata Rave. 
 
The OPEN system will provide the site with a printable confirmation of registration and 
treatment information. Please print this confirmation for your records. 
 
For trials with slot reservation requirements, OPEN will connect to IWRS at enrollment 
initiation to check slot availability. Registration staff should ensure that a slot is 


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available and secured for the patient before completing an enrollment. 
 
4.3.2 OPEN/IWRS User Requirements 
 
OPEN/IWRS users must meet the following requirements: 
x Have a valid CTEP-IAM account (i.e., CTEP username and password). 
x To enroll patients or request slot reservations: Be on an ETCTN Corresponding 
or Participating Organization roster with the role of Registrar. Registrars must 
hold a minimum of an AP registration type. 
x To approve slot reservations or access cohort management: Be identified to 
Theradex as the “Client Admin” for the study. 
x Have regulatory approval for the conduct of the study at their site. 
 
Prior to accessing OPEN/IWRS, site staff should verify the following: 
x All eligibility criteria have been met within the protocol stated timeframes. 
x If applicable, all patients have signed an appropriate consent form and HIPAA 
authorization form. 
 
4.3.3 OPEN/IWRS Questions? 
 
Further instructional information on OPEN is provided on the OPEN tab of the CTSU 
website at https://www.ctsu.org or at https://open.ctsu.org. For any additional questions 
contact the CTSU Help Desk at 1-888-823-5923 or ctsucontact@westat.com. 
 
Theradex has developed a Slot Reservations and Cohort Management User Guide, which 
is available on the Theradex website: 
http://www.theradex.com/clinicalTechnologies/?National-Cancer-Institute-NCI-11. This 
link to the Theradex website is also on the CTSU website OPEN tab. For questions about 
the use of IWRS for slot reservations, contact the Theradex Helpdesk: 609-619-7802 or 
Theradex main number 609-799-7580; CTMSSupport@theradex.com. 
 
4.4 
General Guidelines 
 
Following registration, patients should begin protocol treatment within 28days. Issues that 
would cause treatment delays should be discussed with the Principal Investigator. If a patient 
does not receive protocol therapy following registration, the patient’s registration on the study 
may be canceled. The Study Coordinator should be notified of cancellations as soon as possible. 
 
5. TREATMENT PLAN 
 
5.1 
Agent Administration 
 
Treatment will be administered on an outpatient basis. Reported adverse events and potential 
risks are described in Section 7. Appropriate dose modifications are described in Section 6. 
No investigational or commercial agents or therapies other than those described below may 


32 
NCI Protocol #:10009  
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be administered with the intent to treat the patient's malignancy. 
 
ENT has been previously evaluated as a single agent therapy in patients with MDS at these 
doses and has been well tolerated as in below table: 
 
Clinical trial 
Reference 
Phase 
Treated 
patient 
population 
Doses and schedule used
Safety profile 
Liquid Malignancies 
Gojo et al, 
Blood 2007 
[1] 
I 
38 AML 
patients 
18 primary 
refractory 
AML 
20 
relapsed 
AML 
Entinostat 4 mg/m2 Days 1 
and 8 every 4 weeks 
escalated at increments of 
2 mg/m2 up to 10 mg/m2 
MTD: 8 mg/m2 weekly for 4 weeks every 
6 weeks. 
DLT: infections and neurologic toxicity 
manifesting as unsteady gait and 
somnolence 
Frequent non-DLTs: fatigue, anorexia, 
nausea, vomiting, hypoalbuminemia, and 
hypocalcemia. 
Ryan et al JCO 
2015 
[2] 
I 
28 patients 
with 
advanced 
solid 
tumors and 
lymphoma 
Part 1: Entinostat 2 
mg/m2 daily for 28 days, 
followed by 2 weeks off 
(6-week cycle) 
 
 
Part 2: Entinostat 2, 4, 6, 
8, 10, or 12 mg/m2 every 
2 weeks 
MTD: 10 mg/m2 every 2 weeks 
daily schedule was Intolerable at a dose 
and schedule explored. 
DLT: Nausea, vomiting, anorexia, and 
fatigue 
DLT daily schedule, dose 2 mg/m2 (only 
2 patients): 
MTD was exceeded at the first dose level. 
abdominal/epigastric pain in one patient, 
and cardiac arrhythmia (supraventricular 
tachycardia), elevated AST/ALT, 
hypotension, hypoalbuminemia, and 
hypophosphatemia in a second patient. 
 
 
 
Part 3: Entinostat 2, 4, 6, 
8 mg/m2 weekly for 4 
weeks, followed by 2 
weeks of rest (fasting or 
fed) 
DLT every 14-day schedule, dose 10 
mg/m2 (28 patients): 
DLT were nausea, vomiting, anorexia, and 
fatigue. The first patients with first course 
DLTs were observed at dose level 3 (6 
mg/m2). After five patients tolerated dose 
level 4 without DLT, dose escalation 
continued to level 5 (10 mg/m2). One 
patient experienced similar DLTs at level 5 
as had been seen at level 3. At dose level 
6 (12 mg/ m2), two patients experienced 
similar DLTs. 
Gore et al, Clin 
I 
27 patients 
Part 1: Entinostat 2, 4, or 
MTD: 6 mg/m2 every other week or 4 
Cancer 
 
with 
6 mg/m2 PO every 2 
mg/m2 weekly for 3 weeks followed by 1 
Research [3] 
 
refractory 
weeks 
week of rest 
 
 
solid 
Part 2: Entinostat 2 
DLT: Hypophosphatemia and asthenia 
 
 
tumors and 
mg/m2 twice weekly for 3 
were dose limiting on the weekly and 
 
 
lymphomas 
of 4 weeks 
twice-weekly dosing schedules no dose- 
 
 
 
Part 3: Entinostat 4 or 5 
limiting toxicity on every other week 
 
 
 
mg/m2 weekly for 3 of 4 
schedule 
 
 
 
weeks 
 


33 
NCI Protocol #:10009  
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Batlevi et al, 
Haematologica 
[4] 
II 
49 patients 
with 
relapsed 
/refractory 
Hodgkin 
lymphoma 
Part 1: Entinostat 10 to 15 
mg PO every 2 weeks 
(Days 1 and 15) of a 4- 
week cycle 
Part 2: Entinostat 15 mg 
Days 1, 8, and 15 of a 4- 
week cycle 
Most frequent grade 3 and 4 AE: 
thrombocytopenia (63%), anemia (47%), 
neutropenia (41%), leukopenia (10%), 
hypokalemia (8%), and hypophosphatemia 
(6%). 
Does reduction/delays: 51% of patients 
Solid malignancies 
Denis et al, 
JCO 2014 
(abstract) 
[5] 
I/ 
ER+ breast 
cancer or 
NSCLC 
Cycle 1: All patients: 
Entinostat 10 mg PO on 
Day 1 under fed 
conditions and on Day 15 
under fasted conditions or 
on Day 1 under fasted 
conditions and on Day 15 
under fed conditions. 
Entinostat was well tolerated with no 
unexpected toxicities 
Results only published in abstract form 
without many details provided 
 
 
 
Cycle2: Breast cancer: 
Entinostat 10 mg PO on 
Days 1 and 15 at least 2 
hours after breakfast 
IROORZHGE\D-hour fast 
every 28 days + 
exemestane 25 mg QD 
PO 
 
 
 
 
NSCLC: Entinostat 10 mg 
PO on Days 1 and 15 at 
least 2 hours after 
breakfast followed by a 
B-hour fast every 28 
days + erlotinib 150 mg 
QD PO 
 
Hausschild et 
al, Melanoma 
Research [6] 
II 
28 patients 
with non- 
resectable 
metastatic 
melanoma 
Arm A: Entinostat 3 mg 
Days 1 and 15 of 4-week 
cycle 
 
Arm B: 7 mg Days 1, 8, 15 
of 4-week cycle 
No treatment-related serious adverse 
events occurred. 
 
Toxicity was mild to moderate with nausea 
(39%) and hypophosphatemia (29%) as the 
most frequently reported events. 
occurrence of the most common adverse 
events was similar in both treatment arms 
 
 
 
As can be seen from the table, the dose we proposed (4mg/m2 weekly) has been widely used 
in other studies and has been well tolerated (see especially Hausschild et al, Gore et al and 
Batlevi et al) and therefore was chosen as the RP2D. This dose was already used in combination 
with azacitidine (a drug which has more myelosuppressive potential than pembrolizumab). 
This E1905 study was a randomized phase 2 study (NCT00313586) of 10 days of AZA (50 
mg/m(2)/d) +/- entinostat (4 mg/m2) given on days 3 and 10 of each cycle and enrolled 149 
patients with MDS and the combination was well tolerated 23. Given that the entinostat 
manufacturer recommended the use of a flat dose of entinostat of 8 mg Po instead 


34 
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of 4mg/m2 based on a study that showed BSA-based dosing is not more accurate than fixed 
dosing 47. 
 
PART 1: 
 
 
The ENT monotherapy run-in part (cycle 1) will allow to study the changes in myeloid 
derived suppressor cells (MDSCs) at pre- and post- ENT treatment (the end of cycle 1) to 
dissect the effects of ENT monotherapy from those of the combined ENT/anti-PD1 therapy. 
 
We will perform ‘3+3’ classical dose escalation design to determine dose limiting toxicity 
(DLT). The first 3 patients will receive ENT at dose level 1 (DL1: 8mg on D1, 8 of 21-day 
cycles). pembrolizumab (MK-3475) will be added starting day 1 cycle 2 at the approved dose of 
200mg intravenously [IV] over 30 minutes on D1 of 21-day cycles) for 4 cycles (cycle 2-5). A 
safety committee that includes the study chair, the principal investigator from any study site that 
has enrolled a patient on the specific DL, as well as two sub-investigators from Yale will review 
safety data after enrollment of every 3 patients during the dose escalation phase. The committee 
will have the discretion to decide to enroll 3 more patients in the specific dose level even if no 
DLT occurred in the first 3 treated patients in that dose level if there are toxicity/safety concerns 
that did not qualify as DLT (maximum of 6 evaluable patients to be enrolled at any dose level). 
The committee can also decide to enroll patients at a lower dose level after the first 3 or after the 
first 6 patients enrolled in any dose level even if no or one DLT occurred at that dose level if 
there are toxicity/safety concerns that did not qualify as DLT. The following regarding 
enrollment in different dose levels will be considered guidance for the safety committee but can 
be modified in accordance of above: If no patient of the 3 patients treated with combined therapy 
at DL1 develops DLT, then dose level 2 (DL2) will be tested. For instance, DL2 will be with 
ENT at 8mg on D1, 8 and 15 and pembrolizumab (MK-3475) at 200mg IV on D1 of 21-day 
cycles. If 1 patient out of the 3 patients has DLT at DL1, we will expand cohort to 3 more 
patients and if 1 patient of the 6 patients has DLT (i.e. only 0/3 or 1/6), we will test next dose 
(DL2). If no patient of the 3 patients treated with combined therapy at DL2 develop DLT, then 
DL2 will be used for part 2 (expansion cohort). If 1 patient out of the 3 patients has DLT at DL2, 
we will expand cohort to 3 more patients and if 1 patient of the 6 patients has DLT (i.e. only 0/3 
or 1/6), dose level 2 (DL2) will be used for part 2 (expansion cohort). If 2 or more patients of the 
6 patients have DLT, DL1 will be used for expansion cohort. The cycle duration will be 21 days, 
the maximum number of treatment cycles of entinostat will be 18 cycles and for pembrolizumab 
(MK-3475) the maximum number of cycles will be 17. Cycle 2 to cycle 18, will have concurrent 
administration. In all the phases of the study, entinostat will be administered in clinic within 2 
hours before administration of pembrolizumab (MK-3475). Entinostat will be stored at the 
pharmacy and it will not be given for the patient to take home. 
 
 
An additional dose level (DL -1) in which day 8 of ENT will be omitted (i.e. ENT at 8mg on 
D1, and pembrolizumab (MK-3475) at 200mg IV on D1 of 21-day cycles) will be used if 2 
or more patients develop DLT during the first 2 cycles of combined therapy at DL1. If 0 or 1 
patients of the 6 patients treated with combined therapy at DL-1 develop DLT then DL-1 will 
be used in the second part of the study. If 2 or more 6 patients enrolled in DL -1 develop 


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DLT, then the study will be terminated. 
 
Toxicities will be tabulated and graded according to the Common Terminology Criteria for 
Adverse Events Version 5 (CTCAE-5). DLT will be assessed after the first 2 cycles of 
combined therapy. Responses will be as defined by IWG-2006 criteria (CR, PR, or HI) and 
assessed at the end of the combined first 2 cycles, and every 2 cycles afterwards. Patients 
who achieve an objective response or maintain a stable disease (SD) status after the first 4 
combination cycles will continue receiving both drugs at the same schedule till progression 
up to 1 year of combined therapy. 
 
PART 2: After the safe dose of the combination is established, 15 patients will be treated 
with ENT+ pembrolizumab (MK-3475) combination at that same dose and schedule. 
Assessment of response will occur after 4 combination cycles and similar to part 1, those 
who achieve an objective response or SD will continue to receive the therapy till progression. 
Patients who progress or respond and subsequently lose response will be withdrawn from 
study. BM biopsy/aspirate and PB samples will be done at baseline, after first cycle of ENT 
monotherapy, every 2 cycles of combined therapy, and at time of progression or study 
discontinuation. Formal safety assessment will be performed after the first 6 patients are 
enrolled and each have received at least 5 cycles (i.e. 1 ENT monotherapy cycle and 4 cycles 
of combined therapy (DL1, DL2) for each patient) to assess for any unexpected autoimmune 
or other toxicity. In all the phases of the study, entinostat will be administered in clinic 
within 2 hours before administration of pembrolizumab (MK-3475). Entinostat will be stored 
at the pharmacy and it will not be given for the patient to take home. 
 
 
 


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Dose Escalation Schedule 
Dose Level 
Dose* 
Entinostat 
MK-3475 - Pembrolizumab 
Level -1(Backup dose 
level) 
8mg po D1 
200mg 
Level 1 (starting dose 
level) 
8mg po D1,8 
200mg 
Level 2 
8mg po D1,8,15 
200mg 
*Doses are stated as exact dose in units (e.g., mg/m2, mcg/kg, etc.) rather than as a percentage. 
 
5.1.1 Pembrolizumab (MK-3475) 
 
Trial treatment should be administered on Day 1 of each cycle after all 
procedures/assessments have been completed. Trial treatment may be administered up to 
1 day (calendar states +/- 1 day window, so does Entinostat dosing, and needs to be 
administered within 2 hours after entinostat dose so needs to be 1 day window) before or 
after the scheduled Day 1 of each cycle due to administrative reasons. 
 
Pembrolizumab (MK-3475) is added on day one of each subsequent cycle starting 
Cycle 2. Pembrolizumab (MK-3475) treatment will be administered on an 
outpatient basis. 
 
Pembrolizumab (MK-3475) will be administered as a 30 minute IV infusion (treatment 
cycle intervals may be increased due to toxicity as described in Section 6.1). Infusion 
timing should be as close to 30 minutes as possible; however, a window of -5 minutes 
and +10 minutes is permitted (i.e., infusion time is 30 minutes: -5 min/+10 min). 
 
5.1.2 Entinostat 
 
Entinostat is to be taken in the clinic on an empty stomach, at least 1 hour before or 2 
hours after a meal. Entinostat is supplied as pink to light red (1 mg) or yellow (5 mg) 
coated tablets. Entinostat is to be stored at controlled room temperature (15ºC to 25ºC) in 
a secure, locked storage area to which access is limited. Entinostat is to be protected from 
light. Entinostat is not to be exposed to extremes of temperature (greater than 30ºC or less 
than 5ºC). The medication will be dispensed in the clinic. Administration one day prior to 
or one day after the scheduled day will be allowed. 
 
5.1.3 Other Modality(ies) or Procedures: N/A 
 
5.2 
Definition of Dose-Limiting Toxicity 
 
The primary endpoint is the maximum tolerated dose (MTD), which is the highest dose level 
where 1 or fewer of 6 participants experience a dose limiting toxicity (DLT) during the first two 
combined cycles of treatment. The recommend phase 2 dose (RP2D) is the same as the MTD. 


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The maximally administered dose is the dose at which two or more of 6 participants experience a 


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DLT. 
 
DLT will be defined as the occurrence of any of the following during the first 2 cycles of combined 
therapy unless the event can clearly be determined to be unrelated to the study drugs. 
 
 Any grade 3 or 4 non-hematologic toxicity with the following exceptions: A) Transient 
laboratory abnormalities that can be treated or resolve to grade 2 or less within 72 hours. 
 Grade 4 hematologic toxicity, including treatment-associated aplasia lasting >42 days from 
the last dose of study drug. Given that severe anemia, neutropenia and thrombocytopenia 
are features of advanced MDS and commonly encountered in this patient population, they 
will not be used to define DLT except if associated with prolonged treatment-associated 
aplasia as described above. 
x Patients with grade 3 immune related adverse events will be removed from the treatment 
and this will be considered to be a DLT as well as any other immune-related adverse 
event that results in pembrolizumab interruption, dose modification, or discontinuation of 
therapy. 
 
Participants who do not complete the first 2 cycles of combined therapy without experiencing a 
DLT (as DLT period is defined in the first 2 combined therapy cycles) during dose escalation will 
also be replaced. To be DLT-evaluable, the patient must complete the first two cycles of combined 
therapy without experiencing a DLT. Any patient who is dose reduced during the entinostat run- 
in first cycle due to entinostat-related toxicity will be analyzed on the dose level that corresponds 
to the reduced dose level delivered during the first cycle of combined therapy. 
 
A safety committee that includes the study chair, the principal investigator from any study site 
that has enrolled a patient on the specific DL, as well as two sub-investigators from Yale will 
review safety data after enrollment of every 3 patients during the dose escalation phase. The 
committee will have the discretion to decide to enroll 3 more patients in the specific dose level 
even if no DLT occurred in the first 3 treated patients in that dose level if there are toxicity/safety 
concerns that did not qualify as DLT (maximum of 6 evaluable patients to be enrolled at any 
dose level). The committee can also decide to enroll patients at a lower dose level after the first 3 
or after the first 6 patients enrolled in any dose level even if no or one DLT occurred at that dose 
level if there are toxicity/safety concerns that did not qualify as DLT. The following regarding 
enrollment in different dose levels will be considered guidance for the safety committee but can 
be modified in accordance of above. Dose escalation will proceed within each cohort according 
to the following scheme. 
 
Number of Patients with DLT at a 
Given Dose Level 
 
Escalation Decision Rule 
0 out of 3 
Enter 3 patients at the next dose level. 
 
Dose escalation will be stopped. This dose level will be 
declared the maximally administered dose (highest dose 
administered). Three (3) additional patients will be 
entered at the next lowest dose level if only 3 patients 


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were treated previously at that dose. 
1 out of 3 
Enter at least 3 more patients at this dose level. 
x If 0 of these 3 patients experience DLT, proceed to 
the next dose level. 
x If 1 or more of this group suffer DLT, then dose 
escalation is stopped, and this dose is declared the 
maximally administered dose. Three (3) additional 
patients will be entered at the next lowest dose level 
if only 3 patients were treated previously at that dose. 
RXWRIDWKLJKHVWGRVHOHYHO
below the maximally administered 
dose 
This is generally the recommended phase 2 dose 
(RP2D)= MTD. At least 6 patients must be entered at 
the recommended phase 2 dose (RP2D). 
 
Management and dose modifications associated with the above adverse events are outlined in 
Section 6. 
 
5.3 
Dose Expansion Cohorts 
 
Once the RP2D is reached, an additional 15 patients will be treated at this dose. For the expansion 
cohort, patients will continue to be monitored for occurrence of DLT. Participants who go off- 
study during the 1st cycle of entinostat will be replaced in the escalation phase. Participants who 
do not complete the first 2 cycles of combined therapy without experiencing a DLT (as DLT period 
is defined in the first 2 combined therapy cycles) will also be replaced. To be DLT-evaluable, the 
patient must complete the first two cycles of combined therapy without experiencing a DLT. Any 
patient who is dose reduced during the entinostat run-in first cycle due to entinostat-related toxicity 
will be analyzed on the dose level that corresponds to the reduced dose level delivered during the 
first cycle of combined therapy. 
 
The following early stopping rule for excess toxicity in the expansion cohort will be used: We 
will cease enrollment in the expansion phase at the dose determined from dose escalation part of 
the study if we observe two or more DLTs among the first six evaluable patients enrolled in the 
expansion cohort. An evaluable patient is any patient who received a study drug. If the true 
toxicity rate is 50% or greater at the MTD then this rule will stop the trial with probability at 
least .89. Similarly, if the true toxicity rate is 33% at the MTD then this stopping rule will end 
the trial with probability .65. In that event, a new expansion cohort will start at the dose level 
immediately below the dose level that was expanded. If the dose level being expanded is dose 
level -1, then the study will be terminated. 
Monitoring of all safety and toxicity data is done by the Principal Investigator and the 
Corresponding Organization on a real-time basis as data are entered into Medidata Rave using 
the Web Reporting Module. All participating sites are expected to notify the Principal 
Investigator when a DLT has occurred. 
 
 
5.4 
General Concomitant Medication and Supportive Care Guidelines 


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The patients will receive appropriate standard of care, supportive measures as per the 
institutional guidelines of each participating institution, including but not limited to blood and 
platelet transfusions, antibiotics, anti-emetics, etc. Hematopoietic growth factors will not be 
routinely used. 
 
Because there is a potential for interaction of entinostat with other concomitantly administered 
drugs, the case report form must capture the concurrent use of all other drugs, over-the-counter 
medications, or alternative therapies. The Principal Investigator should be alerted if the patient is 
taking any agent known to affect or with the potential for drug interactions. The study team 
should check a frequently-updated medical reference for a list of drugs to avoid or minimize use 
of. Appendix F (Patient Drug Information Handout and Wallet Card) should be provided to 
patients if available. 
 
5.4.1 Pembrolizumab (MK-3475) 
 
5.4.1.1 
Pembrolizumab (MK-3475) Concomitant Medication 
 
Medications or vaccinations specifically prohibited in the exclusion criteria are not allowed 
during the ongoing trial. If there is a clinical indication for one of these or other medications or 
vaccinations specifically prohibited during the trial, discontinuation from trial therapy or 
vaccination may be required. The investigator should discuss any questions regarding this with 
CTEP. The final decision on any supportive therapy or vaccination rests with the investigator 
and/or the subject's primary physician; however, the decision to continue the subject on trial 
therapy or vaccination schedule requires the mutual agreement of the Investigator, CTEP, and 
the patient. 
 
Acceptable Concomitant Medications 
All treatments that the investigator considers necessary for a patient’s welfare may be 
administered at the discretion of the investigator in keeping with the community standards of 
medical care. All concomitant medication will be recorded on the case report form (CRF) 
including all prescription, over-the-counter (OTC), herbal supplements, and IV medications and 
fluids. If changes occur during the trial period, documentation of drug dosage, frequency, route, 
and date may also be included on the CRF. 
 
All concomitant medications received within 28 days before the first dose of trial treatment and 
30 days after the last dose of trial treatment should be recorded. Concomitant medications 
administered after 30 days after the last dose of trial treatment should be recorded for SAEs. 
 
Prohibited Concomitant Medications 
Patients are prohibited from receiving the following therapies during the Screening and 
Treatment Phase (including retreatment for post-complete response relapse) of this trial: 
 
Anti-cancer systemic chemotherapy or biological therapy. 
 
Immunotherapy not specified in this protocol. 
 
Chemotherapy not specified in this protocol. 
 
Investigational agents other than pembrolizumab (MK-3475). 
 
Radiation therapy 


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o Note: Radiation therapy to a symptomatic solitary lesion or to the brain may be 
allowed after consultation with Sponsor. 
 
Live vaccines within 30 days prior to the first dose of trial treatment and while 
participating in the trial. Examples of live vaccines include, but are not limited to, the 
following: measles, mumps, rubella, chicken pox, yellow fever, rabies, BCG, and typhoid 
(oral) vaccine. Seasonal influenza vaccines for injection are generally killed virus 
vaccines and are allowed; however, intranasal influenza vaccines (e.g. Flu-Mist®) are live 
attenuated vaccines, and are not allowed. 
 
Glucocorticoids for any purpose other than to modulate symptoms from an event of 
suspected immunologic etiology. The use of physiologic doses of corticosteroids may be 
approved after consultation with the Sponsor. 
 
Patients who, in the assessment by the investigator, require the use of any of the aforementioned 
treatments for clinical management should be removed from the trial. Patients may receive other 
medications that the investigator deems to be medically necessary. 
 
The Exclusion Criteria describes other medications which are prohibited in this trial. 
There are no prohibited therapies during the Post-Treatment Follow-up Phase. 
Co-administration of drugs sensitive substrates of CYPs 1A2, 2B6, 2C8, 2C9, 2C19, and 2D6 
with a narrow therapeutic window are permitted, as it appears that entinostat is unlikely to inhibit 
these enzymes in humans. 
 
 
5.5 
Duration of Therapy 
 
In the absence of treatment delays due to adverse event(s), treatment may continue for one year 
(maximum 18 cycles or entinostat and maximum 17 cycles of pembrolizumab (MK-3475) or 
until one of the following criteria applies: 
 
x 
Disease progression as defined in modified IWG-2006 (Appendix A1, Appendix 
A2) for MDS 
 
x 
If disease has shown evidence of progression based on an increase in the blast counts 
according to the modified IWG-2006, but the patient is otherwise clinically stable and 
his transfusion requirement have not changed, the investigator can give two additional 
cycles of combined treatment and bone marrow biopsy will be repeated after that 
cycle. If repeat bone marrow biopsy confirms disease progression, the patient will be 
taken off the trial. 
 
x 
Intercurrent illness that prevents further administration of treatment 
 
x 
Patient decides to withdraw from the study 
 
x 
General or specific changes in the patient's condition render the patient unacceptable 
for further treatment in the judgment of the investigator 


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x 
Clinical progression 
 
x 
Patient non-compliance 
 
x 
Pregnancy 
 
o All women of child bearing potential should be instructed to contact the 
investigator immediately if they suspect they might be pregnant (e.g., missed 
or late menstrual period) at any time during study participation. 
 
o The investigator must immediately notify CTEP in the event of a confirmed 
pregnancy in a patient participating in the study. 
 
x 
Termination of the study by sponsor 
 
x 
The drug manufacturer can no longer provide the study agent 
 
x 
Unacceptable adverse event(s), including: 
 
x 
Any Grade 2 drug-related uveitis or eye pain or blurred vision that does not 
respond to topical therapy and does not improve to Grade 1 severity within the re- 
treatment period OR requires systemic treatment. 
 
x 
Grade 3 drug-related autoimmune or inflammatory event including uveitis, 
pneumonitis, diarrhea, colitis, neurologic adverse events, hypersensitivity 
reaction, or infusion reaction of any duration requires discontinuation. 
 
x 
Any Grade 3 or 4 drug-related laboratory imbalance or electrolyte abnormality, 
not associated with underlying organ pathology and that do not require treatment 
except for electrolyte replacements, do not require treatment discontinuation, with 
the following exceptions with approval of the Principal Investigator: 
 
x 
Grade 2-4 hypophysitis or pan-hypopituitarism requires holding 
pembrolizumab (MK-3475) and discontinuing treatment if unable to reduce 
FRUWLFRVWHURLGGRVHWRPJRISUHGQLVRQHRUHTXLYDOHQWSHUGD\ZLWKLQ 
weeks. 
 
x 
Grade 4 amylase or lipase abnormalities that are not associated with diabetes 
mellitus, associated liver or gall bladder inflammation clinical manifestations 
of pancreatitis and which decrease to <Grade 4 within 1 week of onset may 
stay on study. 
 
x 
Any drug-related liver function test (LFT) abnormality that meets the 
following criteria requires discontinuation: Grade 3 AST or ALT (>5 x ULN) 
and total bilirubin >3 x ULN. 
 
x 
Grade 3 drug-related thrombocytopenia >7 days or associated with bleeding 


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requires discontinuation. 
 
x 
Any patient requiring systemic steroid or other immunosuppressive treatment. 
 
x 
For patients with skin-only toxicity, when symptoms improve to Grade 1, steroid 
taper should be started and continued over no less than 4 weeks. Discontinue MK- 
LIXQDEOHWRUHGXFHFRUWLFRVWHURLGGRVHIRULU$(VWRPJpembrolizumab 
(MK-3475) treatment may be restarted and the dose modified as specified in the 
protocol. 
 
x 
Patients with peripheral thyroiditis and no other autoimmune/inflammatory event may 
be restarted after a short course of steroids on a stable replacement regimen. 
 
x 
Any dosing interruption lasting >12 weeks with the following exceptions: 
x 
Dosing interruptions >12 weeks that occur for non-drug-related reasons may 
be allowed if approved by the Principal Investigator. Prior to re-initiating 
treatment in a subject with a dosing interruption lasting >12 weeks, the 
Principal Investigator must be consulted. 
x 
Tumor assessments should continue as per protocol even if dosing is 
interrupted. 
 
The reason(s) for protocol therapy discontinuation, the reason(s) for study removal, and the 
corresponding dates must be documented in the Case Report Form (CRF). 
 
 
5.6 
Duration of Follow Up 
 
Patients will be followed closely throughout the study period and for 6 months after the last dose 
of entinostat in combination with pembrolizumab (MK-3475) or 6 months after they are removed 
for the presence of any toxicity (early or late). 
 
During maintenance: Patients whose primary oncologist is at one of the study sites will be 
followed and seen by a member of the study team at least monthly in person or by phone when 
study visits are not required. For patients who are followed by an oncologist outside of one of 
the study sites, a study team member will evaluate the patients at least monthly in person or by 
phone. 
 
During 6 months post last dose or after removal from study: all patients will be evaluated by the 
study team at least monthly. Evaluations may be done during routine clinic visits in patients 
followed longitudinally at the study Cancer Center or via phone if in person visits are not 
possible due to logistical reasons. The study team will note the follow-up in the medical records 
and source documents will be sought for any change in condition related to the study or study 
drug. 
 
Patients removed from study for unacceptable adverse event(s) will be followed in person or by 
phone until resolution or stabilization of the adverse event OR for at least 6 months after removal 
from study. Prior data have showed a median OS of 5 months for patients with HR-MDS who 


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failed 5-AZA or Decitabine. Therefore, we expect to capture most events of progression, relapse 
or death during the maintenance and follow-up phases of the study. For patients who are still 
alive at end of follow-up period, we will check social security Death Index every 3 months for 2 
more years to obtain death dates for OS calculations 
 
 
5.7 
Criteria for Removal from Study 
 
Patients will be removed from study when any of the applicable criteria, including progressive 
disease, withdrawal, or inability to follow study protocol as listed in Section 5.5. The reason for 
study removal and the date the patient was removed must be documented in the Case Report 
Form. 
 
5.8 
Criteria to Resume Treatment 
 
For non-autoimmune or inflammatory events, patients may resume treatment with study drug 
when the drug-UHODWHG$(VUHVROYHWR*UDGHRUEDVHOLQHYDOXHZLWKWKHIROORZLQJ
exceptions: 
x Patients may resume treatment in the presence of Grade 2 fatigue. 
x Patients with baseline Grade 1 AST/ALT or total bilirubin who require dose delays for 
reasons other than a 2-grade shift in AST/ALT or total bilirubin may resume treatment in 
the presence of Grade 2 AST/ALT OR total bilirubin. 
x Patients with combined Grade 2 AST/ALT AND total bilirubin values meeting study 
parameters outlined in Section 5.4 should have treatment permanently discontinued. 
x Non-drug-related toxicity including hepatic, pulmonary toxicity, diarrhea, or colitis, must 
have resolved to baseline before treatment is resumed. 
x Drug-related endocrinopathies (not including drug-related adrenal insufficiency or 
hypophysitis) adequately controlled with only physiologic hormone replacement may 
resume treatment after replacement correction and clinically stable regimen. 
 
If the criteria to resume treatment are met, the patient should restart treatment no sooner than the 
next scheduled time point per protocol. However, if the treatment is delayed past the next 
scheduled time point per protocol, the treatment should resume at the earliest convenient point 
that is within the 12 week delay period. 
 
If treatment is delayed >12 weeks, the patient must be permanently discontinued from study 
therapy, except as specified in Section 5.4 (Duration of Therapy). 
 
5.9 
Treatment Beyond Progression 
 
Immunotherapeutic agents such as pembrolizumab (MK-3475) may produce antitumor 
effects by potentiating endogenous cancer-specific immune responses. The response patterns 
seen with such an approach may extend beyond the typical time course of responses seen 
with cytotoxic agents, and can manifest as a clinical response after an initial increase in 
tumor burden. 
 
If bone marrow biopsy shows progressive disease (PD) but patient is clinically stable and blood 


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counts have not significantly worsened, tumor assessment may be repeated by the site 
approximately 4 weeks later in order to confirm PD with the option of continuing treatment per 
below while awaiting confirmation of progression. If repeat bone marrow biopsy shows a 
reduction in the blast counts compared to the initial bone marrow biopsy demonstrating PD, 
treatment may be continued as per treatment calendar. If repeat bone marrow biopsy confirms 
PD, patients will be discontinued from study therapy. The decision to continue study treatment 
after the 1st evidence of disease progression determined by (bone marrow biopsy) is at the 
Investigator’s discretion based on the clinical status of the patient as described in the table below. 
 
 
Patients may receive study treatment while waiting for confirmation of PD if they are clinically 
stable as defined by the following criteria: 
x Absence of signs and symptoms (including worsening of laboratory values) indicating 
disease progression 
x No decline in ECOG performance status 
x Absence of rapid progression of disease 
 
 
Clinically Stable 
Clinically Unstable 
 
- Bone Marrow 
Biopsy 
Treatment 
Bone Marrow 
Biopsy 
Treatment 
1st bone marrow 
evidence of PD 
Repeat bone 
marrow biopsy 
at approximately 
4 weeks to 
confirm PD 
May continue 
study treatment 
at the 
Investigator’s 
discretion while 
awaiting 
confirmatory 
scan 
Repeat bone 
marrow biopsy 
at approximately 
4 weeks to 
confirm PD if 
possible 
Discontinue 
treatment 
Repeat bone 
marrow biopsy 
confirms PD 
No additional 
bone marrow 
biopsyrequired 
Discontinue 
treatment 
No additional 
bone marrow 
biopsy required 
N/A 
Repeat bone 
marrow biopsy 
shows SD, PR, 
or CR 
Continue 
regularly 
scheduled bone 
marrow biopsies 
every 6 weeks 
Continue study 
treatment at the 
Investigator’s 
discretion 
Continue 
regularly 
scheduled bone 
marrow biopsies 
every 6 weeks 
May restart 
study treatment 
if condition has 
improved and/or 
clinically stable 
per 
Investigator’s 
discretion 
Bone marrow biopsies will occur at the end of Cycle 1 and at the end of every 2 cycles 
thereafter. 
 
5.10 
Discontinuation of Treatment Following Complete Response 
 
Discontinuation of treatment may be considered for patients who have attained a confirmed 
complete response (CR) that have been treated for at least 24 weeks with entinostat and MK- 


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3475 and had at least two treatments with entinostat and pembrolizumab (MK-3475) beyond 
the date when the initial CR was declared. 
 
5.11 
Treatment Up to 1 Years 
 
Treatment with entinostat and pembrolizumab (MK-3475) will continue for up to one year of 
combined therapy administered, documented disease progression, unacceptable adverse 
event(s), intercurrent illness that prevents further administration of treatment, investigator’s 
decision to withdraw the patient, patient withdraws consent, pregnancy of the patient, 
noncompliance with trial treatment or procedure requirements, or administrative reasons. 
 
 
6. DOSING DELAYS/DOSE MODIFICATIONS 
 
Although entinostat and pembrolizumab (MK-3475) have distinct toxicity profiles, they do share 
some AEs 
such as fatigue and nausea. There is the theoretical possibility that 1 agent may potentiate the 
other and hence drug causality will not always be clear. In the event of uncertainty, dose 
reductions and/or delays will follow the most conservative approach (i.e., delays and/or dose 
reductions for both drugs) until resolution of the event. No dose reductions will be performed for 
pembrolizumab (MK-3475). Guidance for delays and or dose reductions for entinostat are 
presented. 
 
 
6.1 
Entinostat Dose Modifications and Supportive Care Guidelines for Drug-Related 
Adverse Events 
 
6.1.1 Dose Modifications: see tables below 
 
6.1.2 Dose Modifications: see tables below 
 
6.1.3 Table of Dose Modifications and Supportive Care Guidelines 
 
Dose Level 
Entinostat Dose 
-1 
8 mg, po D1 
+1 
8 mg, po D1, D8 
+2 
8 mg, po D1, D8, D15 
Note: All treatment modifications must be expressed as a specific dose or amount rather than as 
a percentage of the starting or previous dose. 
 
Nausea 
Management/Next Dose for Entinostat 
*UDGH 
No change in dose 
Grade 2 
+ROGXQWLO*UDGH5HVXPHDWVDPHGRVHOHYHO 
Grade 3 
Hold* until < Grade 2. Resume at one dose level lower, if indicated.** 
Grade 4 
Off protocol therapy 
*Patients requiring a delay of >2 weeks should go off protocol therapy. 


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Version Date: 09/04/2020
Nausea
Management/Next Dose for Entinostat
**Patients requiring > two dose reductions should go off protocol therapy.
Recommended management: antiemetics.
Vomiting
Management/Next Dose for entinostat
*UDGH
No change in dose
Grade 2
+ROGXQWLO*UDGH5HVXPHDWVDPHGRVHOHYHO
Grade 3
Hold* until < Grade 2. Resume at one dose level lower, if indicated.**
Grade 4
Off protocol therapy
*Patients requiring a delay of >2 weeks should go off protocol therapy.
**Patients requiring > two dose reductions should go off protocol therapy.
Recommended management: antiemetics.
Diarrhea
Management/Next Dose for entinostat
*UDGH
No change in dose
Grade 2
+ROGXQWLO*UDGH5HVXPHDWVDPHGRVHOHYHO
Grade 3
Hold* until < Grade 2. Resume at one dose level lower, if indicated.**
Grade 4
Off protocol therapy
*Patients requiring a delay of >2 weeks should go off protocol therapy.
**Patients requiring > two dose reductions should go off protocol therapy.
Recommended management: Loperamide antidiarrheal therapy
Dosage schedule: 4 mg at first onset, followed by 2 mg with each loose motion until diarrhea- 
free for 12 hours (maximum dosage: 16 mg/24 hours) 
Adjunct anti-diarrheal therapy is permitted and should be recorded when used.
For other non-hematological toxicities that are grade 2 and do not resolve within a week, the 
investigator can reduce the entinostat dosing by one dose level (e.g. go from DL2 dosing to DL1 
dosing). 
Given the frequent occurrence of severe cytopenias among patients with MDS and AML which 
are disease and not drug related and that worsening of cytopenias is a common occurrence during 
the disease course, the below dose modifications of entinostat for hematologic adverse events are 
considered guidance for the treating investigator and not mandatory. The decision to modify/hold 
dose will be at the discretion of the treating investigator and should include consideration of the 
clinical situation including severity consideration of baseline counts. 
Dose Modifications 
Hematologic
adverse
events 


48 
NCI Protocol #:10009  
Version Date: 09/04/2020 
 
 
 
*UDGHQHXWURSHQLD 
*UDGH
thrombocytopenia, 
Administer symptomatic remedies/ start prophylaxis. 
Hold dose1 until recovery to Grade 1 or study baseline under the 
following direction: 
1. If not recovered by next scheduled dose, skip the dose. If 
recovered by next scheduled dose, resume study drug at 
prior dose. 
2. If receiving 8 mg dose on day 1 and 8, and not recovered by 
either of the next 2 scheduled doses, permanently 
discontinue study treatment. Otherwise, skip each dose. If 
recovered for either of these doses, resume study drug as 
follows: 
x 
If receiving 8 mg on days 1 and 8, restart study drug at 8 
mg only on day 1. 
x 
If receiving 8 mg on days 1, 8, and 21, restart study drug at 
8 mg only on days 1 and 8. 
3. If not recovered within 4 weeks, permanently discontinue 
study drug. 
Recurrence of the same 
hematologic toxicity 
1. If the same hematologic toxicity recurs: 
x Administer symptomatic remedies/ start prophylaxis. 
Hold1 dose until recovery to Grade 1 or baseline. 
2. If recovered within 2 weeks, resume study drug as follows: 
x If receiving 8 mg on days 1 and 8, restart study drug 
at 8 mg on day 1 only 
x If receiving 8 mg on day 1 only, permanently 
discontinue study drug 
3. ,IWKHVDPH*UDGHHYHQWUHFXUVLHWKLUGRFFXUUHQFH
despite entinostat dose reduction to 8 mg on day 1 only, as 
described above, permanently discontinue study drug. 
* Therapy emergent neutropenia or thrombocytopenia is defined as 50% or more reduction from 
the baseline. 
1 If greater than 50% of doses are missed during any 6-week period, discontinue from study drug 
treatment. 
# There will be no dose modification for anemia 
 
6.2 
Pembrolizumab (MK-3475) Dose Modifications and Supportive Care 
Guidelines for Drug-Related Adverse Events 
 
6.2.1 Dose Modifications 
 
Immune-related adverse events (irAEs), defined as AEs of unknown etiology, associated with 
drug exposure and consistent with an immune phenomenon, may be predicted based on the 


49 
NCI Protocol #:10009  
Version Date: 09/04/2020 
 
 
nature of the pembrolizumab (MK-3475) compound, its mechanism of action, and reported 
experience with immunotherapies that have a similar mechanism of action. Special attention 
should be paid to AEs that may be suggestive of potential irAEs. An irAE can occur shortly 
after the first dose or several months after the last dose of treatment. All AEs of unknown 
etiology associated with drug exposure should be evaluated to determine if they are possibly 
immune-related. If an irAE is suspected, efforts should be made to rule out neoplastic, 
infectious, metabolic, toxin or other etiologic causes prior to labeling an adverse event as an 
irAE. 
 
6.2.2 Supportive care guidelines 
 
Based on the severity of the adverse reaction, KEYTRUDA will be withheld and corticosteroids 
administered. Upon improvement to Grade 1 or less, corticosteroid taper will be initiated and 
continued to taper over at least 1 month. Based on limited data from clinical studies in patients 
whose immune-related adverse reactions could not be controlled with corticosteroid use, 
administration of other systemic immunosuppressants can be considered. KEYTRUDA will be 
resumed when the immune-mediated adverse reaction remains at Grade 1 or less following 
corticosteroid taper. KEYTRUDA will be permanently discontinue for any Grade 3 immune- 
mediated adverse reaction that recurs and for any life-threatening immune-mediated adverse 
reaction. The following clinically significant, immune-mediated adverse reactions occurred in 
less than 1% (unless otherwise indicated) of 2799 patients treated with KEYTRUDA: arthritis 
(1.5%), exfoliative dermatitis, bullous pemphigoid, rash (1.4%), uveitis, myositis, Guillain-Barré 
syndrome, myasthenia gravis, vasculitis, pancreatitis, hemolytic anemia, and partial seizures 
arising in a patient with inflammatory foci in brain parenchyma.” 
See complete Keytruda prescribing information, especially regarding permanent discontinuation 
for any Grade 3 immune-mediated adverse reaction that recurs. 
 
6.2.3 Table of Dose Modifications and Supportive Care Guidelines 
 
The table below includes guidelines for managing irAEs that are not listed in the AE-specific 
table 
 
General Dose Modification Guidelines for Drug-Related Immune-Related Adverse Events 
irAE 
Withhold/Discontinue MK- 
3475? 
Supportive Care 
Grade 1 
No action 
Provide symptomatic treatment 
Grade 2 
May withhold pembrolizumab 
(MK-3475) 
Consider systemic corticosteroids in addition to 
appropriate symptomatic treatment 
Grade 3 and Grade 4 
Withhold pembrolizumab (MK-
3475) 
Discontinue if unable to reduce 
corticosteroid dose to < 10 mg 
per day prednisone equivalent 
within 12 weeks of toxicity 
Systemic corticosteroids are indicated in addition to 
appropriate symptomatic treatment. May utilize 1 to 2 
mg/kg prednisone or equivalent per day. 
Steroid taper should be considered once symptoms 
improve to Grade 1 or less and tapered over at least 4 
weeks. 
 
Additionally, pembrolizumab (MK-3475) will be withheld for other drug-related Grade 4 
hematologic toxicities, non-KHPDWRORJLFDOWR[LFLW\*UDGHLQFOXGLQJODERUDWRU\
abnormalities, and severe or life- threatening AEs. 


50 
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The table below includes dose modification guidelines for other toxicities that do not appear to 
be irAEs and are not listed in the AE-specific table. 
 
Dose Modification Guidelines for Other Drug-Related Adverse Events 
 
 
Toxicity 
 
 
Grade 
Hold 
Treatment 
(Y/N) 
Timing for 
restarting 
treatment 
Dose/Schedule 
for restarting 
treatment 
 
Discontinue 
Subject 
Hematological Toxicity 
1, 2, 3 
No 
N/A 
N/A 
N/A 
4 
Yes 
Toxicity 
resolves to 
Grade 0-1 or 
baseline 
May increase the 
dosing interval by 
1 week 
Toxicity does 
not resolve 
within 12 
weeks of last 
infusion 
Permanent 
discontinuation 
should be 
considered for 
any severe or 
life-threatening 
event 
Non-hematological toxicity 
1 
No 
N/A 
N/A 
N/A 
 
Note: Exception to be treated 
similar to grade 1 toxicity 
x Grade 2 alopecia 
x Grade 2 fatigue 
 
For 
additional 
information 
regarding Adverse Events with 
a potential Immune-Etiology 
reference Section 6.1.2. 
2 
Consider 
withholding 
for 
persistent 
symptoms 
Toxicity 
resolves to 
Grade 0-1 or 
baseline 
Clinical AE 
resolves within 4 
weeks:  Same 
dose and schedule 
Clinical AE does 
not resolve within 
4 weeks: May 
increase the 
dosing interval by 
1 week for each 
occurrence 
Toxicity does 
not resolve 
within 12 
weeks of last 
infusion 
 
3 
Yes 
Toxicity 
resolves to 
Grade 0-1 or 
baseline 
May increase the 
dosing interval by 
1 week for each 
occurrence 
Toxicity does 
not resolve 
within 12 
weeks of last 
infusion 
 
4 
Yes 
N/A 
N/A 
Subject must be 
discontinued 
 
In case toxicity does not resolve to Grade 0-1 within 12 weeks after last infusion, trial treatment 
should be discontinued. With Principal Investigator agreement, subjects with a laboratory 
adverse event still at Grade 2 after 12 weeks may continue treatment in the trial only if 
asymptomatic and controlled. Patients who experience a recurrence of the same severe or life- 
threatening event at the same grade or greater with re-challenge of pembrolizumab (MK-3475) 
should be discontinued from trial treatment. 


51 
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AE-specific pembrolizumab (MK-3475) Dose Modifications and Supportive Care Guidelines: 
The table below includes recommendations on the management of specific AEs and when to 
hold and/or discontinue pembrolizumab (MK-3475). These guidelines are intended to be applied 
when the investigator determines the events to be treatment-related. Note: if after the evaluation 
the event is determined not to be related, the investigator does not need to follow the treatment 
guidance. Therefore, these recommendations should be seen as guidelines and the treating 
physician should exercise individual clinical judgment based on the patient. 
Given the frequent occurrence of severe cytopenias among patients with MDS and AML which 
are disease and not drug related and that worsening of cytopenias is a common occurrence during 
the disease course, the below dose modifications of pembrolizumab (MK-3475) for hematologic 
adverse events are considered guidance for the treating investigator and not mandatory. The 
decision to modify/hold dose will be at the discretion of the treating investigator and should 
include consideration of the clinical situation including severity consideration of baseline counts. 


NCI Protocol #:10009  
Version Date: 09/04/2020 
49 
 
 
 
 
AE-Specific Dose Modification Guidelines for Drug-Related Adverse Events 
Event(s) 
CTCAE v4.0 
Grade 
Management / 
Next Dose for 
pembrolizu
mab (MK-
3475) 
 
Action / Supportive Care Guidelines 
 
Colitis 
x Colitis 
x Colitis microscopic 
x Enterocolitis 
x Enterocolitis, 
hemorrhagic 
x Gastrointestinal (GI) 
perforation 
x Intestinal obstruction 
x Necrotizing colitis 
x Diarrhea 
Grade 2 
Diarrhea/Colitis (4- 
6 stools/day over 
baseline, 
dehydration 
requiring 
intravenous (IV) 
fluids <24 hours, 
abdominal 
pain, mucus or 
blood in stool) 
Hold 
pembrolizumab 
(MK-3475). 
x Symptomatic treatment 
x For Grade 2 diarrhea that persists for >3 days, and for diarrhea with 
blood and/or mucus: 
- 
Consider GI consultation and endoscopy to confirm or rule out 
colitis; and 
- 
Administer oral corticosteroids (prednisone 1-2 mg/kg once daily or 
equivalent). 
x :KHQV\PSWRPVLPSURYHWR*UDGHVWHURLGWDSHUVKRXOGEH started 
and continued over no less than 4 weeks. 
x Permanently discontinue for inability to reduce corticosteroid dose to 
PJRIprednisone or equivalent per day within 12 weeks. 
x If symptoms worsen or persist >3 days treat as Grade 3 
Grade 3 
Diarrhea/Colitis (or 
Grade 2 diarrhea 
that persists for >1 
week) 
Hold 
pembrolizumab 
(MK-3475). 
x Rule out bowel perforation. Imaging with plain films or computed 
tomography (CT) can be useful. 
x Recommend consultation with Gastroenterologist and confirmation 
biopsy with endoscopy. 
x Treat with IV steroids (methylprednisolone 125 mg) followed by high 
dose oral steroids (prednisone 1 to 2 mg/kg once per day or 
dexamethasone 4 mg every 4 hours). When symptoms improve to 
*UDGHVWHURLGWDSHUVKRXOGEHVWDUWHGDQGFRQWLQXHGRYHUQRless than 
4 weeks. Taper over 6 to 8 weeks in patients with diffuse and severe 
ulceration and/or bleeding. 
x Permanently discontinue for inability to reduce corticosteroid dose to 
PJRISUHGQLVRQHRUHTXLYDOHQWSHUGD\ZLWKLQZHHNV 
x If IV steroids followed by high dose oral steroids does not reduce initial 
symptoms within 48 to 72 hours, consider treatment with additional anti- 
inflammatory measures as described in the literaturea. Discontinue 
additional anti-inflammatory measures upon symptom relief and initiate 
a prolonged steroid taper over 45 to 60 days.  If symptoms worsen 
during steroid reduction, initiate a retapering of steroids starting at a 
higher dose of 80 or 100 mg followed by a more prolonged taper and 
administer additional anti-inflammatory measures as needed. 
Grade 4 
Permanently 
discontinue MK- 
3475 
x Manage as per Grade 3. 


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50 
 
 
 
Event(s) 
CTCAE v4.0 
Grade 
Management / 
Next Dose for 
pembrolizu
mab (MK-
3475) 
 
Action / Supportive Care Guidelines 
 
Endocrine 
 
Hyperthyroidism and 
Hypothyroidism 
Grade 2 
hyperthyroidism 
 
Grade 2-4 
hypothyroidism 
No change in dose x Monitor thyroid function or other hormonal level tests and serum 
chemistries more frequently until returned to baseline values. 
x Thyroid hormone and/or steroid replacement therapy to manage adrenal 
insufficiency. 
x Therapy with pembrolizumab (MK-3475) can be continued while 
treatment for the thyroid disorder is instituted. 
x In hyperthyroidism, non-selective beta-blockers (e.g. propranolol) are 
suggested as initial therapy. 
x In hypothyroidism, thyroid hormone replacement therapy, with 
levothyroxine or liothyroinine, is indicated per standard of care. 
x Consultation with an endocrinologist may be considered. 
Thyroid disorders can 
occur at any time during 
treatment. Monitor 
patients for changes in 
thyroid function (at the 
start of treatment, 
periodically during 
treatment, and as 
indicated based on 
clinical evaluation) and 
for clinical signs and 
symptoms of thyroid 
disorders. 
 
 
 
Grade 3 
hyperthyroidism 
Hold 
pembrolizumab 
(MK-3475). 
x Rule out infection and sepsis with appropriate cultures and imaging. 
x Treat with an initial dose of methylprednisolone 1 - 2 mg/kg IVfollowed 
by oral prednisone 1 - 2 mg/kg per day. When symptoms improve to 
*UDGHVWHURLGWDSHUVKRXOGEHVWDUWHGDQGFRQWLQXHGRYHUQROHVVWKDQ
4 weeks. Replacement of appropriate hormones may be required as the 
steroid dose is tapered. 
x Permanently discontinue for inability to reduce corticosteroid dose to 
PJRISUHGQLVRQHRUHTXLYDOHQWSHUGD\ZLWKLQZHHNV 
 
Grade 4 
hyperthyroidism 
Discontinue MK- 
3475. 
Manage as per Grade 3 
 
Endocrine 
Grade 2-4 
Hold 
pembrolizumab 
(MK-3475). 
x Rule out infection and sepsis with appropriate cultures and imaging. 
x Monitor thyroid function or other hormonal level tests and serum 
chemistries more frequently until returned to baseline values. 
x 
Pituitary gland imaging should be considered (magnetic resonance 
imaging [MRIs] with gadolinium and selective cuts of the pituitary can 
show enlargement or heterogeneity and confirm the diagnosis). 
x Treat with prednisone 40 mg by mouth (PO) or equivalent per day. 
:KHQV\PSWRPVLPSURYHWR*UDGHVWHURLGWDSHUVKRXOGEH started 
and continued over no less than 4 weeks. Replacement of appropriate 
hormones may be required as the steroid dose is tapered. 
x Permanently discontinue for inability to reduce corticosteroid dose to 
PJRISUHGQLVRQHRUHTXLYDOHQWSHUGD\ZLWKLQZHHNV 
x Hypophysitis with clinically significant adrenal insufficiency and 
hypotension, dehydration, and electrolyte abnormalities (such as 
hyponatremia and hyperkalemia) constitutes adrenal crisis. 
x Consultation with an endocrinologist may be considered. 
 
Hypophysitis or other 
symptomatic 
endocrinopathy other 
than hypo- or 
hyperthyroidism 
 
 
 


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51 
 
 
 
Event(s) 
CTCAE v4.0 
Grade 
Management / 
Next Dose for 
pembrolizu
mab (MK-
3475) 
 
Action / Supportive Care Guidelines 
 
Endocrine 
 
Type 1 diabetes mellitus 
(T1DM), if new onset, 
including diabetic 
ketoacidosis (DKA) and 
*UDGH
hyperglycemia, if 
associated with ketosis 
(ketonuria) or metabolic 
acidosis (DKA). 
 
The patients may present 
with hyperglycemia 
(abrupt onset or abrupt 
decompensation) with 
clinical evidence of 
diabetic ketoacidosis or 
laboratory evidence of 
insulin deficiency, such 
as ketonuria, laboratory 
evidence of metabolic 
acidosis, or low or 
undetected c- peptide. 
T1DM or Grade 3-4 
hyperglycemia 
Hold 
pembrolizumab 
(MK-3475) for 
new onset T1DM 
or Grade 3-4 
hyperglycemia 
associated with 
evidence of beta 
cell failure, and 
resume 
pembrolizumab 
(MK-3475) when 
patients are 
clinically and 
metabolically 
stable. 
x T1DM should be immediately treated with insulin. 
x Insulin replacement therapy is recommended for T1DM and for Grade 3- 
4 hyperglycemia associated with metabolic acidosis or ketonuria. 
x Evaluate patients with serum glucose and a metabolic panel, urine 
ketones, glycosylated hemoglobin, and C-peptide. 
x Consultation with an Endocrinologist is recommended. 
x Consider local testing for islet cell antibodies and antibodies to GAD, 
IA-2, ZnT8, and insulin may be obtained. 
Hematologic 
x Autoimmune 
hemolytic anemia 
x Aplastic anemia 
x Disseminated 
Intravascular 
Coagulation (DIC) 
x Hemolytic Uremic 
Syndrome (HUS) 
x Idiopathic (or 
immune) 
Grade 2 
Hold 
pembrolizumab 
(MK-3475) 
x Prednisone 1-2 mg/kg daily may be indicated. 
x Consider Hematology consultation. 
x Permanently discontinue for inability to reduce corticosteroid dose to 
PJRISUHGQLVRQHRUHTXLYDOHQWSHUGD\ZLWKLQZHHNV 
Grade 3 
Hold 
pembrolizumab 
(MK-3475). 
Discontinuation 
should be 
considered as per 
specific protocol 
guidance. 
x Hematology consultation. 
x Discontinuation should be considered as per specific protocol guidance. 
x Treat with methylprednisolone 125 mg IV or prednisone 1-2 mg/kg PO 
(or equivalent) as appropriate 
x Permanently discontinue for inability to reduce corticosteroid dose to 
PJRISUHGQLVRQHRUHTXLYDOHQWSHUGD\ZLWKLQZHHNV 


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52 
 
 
 
Event(s) 
CTCAE v4.0 
Grade 
Management / 
Next Dose for 
pembrolizu
mab (MK-
3475) 
 
Action / Supportive Care Guidelines 
 
Thrombocytopenia 
Purpura (ITP) 
x Thrombotic 
Thrombocytopenic 
Purpura (TTP) 
x Any Grade 4 anemia 
regardless of 
underlying 
mechanism 
Grade 4 
 
x Hematology consultation. 
x Discontinue MK-3475 for all solid tumor indications; refer to protocol 
for hematologic malignancies. 
x Treat with methylprednisolone 125 mg IV or prednisone 1-2 mg/kg PO 
(or equivalent) as appropriate. 
Hepatic 
x Autoimmune hepatitis 
x Hepatitis 
x Transaminase 
elevations 
Grade 2 
Hold 
pembrolizumab 
(MK-3475) 
when AST or ALT 
>3.0 to 5.0 × ULN 
and/or total 
bilirubin >1.5 to 
3.0 × ULN. 
x Monitor liver function tests (LFT) more frequently until returned to 
baseline values (consider weekly). 
- 
Treat with 0.5-1 mg/kg/day methylprednisolone or oral equivalent 
and when LFT returns to Grade 1 or baseline, taper steroids over at 
least 1 month, consider prophylactic antibiotics for opportunistic 
infections, and resume MK-3475 per protocol. 
x Permanently discontinue for inability to reduce corticosteroid dose to 
PJRISUHGQLVRQHRUequivalent per day within 12 weeks. 
x Permanently discontinue MK-3475 for patients with liver metastasis 
who begin treatment with Grade 2 elevation of AST or ALT, and AST or 
$/7LQFUHDVHVUHODWLYHWREDVHOLQHDQGODVWV week. 
Grade 3 
Discontinue 
pembrolizumab 
(MK-3475) when 
AST or ALT >5.0 
× ULN 
and/or total 
bilirubin >3.0 × 
ULN. 
x Consider appropriate consultation and liver biopsy to establish etiology 
of hepatic injury, if necessary. 
x Treat with high-dose IV glucocorticosteroids for 24-48 hours. When 
V\PSWRPVLPSURYHWR*UDGHDVWHURLGWDSHUZLWKGH[DPHWKDVRQH 4 
mg every 4 hours or prednisone at 1-2 mg/kg should be started and 
continued over no less than 4 weeks. 
x If serum transaminase levels do not decrease 48 hours after initiation of 
systemic steroids, oral mycophenolate mofetil 500 mg every 12 hours 
may be given. Infliximab is not recommended due to its potential for 
hepatotoxicity. 
x Several courses of steroid tapering may be necessary as symptoms may 
worsen when the steroid dose is decreased. 
x Permanently discontinue for inability to reduce corticosteroid dose to 
PJRISUHGQLVRQHRUHTXLYDOHQWSHUGD\ZLWKLQweeks. 
Grade 4 
Permanently 
discontinue MK- 
3475 
x Manage patient as per Grade 3 above. 
Nausea 
*UDGH 
No change in dose x Nausea should be treated aggressively, and consideration should be 
 


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53 
 
 
 
Event(s) 
CTCAE v4.0 
Grade 
Management / 
Next Dose for 
MK-3475 
 
Action / Supportive Care Guidelines 
 
 
Grade 2 
+ROGXQWLO*UDGH 
1. 
 
Resume at same 
dose level. May 
increase dosing 
interval by 1 week 
if it takes more 
than 4 weeks for 
toxicities to 
resolve. 
given in subsequent cycles to the administration of prophylactic 
antiemetic therapy according to standard institutional practice. Patients 
should be strongly encouraged to maintain liberal oral fluid intake. 
 
Grade 3 
Hold until <Grade 
2. May increase 
dosing interval by 
1 week for each 
occurrence. 
 
Discontinue if 
toxicities do not 
resolve within 12 
weeks. 
Grade 4 
Off protocol 
therapy 
Neurologic events 
x Autoimmune 
neuropathy 
x Demyelinating 
polyneuropathy 
x Guillain-Barre 
syndrome 
x Myasthenic syndrome 
Grade 2 
Consider 
withholding 
pembrolizumab 
(MK-3475). 
x Consider treatment with prednisone 1-2 mg/kg PO daily as appropriate 
x Consider Neurology consultation. Consider biopsy for confirmation of 
diagnosis. 
x Permanently discontinue for inability to reduce corticosteroid dose to 
PJRISUHGQLVRQHRUHTXLYDOHQWSHUGD\ZLWKLQZHHNV 
x :KHQV\PSWRPVLPSURYHWR*UDGHVWHURLGWDSHUVKRXOGEHVWDUWHG
and continued over no less than 4 weeks. 
Grade 3-4 
Discontinue 
pembrolizumab 
(MK-3475) 
x Obtain neurology consultation. Consider biopsy for confirmation of 
diagnosis 
x 
Treat with systemic corticosteroids at a dose of 1 to 2 mg/kg 
prednisone or equivalent once per day. If condition worsens consider 
IV immunoglobulin (IVIG) or other immunosuppressive therapies as 
per local guidelines 
x :KHQV\PSWRPVLPSURYHWR*UDGHVWHURLGWDSHUVKRXOGEH started 
and continued over no less than 4 weeks. 
Neutropenia 
*UDGH 
No change in dose  
 
 
 


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54 
 
 
 
Event(s) 
CTCAE v4.0 
Grade 
Management / 
Next Dose for 
pembrolizu
mab (MK-
3475) 
 
Action / Supportive Care Guidelines 
 
 
Grade 2 
No change in dose  
 
Grade 3 
No change in dose 
Grade 4 
Hold until resolves 
WR *UDGH  0D\
increase the dosing 
interval by 1 week. 
 
Discontinue if 
toxicities do not 
resolve within 12 
weeks. 
Ocular 
x Uveitis 
x Iritis 
Grade 2 
Discontinue MK- 
3475 if symptoms 
persist despite 
treatment with 
topical immune- 
suppressive 
therapy 
x Evaluation by an ophthalmologist is strongly recommended. 
x Treat with topical steroids such as 1% prednisolone acetate suspension 
and iridocyclitics. 
Grade 3 
Hold 
pembrolizumab 
(MK-3475) 
and consider 
permanent 
discontinuation per 
specific protocol 
guidance. 
x Evaluation by an ophthalmologist is strongly recommended. 
x Treat with systemic corticosteroids such as prednisone at a dose of 1 to 2 
PJNJSHUGD\:KHQV\PSWRPVLPSURYHWR*UDGHVWHURLGWDSHU
should be started and continued over no less than 4 weeks. 
x Permanently discontinue for inability to reduce corticosteroid dose to 
PJRISUHGQLVRQHRUHTXLYDOHQWSHUGD\ZLWKLQZHHNV 
Grade 4 
Permanently 
discontinue 
pembrolizumab 
(MK-3475). 
x Evaluation by an ophthalmologist is strongly recommended. 
x Treat with corticosteroids as per Grade 3 above. 


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55 
 
 
 
Event(s) 
CTCAE v4.0 
Grade 
Management / 
Next Dose for 
pembrolizu
mab (MK-
3475) 
 
Action / Supportive Care Guidelines 
 
Pneumonitis 
x Pneumonitis 
x Interstitial lung 
disease 
x Acute interstitial 
pneumonitis 
 
If symptoms indicate 
possible new or worsening 
cardiac abnormalities 
additional testing and/or a 
cardiology consultation 
should be considered. 
Grade 2 
Hold 
pembrolizumab 
(MK-3475)  
x Consider pulmonary consultation with bronchoscopy and 
biopsy/bronchoalveolar lavage (BAL). 
x Consider infectious disease consult. 
x Conduct an in person evaluation approximately twice per week 
x Consider frequent chest X-ray as part of monitoring 
x Treat with systemic corticosteroids at a dose of 1-2 mg/kg/day 
SUHGQLVRQHRUHTXLYDOHQW:KHQV\PSWRPVLPSURYHWR*UDGH steroid 
taper should be started and continued over no less than 4 weeks. 
x Permanently discontinue for inability to reduce corticosteroid dose to 
PJRISUHGQLVRQHRUHTXLYDOHQWSHUGD\ZLWKLQZHHNV 
x Second episode of pneumonitis – discontinue MK-3475 if upon re- 
challenge the patient develops a second episode of Grade 2 or higher 
pneumonitis. 
Grade 3-4 
Discontinue 
pembrolizumab 
(MK-3475) 
x Hospitalize patient 
x Bronchoscopy with biopsy and/or BAL is recommended. 
x Immediately treat with IV steroids (methylprednisolone 125 mg IV). 
:KHQV\PSWRPVLPSURYHWR*UDGHDKLJKGRVHRUDOVWHURLG
(prednisone 1 to 2 mg/kg once per day or dexamethasone 4 mg every 4 
hours) taper should be started and continued over no less than 4 weeks. 
x If IV steroids followed by high dose oral steroids does not reduce initial 
symptoms within 48 to 72 hours, treat with additional anti-inflammatory 
measures. Discontinue additional anti-inflammatory measures upon 
symptom relief and initiate a prolonged steroid taper over 45 to 60 days. 
If symptoms worsen during steroid reduction, initiate a retapering of 
steroids starting at a higher dose of 80 or 100 mg followed by a more 
prolonged taper and administer additional anti-inflammatory measures, 
as needed 
x Add prophylactic antibiotics for opportunistic infections. 
 
 
 
Renal events 
x Nephritis 
x Nephritis autoimmune 
x Renal failure 
x Renal failure acute 
Grade 2 
Hold 
pembrolizumab 
(MK-3475). 
x 
Treatment with prednisone 1-2 mg/kg PO daily. 
x 
Permanently discontinue for inability to reduce corticosteroid dose to 
PJRISUHGQLVRQHRUHTXLYDOHQWSHUGD\ZLWKLQZHHNV 
x 
:KHQV\PSWRPVLPSURYHWR*UDGHVWHURLGWDSHUVKRXOGEH started 
and continued over no less than 4 weeks. 
Grade 3-4 
Discontinue 
pembrolizumab 
(MK-3475). 
x Renal consultation with consideration of ultrasound and/or biopsy as 
appropriate. 
x Treat with systemic corticosteroids at a dose of 1-2 mg/kg prednisone IV 
or equivalent once per day. 
x :KHQV\PSWRPVLPSURYHWR*UDGHVWHURLGWDSHUVKRXOGEH started 
and continued over no less than 4 weeks. 


NCI Protocol #:10009  
Version Date: 09/04/2020 
56 
 
 
 
Event(s) 
CTCAE v4.0 
Grade 
Management / 
Next Dose for 
pembrolizu
mab (MK-
3475) 
 
Action / Supportive Care Guidelines 
 
Skin events 
Grade 2 
No change in dose 
x Symptomatic treatment should be given such as topical 
glucocorticosteroids (e.g., betamethasone 0.1% cream or hydrocortisone 
1%) or urea-containing creams in combination with oral anti-pruritics 
(e.g., diphenhydramine HCl or hydroxyzine HCl). 
x Treatment with oral steroids is at investigator discretion for Grade 2 
events. 
 
x Pruritus 
x Rash 
x Rash generalized 
x Rash maculo-papular 
x Dermatitis exfoliative 
x Erythema multiforme 
x Steven’s Johnson 
syndrome 
x Toxic epidermal 
necrolysis 
 
 
Grade 3 
Hold 
pembrolizumab 
(MK-3475). 
x Consider Dermatology consultation and biopsy for confirmation of 
diagnosis. 
x Treatment with oral steroids is recommended, starting with 1 mg/kg 
prednisone or equivalent once per day or dexamethasone 4 mg four 
times orally GDLO\:KHQV\PSWRPVLPSURYHWR*UDGHVWHURLG taper 
should be started and continued over no less than 4 weeks. 
x Permanently discontinue for inability to reduce corticosteroid dose to 
PJRISUHGQLVRQHRUHTXLYDOHQWSHUGD\ZLWKLQZHHNV 
See 6.1.3 for immediate 
evaluation for skin 
events 
 
 
Grade 4 
Permanently 
discontinue 
pembrolizumab 
(MK-3475). 
x Dermatology consultation and consideration of biopsy and clinical 
dermatology photograph. 
x Initiate steroids at 1-2 mg/kg prednisone or equivalent. When symptoms 
LPSURYHWR*UDGHVWHURLGWDSHUVKRXOGEHVWDUWHGDQGFRQWLQXHGRYHU
no less than 4 weeks. 
Thrombocytopenia 
*UDGH 
No change in dose  
 
Grade 2 
No change in dose  
Grade 3 
No change in dose 
x Grade 3 drug-related thrombocytopenia >7 days or associated with 
bleeding requires discontinuation. 
Grade 4 
Hold 
pembrolizumab 
(MK-3475) 
until resolves to 
*UDGH0D\
increase the dosing 
interval by 1 week. 
x Grade 4 drug-related thrombocytopenia >7 days or associated with 
bleeding requires discontinuation. 
Vomiting 
*UDGH 
No change in dose 
x Vomiting should be treated aggressively, and consideration should be 
given in subsequent cycles to the administration of prophylactic 
antiemetic therapy according to standard institutional practice. Patients 
should be strongly encouraged to maintain liberal oral fluid intake. 
 
Grade 2 
+ROGXQWLO*UDGH 
1. Resume at same 
dose level. May 
increase dosing 
interval by 1 week 
if it takes more 
than 4 weeks for 
toxicities to 
resolve. 


NCI Protocol #:10009  
Version Date: 09/04/2020 
57 
 
 
 
Event(s) 
CTCAE v4.0 
Grade 
Management / 
Next Dose for 
pembrolizu
mab (MK-
3475) 
 
Action / Supportive Care Guidelines 
 
 
Grade 3 
Hold until <Grade 
2. May increase 
dosing interval by 
1 week for each 
occurrence. 
 
 
 
Discontinue if 
toxicities do not 
resolve within 12 
weeks. 
Grade 4 
Off protocol 
therapy 
Other events 
x Myocarditis 
x Pericarditis 
x Pancreatitis 
Grade 2 or Grade 1 
events that do not 
improve with 
symptomatic 
treatment 
Hold 
pembrolizumab 
(MK-3475) 
x Systemic corticosteroids may be indicated. 
x Consider biopsy for confirmation of diagnosis. 
x If pembrolizumab (MK-3475) is held and corticosteroid required, 
manage as per Grade 3 below. 
Grade 3 
Hold 
pembrolizumab 
(MK-3475). 
x Treat with systemic corticosteroids at a dose of 1 to 2 mg/kg prednisone 
or equivalent once per day. 
x :KHQV\PSWRPVLPSURYHWR*UDGHVWHURLGWDSHUVKRXOGEH started 
and continued over no less than 4 weeks. 
x Permanently discontinue for inability to reduce corticosteroid dose to 
PJRISUHGQLVRQHRUHTXLYDOHQWSHUGD\ZLWKLQZHHNV Otherwise, 
pembrolizumab (MK-3475) treatment may be restarted and dose 
modified as specified in the protocol. 
 
Grade 4 
Discontinue 
pembrolizumab 
(MK-3475). 
x Treat with systemic corticosteroids at a dose of 1-2 mg/kg prednisone or 
equivalent once per day. 
 
aTopalian S.L., F.S. Hodi, J.R. Brahmer, et al. (2012). Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J 
Med. 366:2443-2454. 


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58 
 
 
 
Dose Modification and Toxicity Management Guidelines for Immune-related AEs 
Associated with Pembrolizumab 
 
 
General instructions: 
1. Severe and life-threatening irAEs should be treated with IV corticosteroids followed by oral steroids. Other immunosuppressive tre
irAEs are not controlled by corticosteroids. 
2. Pembrolizumab must be permanently discontinued if the irAE does not resolve or the corticosteroid dose is not  mg/day within
pembrolizumab treatment. 
3. 7KHFRUWLFRVWHURLGWDSHUVKRXOGEHJLQZKHQWKHLU$(LV*UDGHDQGFRQWLQXHDWOHDVW weeks. 
4. ,ISHPEUROL]XPDEKDVEHHQZLWKKHOGSHPEUROL]XPDEPD\UHVXPHDIWHUWKHLU$(GHFUHDVHGWR*UDGHDIWHUFRUWLFRVWHURLG taper.
 
irAEs 
Toxicity grade 
(CTCAE V5.0) 
Action with 
pembrolizumab 
Corticosteroid and/or 
other therapies 
 
Monitoring and follow-up 
 
 
 
 
 
Pneumonitis 
Grade 2 
Withhold 
x Administer 
corticosteroids (initial 
dose of 1 - 2 mg/kg 
prednisone or 
equivalent) followed 
by taper 
 
x Add prophylactic 
antibiotics for 
opportunistic 
infections 
x Monitor participants for signs and sym
 
x Evaluate participants with suspected p
imaging and initiate corticosteroid trea
Recurrent Grade 2, 
Grade 3 or 4 
Permanently 
discontinue 
 
 
 
 
 
Diarrhea / Colitis 
Grade 2 or 3 
Withhold 
x Administer 
corticosteroids (initial 
dose of 1 - 2 mg/kg 
prednisone or 
equivalent) followed 
by taper 
x Monitor participants for signs and sym
diarrhea, abdominal pain, blood or mu
fever) and of bowel perforation (ie, per
 
x 3DUWLFLSDQWVZLWK*UDGHGLDUUKHDVX
consider GI consultation and performin
colitis 
 
x Participants with diarrhea/colitis shoul
quantities of clear fluids. If sufficient o
feasible, fluid and electrolytes should b
Recurrent Grade 3 
or Grade 4 
Permanently 
discontinue 
AST or ALT 
elevation or 
Increased Bilirubin 
Grade 2 a 
Withhold 
x Administer 
corticosteroids (initial 
dose of 0.5 - 1 mg/kg 
 


NCI Protocol #:10009  
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59 
 
 
 
 
 
prednisone or 
equivalent) followed 
by taper 
x Monitor with liver function tests (cons
frequently until liver enzyme value ret
Grade 3 b or 4 c 
Permanently 
discontinue 
x Administer 
corticosteroids (initial 
dose of 1 - 2 mg/kg 
prednisone or 
equivalent) followed 
by taper 
 
 
 
Type 1 diabetes 
mellitus (T1DM) or 
Hyperglycemia 
New onset T1DM 
or 
Grade 3 or 4 
hyperglycemia 
associated with 
evidence of E-cell 
failure 
Withhold d 
x Initiate insulin 
replacement therapy 
for participants with 
T1DM 
 
x Administer anti- 
hyperglycemic in 
participants with 
hyperglycemia 
x Monitor participants for hyperglycemi
symptoms of diabetes 
 
 
Hypophysitis 
Grade 2 
Withhold 
x Administer 
corticosteroids and 
initiate hormonal 
replacements as 
clinically indicated 
x Monitor for signs and symptoms of hy
hypopituitarism and adrenal insufficien
Grade 3 or 4 
Withhold or 
permanently 
discontinue d 
 
 
Hyperthyroidism 
Grade 2 
Continue 
x Treat with non- 
selective beta- 
blockers (eg, 
propranolol) or 
thionamides as 
appropriate 
x Monitor for signs and symptoms of thy
Grade 3 or 4 
Withhold or 
permanently 
discontinue d 
 
 
Hypothyroidism 
Grade 2, 3, 4 
Continue 
x Initiate thyroid 
replacement 
hormones (eg, 
levothyroxine or 
liothyronine) per 
standard of care 
x Monitor for signs and symptoms of thy


NCI Protocol #:10009  
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60 
 
 
Nephritis: 
grading according 
to increased 
creatinine or acute 
kidney injury 
Grade 2 
Withhold 
x Administer 
corticosteroids 
(prednisone 
1 – 2 mg/kg or 
equivalent) followed 
by taper 
x Monitor changes of renal function 
Grade 3 or 4 
Permanently 
discontinue 
 
 
Myocarditis 
Grade 1 or 2 
Withhold 
x Based on severity of 
AE administer 
corticosteroids 
x Ensure adequate evaluation to confirm
other causes 
Grade 3 or 4 
Permanently 
discontinue 
 
 
All Other immune- 
related AEs 
Persistent Grade 2 
Withhold 
x Based on severity of 
AE administer 
corticosteroids 
x Ensure adequate evaluation to confirm
causes 
Grade 3 
Withhold or 
discontinue based 
on the event e 
Recurrent Grade 3 
or Grade 4 
Permanently 
discontinue 
a AST/ALT: >3.0 - 5.0 x ULN if baseline normal; >3.0 - 5.0 x baseline, if baseline abnormal; 
bilirubin:>1.5 - 3.0 x ULN if baseline normal; >1.5 - 3.0 x baseline if baseline abnormal 
b AST/ALT: >5.0 to 20.0 x ULN, if baseline normal; >5.0 - 20.0 x baseline, if baseline abnormal; bilirubin:>3.0 - 10.0 x ULN if baseli
baseline if baseline abnormal 
c AST/ALT: >20.0 x ULN, if baseline normal; >20.0 x baseline, if baseline abnormal; 
bilirubin: >10.0 x ULN if baseline normal; >10.0 x baseline if baseline abnormal 
d The decision to withhold or permanently discontinue pembrolizumab is at the discretion of the investigator or treating physician. If co
pembrolizumab may be resumed. 
e Events that require discontinuation include but are not limited to: Guillain-Barre Syndrome, encephalitis, Stevens-Johnson Syndrome
necrolysis. 


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6.2.4 Immediate Evaluation for Potential Skin Events 
 
Photographs 
Every attempt should be made to get a photograph of the actual skin lesion or rash as soon as 
possible. Obtain appropriate consent for subject photographs if a consent form addendum 
is required by your IRB/ERC. 
 
x 
Take digital photographs of: 
o the head (to assess mucosal or eye involvement), 
o the trunk and extremities, and 
o a close-up of the skin lesion/rash. 
x 
If possible, a ruler should be placed alongside the site of a skin occurrence as a fixed marker 
of distance. 
x 
The time/date stamp should be set in the 'ON' position for documentation purposes. 
x 
Photographs should be stored with the patient’s study records. 
 
Dermatology Consult 
Refer the subject to a dermatologist as soon as possible. 
x 
For a “severe rash”, the subject must be seen within 1-2 days of reporting the event. 
x 
For clinically significant rash, the subject should be seen within 3-5 days. 
 
The dermatologist should submit a biopsy sample to a certified dermatopathology laboratory or 
to a pathologist experienced in reviewing skin specimens. 
 
The site should provide the dermatologist with all relevant case history, including copies of 
clinical photographs and laboratory test results. 
 
 
6.2.5 Treatment Guidelines for Infusion Reactions 
 
The table below shows treatment guidelines for subjects who experience an infusion reaction 
associated with administration of pembrolizumab (MK-3475). 


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                NCI Protocol #:10009  
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Treatment Guidelines for Infusion Reactions 
NCI CTCAE Grade 
Treatment 
Premedication at subsequent 
dosing 
Grade 1 
Mild reaction; infusion interruption 
not indicated; intervention not 
indicated 
 
Increase monitoring of vital signs as medically 
indicated until the patient is deemed medically 
stable in the opinion of the investigator. 
 
None 
Grade 2 
Requires infusion interruption but 
responds promptly to symptomatic 
treatment (e.g., antihistamines, 
NSAIDS, narcotics, IV fluids); 
prophylactic medications indicated 
IRUKRXUV 
 
Stop Infusion. 
Additional appropriate medical therapy may 
include but is not limited to: 
IV fluids 
Antihistamines 
NSAIDS 
Acetaminophen 
Narcotics 
Increase monitoring of vital signs as medically 
indicated until the subject is deemed medically 
stable in the opinion of the investigator. 
If symptoms resolve within one hour of 
stopping drug infusion, the infusion may be 
restarted at 50% of the original infusion rate 
(e.g. from 100 mL/hr to 50 mL/hr). Please 
note: prior to restarting the infusion, confirm 
that the 4 hour room temperature stability from 
the time of the IV bag preparation will not be 
exceeded. Otherwise dosing will be held until 
symptoms resolve and the subject should be 
premedicated for the next scheduled dose. 
Subjects who develop Grade 2 toxicity upon 
rechallenge despite adequate premedication 
should be permanently discontinued from 
further trial treatment administration. 
 
Subject may be premedicated 1.5h 
(± 30 minutes) prior to infusion of 
pembrolizumab (MK-3475) with: 
 
Diphenhydramine 50 mg PO (or 
equivalent dose of antihistamine). 
 
Acetaminophen 500-1000 mg PO 
(or equivalent dose of antipyretic). 
Grades 3 or 4 
Grade 3: 
Prolonged (i.e., not rapidly 
responsive to symptomatic 
medication and/or brief interruption 
of infusion); recurrence of symptoms 
following initial improvement; 
hospitalization indicated for other 
clinical sequelae (e.g., renal 
impairment, pulmonary infiltrates) 
 
Grade 4: 
Life-threatening; pressor or 
ventilatory support indicated 
 
Stop Infusion. 
Additional appropriate medical therapy may 
include but is not limited to: 
IV fluids 
Antihistamines 
NSAIDS 
Acetaminophen 
Narcotics 
Oxygen 
Pressors 
Corticosteroids 
Epinephrine 
 
No subsequent dosing 
 
Increase monitoring of vital signs as medically 
indicated until the subject is deemed medically 
stable in the opinion of the investigator. 
Hospitalization may be indicated. 
Subject is permanently discontinued from 
further trial treatment administration. 
 
Appropriate resuscitation equipment should be available in the room and a physician readily available during the period of 
drug administration. 
For Further information, please refer to the Common Terminology Criteria for Adverse Events v4.0 (CTCAE) at 
http://ctep.cancer.gov 


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7. ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS 
 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial. The 
following list of AEs (Section 7.1) and the characteristics of an observed AE (Sections 7.2 and 
7.3) will determine whether the event requires expedited reporting via the CTEP Adverse Event 
Reporting System(CTEP-AERS) in addition to routine reporting 
 
7.1 
Comprehensive Adverse Events and Potential Risks List(s) (CAEPRs) 
 
The Comprehensive Adverse Event and Potential Risks list (CAEPR) provides a single list of 
reported and/or potential adverse events (AE) associated with an agent using a uniform 
presentation of events by body system. In addition to the comprehensive list, a subset of AEs, 
the Specific Protocol Exceptions to Expedited Reporting (SPEER), appears in a separate column 
and is identified with bold and italicized text. The SPEER is a list of events that are protocol- 
specific exceptions to expedited reporting to NCI (except as noted below). Refer to the 'CTEP, 
NCI Guidelines: Adverse Event Reporting Requirements' 
http://ctep.cancer.gov/protocolDevelopment/adverse_effects.htm for further clarification. 
 
NOTE: The highest grade currently reported is noted in parentheses next to the AE in the 
SPEER. Report ONLY AEs higher than this grade expeditiously. If this CAEPR is part of a 
combination protocol using multiple investigational agents and has an AE listed on different 
SPEERs, use the lower of the grades to determine if expedited reporting is required. 
 
7.1.1 CAEPRs for CTEP IND Agent(s) 
 
7.1.1.1 
CAEPR for entinostat 
 
Comprehensive Adverse Events and Potential Risks list (CAEPR) for 
MS-275 (SNDX-275, entinostat, NSC 706995) 
The Comprehensive Adverse Event and Potential Risks list (CAEPR) provides a single list of 
reported and/or potential adverse events (AE) associated with an agent using a uniform 
presentation of events by body system. In addition to the comprehensive list, a subset, the 
Specific Protocol Exceptions to Expedited Reporting (SPEER), appears in a separate column 
and is identified with bold and italicized text. This subset of AEs (SPEER) is a list of events 
that are protocol specific exceptions to expedited reporting to NCI via AdEERS (except as 
noted below). Refer to the 'CTEP, NCI Guidelines: Adverse Event Reporting Requirements' 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/aeguidelines.pdf for 
further clarification. Frequency is provided based on 221 patients. Below is the CAEPR for 
MS-275 (SNDX-275, entinostat). 
NOTE: Report AEs on the SPEER ONLY IF they exceed the grade noted in parentheses 
next to the AE in the SPEER. If this CAEPR is part of a combination protocol using 
multiple investigational agents and has an AE listed on different SPEERs, use the lower of 
the grades to determine if expedited reporting is required. 


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                NCI Protocol #:10009  
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Version 2.5, September 10, 20181 
 
Adverse Events with Possible 
Relationship to MS-275 (SNDX-275, entinostat) 
(CTCAE 5.0 Term) 
[n= 221] 
  
Specific Protocol Exceptions to 
Expedited Reporting (SPEER) 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious 
(<3%) 
  
BLOOD AND LYMPHATIC SYSTEM DISORDERS 
  
Anemia 
 
 
 Anemia (Gr 3) 
GASTROINTESTINAL DISORDERS 
  
 
Abdominal pain 
 
 Abdominal pain (Gr 2) 
 
Constipation 
 
 Constipation (Gr 2) 
 
Diarrhea 
 
 Diarrhea (Gr 3) 
 
Dyspepsia 
 
 Dyspepsia (Gr 2) 
Nausea 
 
 
 Nausea (Gr 3) 
Vomiting 
 
 
 Vomiting (Gr 3) 
GENERAL DISORDERS AND ADMINISTRATION SITE 
CONDITIONS 
  
 
Edema limbs 
 
 Edema limbs (Gr 2) 
Fatigue 
 
 
 Fatigue (Gr 3) 
 
Fever 
 
 Fever (Gr 2) 
INFECTIONS AND INFESTATIONS 
  
 
Infection2 
 
 Infection2 (Gr 3) 
INVESTIGATIONS 
  
 
Alkaline phosphatase 
increased 
 
 Alkaline phosphatase increased 
(Gr 2) 
 
Lymphocyte count 
decreased 
 
 Lymphocyte count decreased 
(Gr 4) 
Neutrophil count 
decreased 
 
 
 Neutrophil count decreased (Gr 
4) 
Platelet count decreased  
 
 Platelet count decreased (Gr 4) 
 
White blood cell decreased  
 White blood cell decreased (Gr 
3) 
METABOLISM AND NUTRITION DISORDERS 
  
Anorexia 
 
 
 Anorexia (Gr 3) 
 
Dehydration 
 
 Dehydration (Gr 2) 
 
Hyperglycemia 
 
 Hyperglycemia (Gr 2) 
Hypoalbuminemia 
 
 
 Hypoalbuminemia (Gr 2) 
 
Hypocalcemia 
 
 Hypocalcemia (Gr 2) 
 
Hypokalemia 
 
 Hypokalemia (Gr 2) 
 
Hyponatremia 
 
 Hyponatremia (Gr 3) 
Hypophosphatemia 
 
 
 Hypophosphatemia (Gr 3) 


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                NCI Protocol #:10009  
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Adverse Events with Possible 
Relationship to MS-275 (SNDX-275, entinostat) 
(CTCAE 5.0 Term) 
[n= 221] 
 
Specific Protocol Exceptions to 
Expedited Reporting (SPEER) 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious 
(<3%) 
 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS 
 
 
Myalgia 
 
Myalgia (Gr 2) 
NERVOUS SYSTEM DISORDERS 
 
 
Dysgeusia 
 
Dysgeusia (Gr 2) 
Headache 
 
 
Headache (Gr 2) 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS 
 
 
Cough 
 
Cough (Gr 2) 
 
Dyspnea 
 
Dyspnea (Gr 3) 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS 
 
 
 
Erythema multiforme 
 
 
1This table will be updated as the toxicity profile of the agent is revised. Updates will be distributed to all 
Principal Investigators at the time of revision. The current version can be obtained by contacting 
PIO@CTEP.NCI.NIH.GOV. Your name, the name of the investigator, the protocol and the agent should 
be included in the e-mail. 
 
2Infection includes all 75 sites of infection under the INFECTIONS AND INFESTATION SOC. 
 
3Gastrointestinal hemorrhage includes Anal hemorrhage, Cecal hemorrhage, Colonic hemorrhage, 
Duodenal hemorrhage, Esophageal hemorrhage, Esophageal varices hemorrhage, Gastric hemorrhage, 
Hemorrhoidal hemorrhage, Ileal hemorrhage, Intra-abdominal hemorrhage, Jejunal hemorrhage, Lower 
gastrointestinal hemorrhage, Oral hemorrhage, Pancreatic hemorrhage, Rectal hemorrhage, 
Retroperitoneal hemorrhage, and Upper gastrointestinal hemorrhage under the GASTROINTESTINAL 
DISORDERS SOC. 
 
 
Adverse events reported on MS-275 (SNDX-275, entinostat) trials, but for which there is 
insufficient evidence to suggest that there was a reasonable possibility that MS-275 (SNDX- 
275, entinostat) caused the adverse event: 
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS - Febrile neutropenia; Hemolysis; 
Leukocytosis 
CARDIAC DISORDERS - Atrial fibrillation; Atrioventricular block complete; Cardiac 
disorders - Other (transient right-side heart failure with worsening tricuspid regurgitation); Chest 
pain - cardiac; Conduction disorder; Heart failure; Left ventricular systolic dysfunction; 
Palpitations; Pericardial effusion; Pericarditis; Sinus tachycardia; Supraventricular tachycardia; 
Ventricular fibrillation 
EAR AND LABYRINTH DISORDERS - Hearing impaired 
EYE DISORDERS - Blurred vision 


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                NCI Protocol #:10009  
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GASTROINTESTINAL DISORDERS - Anal mucositis; Colitis; Dysphagia; Enterocolitis; 
Esophageal pain; Esophagitis; Flatulence; Gastrointestinal disorders - Other (hyperdefecation); 
Gastrointestinal hemorrhage3; Hemorrhoids; Mucositis oral; Pancreatitis; Periodontal disease; 
Rectal mucositis; Rectal pain; Small intestinal mucositis; Typhlitis; Visceral arterial ischemia 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS - Chills; Edema 
face; Generalized edema; Injection site reaction; Multi-organ failure; Non-cardiac chest pain; 
Pain 
IMMUNE SYSTEM DISORDERS - Allergic reaction; Anaphylaxis; Autoimmune disorder 
INJURY, POISONING AND PROCEDURAL COMPLICATIONS - Bruising 
INVESTIGATIONS - Activated partial thromboplastin time prolonged; Alanine 
aminotransferase increased; Aspartate aminotransferase increased; Blood bilirubin increased; 
CPK increased; Creatinine increased; GGT increased; INR increased; Investigations - Other 
(coagulopathy); Investigations - Other (vitamin D deficiency); Lipase increased; Serum amylase 
increased; Weight loss 
METABOLISM AND NUTRITION DISORDERS - Acidosis; Hypercalcemia; Hyperkalemia; 
Hypermagnesemia; Hypernatremia; Hypertriglyceridemia; Hyperuricemia; Hypoglycemia; 
Hypomagnesemia; Tumor lysis syndrome 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS - Arthralgia; Back 
pain; Bone pain; Chest wall pain; Generalized muscle weakness; Muscle cramp; Musculoskeletal 
and connective tissue disorder - Other (thorax pain); Myositis; Pain in extremity 
NEOPLASMS BENIGN, MALIGNANT AND UNSPECIFIED (INCL CYSTS AND 
POLYPS) - Tumor pain 
NERVOUS SYSTEM DISORDERS - Ataxia; Depressed level of consciousness; Dizziness; 
Dysphasia; Intracranial hemorrhage; Neuralgia; Olfactory nerve disorder; Peripheral motor 
neuropathy; Peripheral sensory neuropathy; Seizure; Syncope; Tremor 
PSYCHIATRIC DISORDERS - Anxiety; Confusion; Depression; Insomnia; Libido decreased 
RENAL AND URINARY DISORDERS - Acute kidney injury; Proteinuria; Renal and urinary 
disorders - Other (bladder distension); Renal calculi; Renal hemorrhage; Urinary frequency; 
Urinary retention 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS - Allergic rhinitis; 
Atelectasis; Epistaxis; Hypoxia; Laryngeal mucositis; Pharyngeal mucositis; Pleural effusion; 
Pleuritic pain; Pulmonary edema; Respiratory failure; Tracheal mucositis 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS - Alopecia; Hyperhidrosis; Nail loss; 
Photosensitivity; Pruritus; Purpura; Rash maculo-papular; Skin and subcutaneous tissue 
disorders - Other (hyperkeratotic lesions/squamous cell carcinoma); Urticaria 
SURGICAL AND MEDICAL PROCEDURES - Surgical and medical procedures - Other 
(packed RBC transfusion) 
VASCULAR DISORDERS - Flushing; Hypertension; Hypotension; Thromboembolic event 
 
Note: MS-275 (SNDX-275, entinostat) in combination with other agents could cause an 
exacerbation of any adverse event currently known to be caused by the other agent, or the 
combination may result in events never previously associated with either agent. 


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7.1.1.2 
CAEPR for pembrolizumab (MK-3475) 
 
Comprehensive Adverse Events and Potential Risks list (CAEPR) 
for 
MK-3475 (pembrolizumab, NSC 776864) 
 
The Comprehensive Adverse Events and Potential Risks list (CAEPR) provides a single list of reported and/or 
potential adverse events (AE) associated with an agent using a uniform presentation of events by body system. In 
addition to the comprehensive list, a subset, the Specific Protocol Exceptions to Expedited Reporting (SPEER), 
appears in a separate column and is identified with bold and italicized text. This subset of AEs (SPEER) is a list of 
events that are protocol specific exceptions to expedited reporting to NCI (except as noted below). Refer to the 
'CTEP, NCI Guidelines: Adverse Event Reporting Requirements' 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/aeguidelines.pdf for further clarification. 
Frequency is provided based on 3793 patients. Below is the CAEPR for MK-3475 (pembrolizumab). 
NOTE: Report AEs on the SPEER ONLY IF they exceed the grade noted in parentheses next to the AE in the 
SPEER. If this CAEPR is part of a combination protocol using multiple investigational agents and has an AE 
listed on different SPEERs, use the lower of the grades to determine if expedited reporting is required. 
 
Version 2.5, December 27, 20191 
 
 
 
Adverse Events with Possible 
Relationship to MK-3475 (pembrolizumab) 
(CTCAE 5.0 Term) 
[n= 3793] 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
BLOOD AND LYMPHATIC SYSTEM DISORDERS 
 
Anemia2 
 
 
Lymph node pain2 
 
 
Thrombotic thrombocytopenic purpura2  
CARDIAC DISORDERS 
 
 
Myocarditis2 
 
 
Pericarditis2 
ENDOCRINE DISORDERS 
 
Adrenal insufficiency2 
 
 
Endocrine disorders - Other 
(thyroiditis)2 
 
 
Hyperthyroidism2 
 
 
Hypohysitis2 
 
 
Hypopituitarism2 
 
 
Hypothyroidism2 
 
EYE DISORDERS 
 
 
Uveitis2 
 
 
Eye disorders - Other (Vogt- 
Koyanagi-Harada syndrome) 
GASTROINTESTINAL DISORDERS 
 
Abdominal pain 
 
 
Colitis2 
 
 
Diarrhea2 
 
 
Mucositis oral2 
 
 
Nausea 
 
 
Pancreatitis2 
 
 
Small intestinal mucositis2 
 
 
 
Specific Protocol Exceptions to 
Expedited Reporting (SPEER) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Diarrhea2 (Gr 2) 
 
Nausea (Gr 2) 
 
 


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Adverse Events with Possible 
Relationship to MK-3475 (pembrolizumab) 
(CTCAE 5.0 Term) 
[n= 3793] 
  
 
Specific Protocol Exceptions to 
Expedited Reporting (SPEER) 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS 
 
 
Chills2 
 
 
Fatigue 
 
 
Fatigue (Gr 2) 
 
Fever2 
 
 
HEPATOBILIARY DISORDERS 
 
 
Hepatobiliary disorders - Other 
(autoimmune hepatitis)2 
 
 
IMMUNE SYSTEM DISORDERS 
 
 
 
Anaphylaxis2 
 
 
 
Cytokine release syndrome2 
 
 
 
Immune system disorders - Other 
(acute graft-versus-host-disease)2,3 
 
 
 
Immune system disorders - Other 
(hemophagocytic 
lymphohistiocytosis)2 
 
 
Immune system disorders - Other 
(pseudoprogression/tumor 
inflammation)2 
 
 
 
Immune system disorders - Other 
(sarcoidosis)2 
 
 
 
 
Serum sickness2 
 
INFECTIONS AND INFESTATIONS 
 
 
Infection4 
 
 
INJURY, POISONING AND PROCEDURAL COMPLICATIONS 
 
 
 
Infusion related reaction 
 
INVESTIGATIONS 
 
 
Alanine aminotransferase increased2 
 
 
 
Alkaline phosphatase increased 
 
 
 
Aspartate aminotransferase increased2  
 
 
Blood bilirubin increased 
 
 
 
CPK increased 
 
 
 
 
GGT increased 
 
 
 
Serum amylase increased 
 
METABOLISM AND NUTRITION DISORDERS 
 
 
Anorexia 
 
 
 
Hyponatremia 
 
 
 
 
Metabolism and nutrition 
disorders - Other (diabetic 
ketoacidosis)2 
 
 
 
Metabolism and nutrition 
disorders - Other (type 1 diabetes 
mellitus)2 
 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS 
 
 
Arthralgia2 
 
Arthralgia2 (Gr 2) 
 
Arthritis2 
 
 
 
Avascular necrosis2 
 
 
 
Back pain 
 
 
 
Joint effusion2 
 
 
 
Joint range of motion decreased 
 
 
 
Musculoskeletal and connective tissue 
disorder - Other (tenosynovitis)2 
 
 


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Adverse Events with Possible 
Relationship to MK-3475 (pembrolizumab) 
(CTCAE 5.0 Term) 
[n= 3793] 
  
 
Specific Protocol Exceptions to 
Expedited Reporting (SPEER) 
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
 
 
Myalgia2 
 
 
 
Myositis2 
 
 
NERVOUS SYSTEM DISORDERS 
 
 
 
Guillain-Barre syndrome2 
 
 
 
Nervous system disorders - Other 
(myasthenic syndrome)2 
 
 
 
Nervous system disorders - Other 
(neuromyopathy)2 
 
 
 
Nervous system disorders - Other 
(non-infectious encephalitis)2 
 
 
 
Nervous system disorders - Other 
(non-infectious meningitis)2 
 
 
 
Nervous system disorders - Other 
(non-infectious myelitis) 
 
 
 
Nervous system disorders - Other 
(polyneuropathy)2 
 
 
 
Paresthesia 
 
 
 
Peripheral motor neuropathy2 
 
RENAL AND URINARY DISORDERS 
 
 
 
Renal and urinary disorders - 
Other (autoimmune nephritis)2 
 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS 
 
 
Cough 
 
 
 
Pleuritic pain2 
 
 
 
Pneumonitis2 
 
 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS 
 
 
Bullous dermatitis2 
 
 
 
 
Erythema multiforme2 
 
 
Erythroderma 
 
 
 
 
Palmar-plantar erythrodysesthesia 
syndrome 
 
 
Pruritus2 
 
Pruritus2 (Gr 2) 
 
Rash acneiform2 
 
 
 
Rash maculo-papular2 
 
Rash maculo-papular2 (Gr 2) 
 
Skin and subcutaneous tissue disorders 
- Other (dermatitis)2 
 
 
 
Skin hypopigmentation2 
 
 
 
 
Stevens-Johnson syndrome2 
 
 
 
Toxic epidermal necrolysis 
 
 
Urticaria2 
 
 
VASCULAR DISORDERS 
 
 
 
Vasculitis2 
 
 
 
1This table will be updated as the toxicity profile of the agent is revised. Updates will be distributed to all 
Principal Investigators at the time of revision. The current version can be obtained by contacting 
PIO@CTEP.NCI.NIH.GOV. Your name, the name of the investigator, the protocol and the agent should be 
included in the e-mail. 
 
2Immune-mediated adverse reactions have been reported in patients receiving MK-3475 (pembrolizumab). 


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Adverse events potentially related to MK-3475 (pembrolizumab) may be manifestations of immune-mediated 
adverse events. In clinical trials, most immune-mediated adverse reactions were reversible and managed with 
interruptions of MK-3475 (pembrolizumab), administration of corticosteroids and supportive care. 
 
3Acute graft-versus-host disease has been observed in patients treated with MK-3475 (pembrolizumab) who 
received hematopoeitic stem cell transplants. 
 
4Infection includes all 75 sites of infection under the INFECTIONS AND INFESTATIONS SOC. 
 
Adverse events reported on MK-3475 (pembrolizumab) trials, but for which there is insufficient evidence to 
suggest that there was a reasonable possibility that MK-3475 (pembrolizumab) caused the adverse event: 
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS - Blood and lymphatic system disorders - Other 
(pancytopenia); Disseminated intravascular coagulation; Hemolysis 
CARDIAC DISORDERS - Atrial fibrillation; Cardiac arrest; Chest pain - cardiac; Heart failure; Myocardial 
infarction; Pericardial effusion; Pericardial tamponade; Ventricular arrhythmia 
EYE DISORDERS - Eye pain 
GASTROINTESTINAL DISORDERS - Abdominal distension; Ascites; Constipation; Duodenal hemorrhage; 
Dysphagia; Gastritis; Gastrointestinal disorders - Other (diverticulitis); Gastrointestinal disorders - Other 
(intestinal obstruction); Gastrointestinal disorders - Other (intussusception); Oral pain; Rectal hemorrhage; Small 
intestinal perforation; Upper gastrointestinal hemorrhage; Vomiting 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS - Edema face; Edema limbs; 
Facial pain; Gait disturbance; General disorders and administration site conditions - Other (general physical health 
deterioration); Generalized edema; Malaise; Non-cardiac chest pain; Pain 
INVESTIGATIONS - Cholesterol high; Creatinine increased; Fibrinogen decreased; Lymphocyte count 
decreased; Neutrophil count decreased; Platelet count decreased; Weight loss; White blood cell decreased 
METABOLISM AND NUTRITION DISORDERS - Dehydration; Hypercalcemia; Hyperglycemia; 
Hyperkalemia; Hypertriglyceridemia; Hyperuricemia; Hypoalbuminemia; Hypokalemia; Hypophosphatemia; 
Metabolism and nutrition disorders - Other (failure to thrive); Tumor lysis syndrome 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS - Bone pain; Generalized muscle 
weakness; Musculoskeletal and connective tissue disorder - Other (groin pain); Pain in extremity 
NERVOUS SYSTEM DISORDERS - Aphonia; Depressed level of consciousness; Dysarthria; Edema cerebral; 
Encephalopathy; Headache; Hydrocephalus; Lethargy; Meningismus; Nervous system disorders - Other 
(brainstem herniation); Seizure; Syncope; Tremor 
PSYCHIATRIC DISORDERS - Agitation; Confusion 
RENAL AND URINARY DISORDERS - Acute kidney injury; Nephrotic syndrome; Proteinuria; Renal and 
urinary disorders - Other (hydronephrosis); Urinary incontinence; Urinary tract pain 
REPRODUCTIVE SYSTEM AND BREAST DISORDERS - Pelvic pain 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS - Dyspnea; Hypoxia; Laryngeal 
inflammation; Pleural effusion; Pneumothorax; Respiratory failure 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS - Alopecia; Dry skin; Skin and subcutaneous tissue 
disorders - Other (drug eruption) 
VASCULAR DISORDERS - Hypertension; Peripheral ischemia; Thromboembolic event 
 
Note: MK-3475 (pembrolizumab) in combination with other agents could cause an exacerbation of any adverse 
event currently known to be caused by the other agent, or the combination may result in events never previously 
associated with either agent. 


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7.2 
Adverse Event Characteristics 
 
x CTCAE term (AE description) and grade: The descriptions and grading scales found 
in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
4.0 will be utilized until March 31, 2018 for AE reporting. CTCAE version 5.0 will be 
utilized for AE reporting beginning April 1, 2018. All appropriate treatment areas should 
have access to a copy of the CTCAE version 5.0. A copy of the CTCAE version 5.0 can 
be downloaded from the CTEP web site 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
 
x For expedited reporting purposes only: 
- 
AEs for the agent that are bold and italicized in the CAEPR (i.e., those listed in the 
SPEER column, Section 7.1.1) should be reported through CTEP-AERS only if the 
grade is above the grade provided in the SPEER. 
- 
Other AEs for the protocol that do not require expedited reporting are outlined in 
section 7.3.4. 
 
x Attribution of the AE: 
- 
Definite – The AE is clearly related to the study treatment. 
- 
Probable – The AE is likely related to the study treatment. 
- 
Possible – The AE may be related to the study treatment. 
- 
Unlikely – The AE is doubtfully related to the study treatment. 
- 
Unrelated – The AE is clearly NOT related to the study treatment. 
 
7.3 
Expedited Adverse Event Reporting 
 
7.3.1 Expedited AE reporting for this study must use CTEP-AERS (CTEP Adverse Event 
Reporting System), accessed via the CTEP Web site (https://eapps- 
ctep.nci.nih.gov/ctepaers). The reporting procedures to be followed are presented in the 
“NCI Guidelines for Investigators: Adverse Event Reporting Requirements for DCTD 
(CTEP and CIP) and DCP INDs and IDEs” which can be downloaded from the CTEP 
Web site 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/adverse_events.htm 
). These requirements are briefly outlined in the tables below (Section 7.3.3). 
 
In the rare occurrence when Internet connectivity is lost, a 24-hour notification is to be 
made to CTEP by telephone at 301-897-7497. Once Internet connectivity is restored, the 
24-hour notification phoned in must be entered electronically into CTEP-AERS by the 
original submitter at the site. 
 
7.3.2 CTEP-AERS is programmed for automatic electronic distribution of reports to the 
following individuals: Principal Investigator and Adverse Event Coordinator(s) (if 
applicable) of the Corresponding Organization, the local treating physician, and the 
Reporter and Submitter. CTEP-AERS provides a copy feature for other e-mail recipients. 
 
The Coordinating Center of the Corresponding Organization is responsible for submitting 


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to the CTSU documentation of AEs that they deem reportable for posting on the CTSU 
protocol web page and inclusion on the CTSU bi-monthly broadcast. 
 
7.3.3 Expedited Reporting Guidelines 
 
Use the NCI protocol number and the protocol-specific patient ID assigned during trial 
registration on all reports. 
 
Note: A death on study requires both routine and expedited reporting, regardless of 
causality. Attribution to treatment or other cause must be provided. 
 
Death due to progressive disease should be reported as Grade 5 “Disease progression” 
in the system organ class (SOC) “General disorders and administration site conditions.”. 
Evidence that the death was a manifestation of underlying disease (e.g., radiological 
changes suggesting tumor growth or progression: clinical deterioration associated with a 
disease process) should be submitted. 
 
Phase 1 and Early Phase 2 Studies: Expedited Reporting Requirements for Adverse 
Events that Occur on Studies under an IND/IDE within 30 Days of the Last Administration 
of the Investigational Agent/Intervention 1, 2 
 
FDA REPORTING REQUIREMENTS FOR SERIOUS ADVERSE EVENTS (21 CFR Part 312) 
NOTE: Investigators MUST immediately report to the sponsor (NCI) ANY Serious Adverse Events, whether or not 
they are considered related to the investigational agent(s)/intervention (21 CFR 312.64) 
An adverse event is considered serious if it results in ANY of the following outcomes: 
1) 
Death 
2) 
A life-threatening adverse event 
3) 
An adverse event that results in inpatient hospitalization or prolongation of existing hospitalization for  24 
hours 
4) 
A persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions 
5) 
A congenital anomaly/birth defect. 
6) 
Important Medical Events (IME) that may not result in death, be life threatening, or require hospitalization 
may be considered serious when, based upon medical judgment, they may jeopardize the patient or 
subject and may require medical or surgical intervention to prevent one of the outcomes listed in this 
definition. (FDA, 21 CFR 312.32; ICH E2A and ICH E6). 
ALL SERIOUS adverse events that meet the above criteria MUST be immediately reported to the NCI via electronic 
submission within the timeframes detailed in the table below. 
Hospitalization 
Grade 1 and Grade 2 Timeframes 
Grade 3-5 
Timeframes 
Resulting in 
Hospitalization 
KUV 
 
10 Calendar Days 
 
 
24-Hour 5 Calendar 
Days 
Not resulting in 
Hospitalization 
KUV 
 
Not required 


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7.3.4
Additional Protocol-Specific Expedited Adverse Event Reporting Exclusions 
For this protocol only, the AEs/grades listed below do not require expedited reporting via 
CTEP-AERS. However, they still must be reported through the routine reporting 
mechanism (Section 7.4): 
CTCAE SOC
Adverse Event
Grade
Hospitalization/ 
Prolongation of 
Hospitalization
Attribution
Comments
Blood and 
Lymphatic 
System/Investigati 
ons 
Bone marrow 
cellularity 
Hemoglobin 
Leukocytes 
Lymphopenia 
Neutrophils 
Platelets 
3 and 4
With or without 
hospitalization 
Leukemia 
Entinostat 
Pembrolizumab 
These events do not 
require expedited 
reporting unless in the 
opinion of PI they are out 
of proportion expected 
for patients with acute 
leukemia undergoing 
chemotherapy (i.e. 
prolonged duration of 
myelosuppression)
Gastrointestinal 
Nausea 
Vomiting 
Diarrhea 
Anorexia 
Taste alteration
3 
With or without 
hospitalization 
Leukemia 
Entinostat 
Pembrolizumab 
These events and 
complications directly 
and definitely related to 
these events such as 
electrolyte abnormalities, 
hypotension, or renal 
dysfunction due to 
hypotension from these 
GI toxicities do not 
require expedited 
reporting
Blood and 
lymphatic 
disorder/Infection
Febrile 
neutropenia 
Infection with
3 and 4
With or without 
hospitalization 
Leukemia 
Entinostat 
Pembrolizumab
1Serious adverse events that occur more than 30 days after the last administration of investigational 
agent/intervention and have an attribution of possible, probable, or definite require reporting as follows: 
Expedited 24-hour notification followed by complete report within 5 calendar days for: 
x
All Grade 3, 4, and Grade 5 AEs 
Expedited 10 calendar day reports for: 
x
Grade 2 AEs resulting in hospitalization or prolongation of hospitalization 
2For studies using PET or SPECT IND agents, the AE reporting period is limited to 10 radioactive half-lives, 
rounded UP to the nearest whole day, after the agent/intervention was last administered. Footnote “1” above 
applies after this reporting period.
Effective Date: May 5, 2011 
NOTE: Protocol specific exceptions to expedited reporting of serious adverse events are found in the Specific 
Protocol Exceptions to Expedited Reporting (SPEER) portion of the CAEPR. 
Expedited AE reporting timelines are defined as: 
“24-Hour; 5 Calendar Days” - The AE must initially be submitted electronically within 24 hours of learning 
of the AE, followed by a complete expedited report within 5 calendar days of the initial 24-hourreport.
“10 Calendar Days” - A complete expedited report on the AE must be submitted electronically within 10 
calendar days of learning of the AE. 


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grade 3 and 4 
neutrophils 
 
 
 
 
Blood and 
Lymphatic 
System/Investigati 
ons 
Bone marrow 
cellularity 
Hemoglobin 
Leukocytes 
Lymphopenia 
Neutrophils 
Platelets 
3 and 4 
With or without 
hospitalization 
Leukemia 
Entinostat 
Pembrolizumab 
These events do not 
require expedited 
reporting unless in the 
opinion of PI they are out 
of proportion expected 
for patients with acute 
leukemia undergoing 
chemotherapy (i.e. 
prolonged duration of 
myelosuppression) 
 
 
 
7.4 
Routine Adverse Event Reporting 
 
All Adverse Events must be reported in routine study data submissions. AEs reported 
expeditiously through CTEP-AERS must also be reported in routine study data 
submissions. 
 
Adverse event data collection and reporting, which are required as part of every clinical trial, are 
done to ensure the safety of patients enrolled in the studies as well as those who will enroll in 
future studies using similar agents. AEs are reported in a routine manner at scheduled times 
during the trial using Medidata Rave. For this trial the Adverse Event CRF is used for routine 
AE reporting in Rave. 
 
7.5 
Secondary Malignancy 
 
A secondary malignancy is a cancer caused by treatment for a previous malignancy (e.g., 
treatment with investigational agent/intervention, radiation or chemotherapy). A secondary 
malignancy is not considered a metastasis of the initial neoplasm. 
 
CTEP requires all secondary malignancies that occur following treatment with an agent under an 
NCI IND/IDE be reported expeditiously via CTEP-AERS. Three options are available to 
describe the event: 
 
x Leukemia secondary to oncology chemotherapy (e.g., acute myelocytic leukemia 
[AML]) 
x Myelodysplastic syndrome (MDS) 
x Treatment-related secondary malignancy 
 
Any malignancy possibly related to cancer treatment (including AML/MDS) should also be 
reported via the routine reporting mechanisms outlined in each protocol. 
 
7.6 
Second Malignancy 
 
A second malignancy is one unrelated to the treatment of a prior malignancy (and is NOT a 
metastasis from the initial malignancy). Second malignancies require ONLY routine AE 


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reporting unless otherwise specified. 
 
7.7 
Reporting of Pregnancy and Lactation to the Sponsor 
 
Although pregnancy and lactation are not considered adverse events, it is the responsibility of 
investigators or their designees to report any pregnancy or lactation in a subject (spontaneously 
reported to them) that occurs during the trial or within 120 days of completing the trial, or 30 
days following cessation of treatment if the subject initiates new anticancer therapy, whichever is 
earlier.  All subjects who become pregnant must be followed to the completion/termination of 
the pregnancy. Pregnancy outcomes of spontaneous abortion, missed abortion, benign 
hydatidiform mole, blighted ovum, pregnancy loss, intrauterine death, miscarriage and stillbirth 
must be reported as serious events (Important Medical Events). If the pregnancy continues to 
term, the outcome (health of infant) must also be reported. 
 
 
8. PHARMACEUTICAL INFORMATION 
 
A list of the adverse events and potential risks associated with the investigational agents 
administered in this study can be found in Section 7.1. 
 
 
8.1 
CTEP IND Agent(s) 
 
 
8.1.1 Entinostat (NSC 706995) 
 
Chemical name: 
3-Pyridylmethyl N-55carbamate 
 
Other names: 
MS-27-275, MS-275, SNDX-275 
 
Classification: 
Histone deacetylase inhibitor (HDACi) 
 
Molecular formula: C21H20N4O3 
 
M.W.: 376.41 
 
Mode of Action: 
Histone deacetylases (HDACs) are a family of enzymes that regulate 
chromatin remodeling and gene transcription via the dynamic  
process of acetylation and deacetylation of core histones. Entinostat 
inhibits histone deacetylases, changes chromatin configuration, and 
induces differentiation and apoptosis of cancer cells through an 
epigenetic mechanism. 
 
How Supplied: 
Entinostat is supplied by the Syndax Pharmaceuticals, Inc. and 
distributed by DCTD, NCI as 1 mg (pink to light red, in bottles of 
40), or 5 mg (yellow, in bottles of 40) film-coated tablets (round- 


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biconvex). Each tablet also contains mannitol, sodium starch 
glycolate, hydroxypropyl cellulose, potassium bicarbonate, and 
magnesium stearate. The film coating consists of hypromellose, 
talc, titanium dioxide, and ferric oxide pigments (red and yellow) 
as colorants. 
 
Matching placebo for entinostat has the same appearance as the 
corresponding active tablets and contains the same inactive 
ingredients and film coating. 
 
Storage: 
Store the bottles at controlled room temperature (15-25oC), and 
protect from light. Entinostat is not to be exposed to extremes of 
temperature (greater than 30ºC or less than 5ºC). 
 
Stability: 
Shelf life stability studies of the intact bottles are on-going. 
 
Route of Administration: 
Oral, on an empty stomach, at least 1 hour before or 2 
hours after a meal. Entinostat tablets should not be split, 
crushed, or chewed. 
 
Potential Drug Interactions: Metabolism: Data from in vitro metabolism experiments 
in human tissues demonstrated that entinostat is not metabolized by CYP enzymes 
(Acharya 2006), but UGT 1A4 did metabolize entinostat to its M2 glucuronide 
metabolite. No metabolites could be detected after incubation of entinostat in human liver 
microsomes (Acharya 2006). While inhibition of CYP enzymes 2B6 and 3A4 was seen, 
the data show that the degree of the inhibition makes it unlikely that any in vivo systemic 
interactions would occur. Intestinal CYP 3A4 may be inhibited by entinostat. However, 
entinostat did not inhibit any UGT enzymes tested. Entinostat was found to induce CYP 
1A2, CYP 2C6, and CYP 2B8 as well as UGT 1A4. Finally, entinostat was found to be a 
substrate for P-gp and BCRP transporters, but did not inhibit either of these transport 
proteins. 
 
 
Patient Care Implications: 
 
Entinostat may cause fatigue or malaise; advise patient to exercise caution while driving 
a vehicle or operating machinery. 
 
Administration of entinostat is contraindicated in patients with a history of allergy to 
entinostat or other medications that have a benzamide structure (eg, tiapride, remoxipride, 
clebropride). 
Careful monitoring of patients for signs of infection or reactivation of past infections is 
recommended, as reactivation of infection has been reported in patients treated with 
entinostat, in some cases without evidence of neutropenia. The clinical significance of 
this finding and the potential association with entinostat is unknown. 


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Entinostat must not be used during pregnancy or while breast-feeding. Women and men 
participating in entinostat clinical studies must agree to use acceptable contraceptive 
methods, as indicated in the clinical study protocol, during treatment and for 4 months 
thereafter. 
 
Availability 
 
Entinostat is an investigational agent supplied to investigators by the Division of Cancer 
Treatment and Diagnosis (DCTD), NCI. 
 
Entinostat is provided to the NCI under a Collaborative Agreement between the 
Pharmaceutical Collaborator, Syndax and the DCTD, NCI (see Section 12.3). 
 
 
8.1.2 MK-3475 (SCH 900475, pembrolizumab) (NSC 776864) 
 
Other Names: SCH 900475, pembrolizumab 
 
Classification: Anti-PD-1 MAb 
Molecular Weight: 148.9-149.5 KDa 
CAS Number: 1374853-91-4 
Mode of Action: The programmed cell death 1 (PD-1) receptor is an inhibitory receptor expressed 
by T cells. When bound to either of its ligands, PD-L1 or PD-L2, activated PD-1 negatively 
regulates T-cell activation and effector function. The pathway may be engaged by tumor cells to 
suppress immune control. pembrolizumab (MK-3475) blocks the negative immune regulatory 
signaling by binding to the PD-1 receptor, inhibiting the interaction between PD-1 and its ligands. 
 
Description: Pembrolizumab (MK-3475) is a humanized MAb of the IgG4/kappa isotype. 
 
How Supplied: Pembrolizumab (MK-3475) is supplied by Merck & Co., Inc. and distributed by the 
Pharmaceutical Management Branch, CTEP/DCTD/NCI. Pembrolizumab (MK-3475) injection is a 
sterile, preservative-free, clear to slightly opalescent, colorless to slightly yellow solution for 
intravenous use. Each vial contains 100 mg of pembrolizumab (MK-3475) in 4 mL of solution. Each 
1 mL of solution contains 25 mg of pembrolizumab (MK-3475) and is formulated in: L-histidine (1.55 
mg), polysorbate 80 (0.2 mg), sucrose (70 mg), and Water for Injection, USP.  
 
Preparation: MK-3475 solution for infusion must be diluted prior to administration. Do not shake 
the vials. Do not use if opaque or extraneous particulate matter other than translucent to white 
proteinaceous particles is observed. Do not use if discolored. To prepare the infusion solution add the 
dose volume of MK- 3475 to an infusion bag containing 0.9% Sodium Chloride Injection, USP or 
5% Dextrose Injection, USP. Gently invert the bag 10-15 times to mix the solution. The final 
concentration must be between 1 mg/mL to 10 mg/mL. 


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Compatible IV bag materials: PVC plasticized with DEHP, non-PVC (polyolefin), EVA, 
or PE lined polyolefin 
 
Storage: Store intact vials between 2°C - 8°C (36°F - 46°F). Do not freeze. Protect from light by 
storing in the original box. 
 
If a storage temperature excursion is identified, promptly return MK-3475 to between 2-8°C and 
quarantine the supplies. Provide a detailed report of the excursion (including documentation of 
temperature monitoring and duration of the excursion) to PMBAfterHours@mail.nih.gov for 
determination of suitability. 
 
Stability: Refer to the package label for expiration. . 
 
Administer prepared solutions immediately after preparation. If not administered immediately, 
prepared solutions may be stored refrigerated for up to 24 hours. Pembrolizumab (MK-3475)  
solutions may be stored at room temperature for a cumulative time of up to 6 hours. This includes 
room temperature storage of liquid drug product solution in vials, room temperature storage of infusion 
solution in the Infusion Solution IV bag, and the duration of infusion. 
 
Route of Administration: IV infusion only. Do not administer as an IV push or bolus injection. 
 
Method of Administration: Infuse over approximately 30 minutes (range: 25 - 40 minutes) using an 
infusion set containing a low-protein binding 0.2 to 5 μm in-line filter made of polyethersulfone or 
polysulfone. Infusion rate should not exceed 6.7 mL/min. A central line is not required; however, if 
a subject has a central venous catheter in place, it is recommended that it be used for the infusion. Do 
not co-administer other drugs through the same infusion line. Following the infusion, flush the IV line 
with normal saline. 
 
Compatible infusion set materials: PVC plasticized with DEHP or DEHT, PVC and tri-(2- 
ethylhexyl) trimellitate, polyethylene lined PVC, polyurethane, or polybutadiene 
 
Patient Care Implications: Refer to the protocol for information on evaluation and management 
of potential immune-related adverse events. 
 
 
Availability 
 
Pembrolizumab (MK-3475) is supplied to investigators by the Division of Cancer 
Treatment and Diagnosis (DCTD), NCI. 
 
Pembrolizumab (MK-3475) is provided to the NCI under a Collaborative 
Agreement between the Pharmaceutical Collaborator and the DCTD, NCI (see 
Section 12.3). 
 
8.1.3 
Agent Ordering and Agent Accountability 


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8.1.3.1 
NCI-supplied agents may be requested by eligible participating Investigators (or their 
authorized designee) at each participating institution. The CTEP-assigned protocol 
number must be used for ordering all CTEP-supplied investigational agents. The 
eligible participating investigators at each participating institution must be registered 
with CTEP, DCTD through an annual submission of FDA Form 1572 (Statement of 
Investigator), NCI Biosketch, Agent Shipment Form, and Financial Disclosure Form 
(FDF). If there are several participating investigators at one institution, CTEP- 
supplied investigational agents for the study should be ordered under the name of one 
lead investigator at that institution. 
 
Active CTEP-registered investigators and investigator-designated shipping designees 
and ordering designees can submit agent requests through the PMB Online Agent 
Order Processing (OAOP) application. Access to OAOP requires the -establishment 
of a CTEP Identity and Access Management (IAM) account and the maintenance of 
an “active” account status and a “current” password. For questions about drug orders, 
transfers, returns, or accountability, call or email PMB any time. Refer to the PMB’s 
website for specific policies and guidelines related to agent management. 
In general, sites may order initial agent supplies when a subject is being screened for 
enrollment onto the study 
 
8.1.3.2 
Agent Inventory Records – The investigator, or a responsible party designated by the 
investigator, must maintain a careful record of the receipt, dispensing and final 
disposition of all agents received from the PMB using the appropriate NCI 
Investigational Agent (Drug) Accountability Record (DARF) available on the CTEP 
forms page. Store and maintain separate NCI Investigational Agent Accountability 
Records for each agent, strength, formulation and ordering investigator on this 
protocol 
 
8.1.3.3 
Investigator Brochure Access for CTEP IND agents 
The current version of the Investigator Brochure (IB) will be accessible to site 
investigators and research staff through the PMB Online Agent Order Processing 
(OAOP) application. Access to OAOP requires the establishment of a CTEP Identity 
and Access Management (IAM) account and the maintenance of an “active” account 
status and a “current” password and active person registration status. Questions about 
IB access may be directed via email to IBcoordinator@mail.nih.gov or by phone 
(240) 276-6575 Monday through Friday between 8:30 am and 4:30 pm (ET). 
 
8.1.3.4 
Useful links and contacts – 
CTEP Forms, Templates, Documents: http://ctep.cancer.gov/forms/ 
NCI CTEP Investigator Registration: RCRHelpDesk@nih.gov 
PMB policies and guidelines: 
http://ctep.cancer.gov/branches/pmb/agent_management.htm 
PMB Online Agent Order Processing (OAOP) application: 
https://ctepcore.nci.nih.gov/OAOP


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CTEP Identity and Access Management (IAM) account: 
https://ctepcore.nci.nih.gov/iam/ 
CTEP IAM account help: ctepreghelp@ctep.nci.nih.gov 
PMB email: PMBAfterHours@mail.nih.gov 
PMB phone and hours of service: (240) 276-6575 Monday through Friday between 
8:30 am and 4:30 pm (ET) 
 
 
9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES 
 
9.1 Exploratory/Ancillary Correlative Studies 
 
Site(s) Performing Correlative Study. 
The samples will be analyzed at the Yale Cancer Center 
 
Collection and Handling of Specimens 
All operating procedures for specimen collection, handling and storage will be 
standardized. Common standardized operating procedures (SOPs) will be to collect 15 cc of 
bone marrow (BM) (3 EDTA tubes) and 15 cc of blood (3 EDTA tubes) from the patient at 
baseline (before cycle 1), after cycles 1 (entinostat alone) and then every 2 cycles of 
entinostat and Pembrolizumab (MK-3475) dose. If a patient disease progresses, an additional 
15 cc of blood and 15 cc of the BM will be obtained. For a patient that progresses and is 
removed from study treatment, additional blood or BM may be obtained at the time of 
routine collection. At the time of collection, tubes must be thoroughly mixed to prevent 
clotting. The specimen should be delivered to the Hematology Tissue Bank (PI: Stephanie 
Halene) at Yale University within 24 hours after collection. Typically, fresh specimens 
(before cryopreservation) will be used for experiments. Specimens should be labeled with the 
patient’s initials, study number, sample collection date and time, and sample source 
(peripheral blood or bone marrow). All data should be kept in laboratory log. 
The clot sections will be obtained from each patient at the time of core biopsy and aspiration. 
The clot section will be fixed immediately after harvesting into 10% formalin solution and 
embedded into paraffin to be delivered to Hematology Tissue Bank (PI: Stephanie Halene) at 
Yale University within 24 hours after collection if the sample is from outside Yale, or if the 
sample is from a Yale patient, it will be stored in the Yale Pathology repository until ready 
for further processing. Paraffin blocks will be cut at maximum 3 mm thickness at the 
Research Histology laboratory at Yale University. Hematology Tissue Bank will separate 
mononuclear cells from BM aspirate and blood in EDTA as described at Appendix D. 
Samples will be stored in liquid nitrogen and should be clearly marked with the patient’s 
identification number (given at the time of registration), study number, sample collection 
date, and sample source (blood or BM). Cryopreserved samples will be batch delivered from 
Yale to Dr. Tae Kon Kim’s laboratory at Vanderbilt University at this address: Preston 
Research Building, suite 532, Nashville TN, 37232 for the correlative studies that will be 
performed at Vanderbilt University Medical Center (Kim laboratory) 


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9.1.1 Determine numeric and functional alteration of MDSCs in relation to ENT/anti- 
PD1 therapy and its correlation with the clinical response 
 
9.1.1.1 
Collection and Handling of Specimens 
BM aspirate and blood collected in EDTA tubes and clot sections in 10% formaldehyde 
will be as described above. We will separate mononuclear cells from BM aspirate and 
blood in EDTA as described at Appendix D. Samples will be used for experiments and 
samples leftover will be stored in liquid nitrogen and should be clearly marked with the 
patient’s identification number (given at the time of registration), study number, sample 
collection date, and sample source (blood or BM). 
Formalin-fixed paraffin-embed clot section will be cut at maximum 3 mm thickness at 
Research Histology lab of Yale University. Shipping of Specimen(s) 
Fresh BM aspirate and blood samples will be collected at Yale and other participating 
sites to deliver overnight and will be processed (according to Appendix D) at the 
Hematology Tissue Bank at Yale University. Upon request of the Study Chair they will 
be batch shipped to Yale Cancer Center. Samples will be shipped or delivered to the 
Hematology Tissue Bank: 300 George, Rm 786. Tel: 203.737.4531. 
Formalin-fixed paraffin-embed block of clot section from external sites should be also 
delivered to Hematology Tissue Bank at Yale University, or if the sample is from a Yale 
patient, it will be stored in the Yale Pathology repository until ready for further 
processing.. Sections will be cut at maximum 3 mm thickness at Research Histology lab 
at Yale University. 
9.1.1.3 
Site(s) Performing Correlative Study 
The correlative studies will be performed at Yale Cancer Center (Dr. Stephanie Halene’s 
lab) and Dr. Tae Kon Kim’s lab at Vanderbilt University as described above in 9.1 and in 
Appendix J. 
 
9.1.2 Evaluate whether PD-L1 expression in CD34+ blasts from human MDS BM 
correlates with the response to anti-PD1 therapy 
 
9.1.2.1 
Collection and Handling of Specimens 
BM aspirate and blood collected in EDTA tubes and clot sections in 10% formaldehyde 
will be as described above. We will separate mononuclear cells from BM aspirate and 
blood in EDTA as described at Appendix D. Samples will be used for experiments and 
samples leftover will be stored in liquid nitrogen and should be clearly marked with the 
patient’s identification number (given at the time of registration), study number, sample 
collection date, and sample source (blood or BM). 
Formalin-fixed paraffin-embed clot section will be cut at maximum 3 mm thickness 
9.1.2.2 
Shipping of Specimen(s) 
Fresh BM aspirate and blood samples will be collected at Yale and other participating 
sites to deliver overnight and will be processed (according to Appendix D) at the 
Hematology Tissue Bank at Yale University. Upon request of the Study Chair they will 
be batch shipped to Yale Cancer Center. Samples will be shipped or delivered to the 
Hematology Tissue Bank: 300 George, Rm 786. Tel: 203.737.4531 


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Formalin-fixed paraffin-embed block of clot section from external sites should be 
delivered to Hematology Tissue Bank at Yale University, or if the sample is from a Yale 
patient, it will be stored in the Yale Pathology repository until ready for further 
processing.. Sections will be cut at maximum 3 mm thickness at Research Histology lab 
at Yale University. 
9.1.2.3 
Site(s) Performing Correlative Study 
All correlative studies will be performed at Yale Cancer Center Stephanie Halene’s lab) 
and Dr. Tae Kon Kim’s lab at Vanderbilt University as described above and in Appendix 
J. 
 
9.1.3 Characterize functional dynamics of immune cell subsets in MDS patients pre- 
and post- anti-PD1 therapy 
 
9.1.3.1 
Collection and Handling of Specimens 
BM aspirate and blood collected in EDTA tubes will be as described above. We will 
separate mononuclear cells from BM aspirate in EDTA as described at Appendix D. 
Samples will be used for experiments and samples leftover will be stored in liquid 
nitrogen and should be clearly marked with the patient’s identification number (given at 
the time of registration), study number, sample collection date, and sample source (or 
bone marrow). 
9.1.3.2 
Shipping of Specimen(s) 
Fresh BM aspirate and blood samples will be collected at Yale and other participating 
sites to deliver overnight and will be processed (according to Appendix D) at the 
Hematology Tissue Bank at Yale University. Upon request of the Study Chair they will 
be batch shipped to Yale Cancer Center. Samples will be shipped or delivered to the 
Hematology Tissue Bank: 300 George, Rm 786. Tel: 203.737.4531. 
9.1.3.3 
Site(s) Performing Correlative Study 
All correlative studies will be performed at Yale Cancer Center Stephanie Halene’s lab) 
and Dr. Tae Kon Kim’s lab at Vanderbilt University as described above and in Appendix 
J. 
 
 
9.2 Special Studies: N/A 


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10. STUDY CALENDAR 
 
Baseline evaluations are to be conducted within 2 weeks prior to start of protocol therapy. In the 
event that the patient’s condition is deteriorating, laboratory evaluations should be repeated 
within 48 hours prior to initiation of the next cycle of therapy. An interim history and physical to 
evaluate signs and symptoms of autoimmune events should be done with each visit and prior to 
each infusion. An autoimmune check list will be used. Interim history, evaluation, and 
laboratory assessments will be performed before Administration of entinostat. Patients will have 
weekly blood test (Complete blood counts (CBC)) throughout the study at the clinic. CBC with 
differentials, serum chemistry and Liver function tests (LFTs) will be performed at least every 2 
weeks. 
 
 
 
Procedure 
Screening# 
d 14 days 
from 
Baseline 
(First day 
study drug 
administrati 
on) 
Each Cycle (21 days) 
Discontinuation 
of Study Drug or 
progression 
Monthly after 
removal from 
study for any 
reason for 6 
months 
D1 
+/- 1 
D8 
+/- 1 
D15 
+/- 1 
D17-D20 
Entinostat 
(depending on 
dose level) 
 
X 
X 
X 
 
 
 
Pembrolizumab 
(MK-3475) 
 
X 
(Starting 
in C2) 
 
 
 
 
 
Age, gender, 
height, baseline 
medications, 
record prior 
therapy, 
informed 
consent , EKG 
X 
 
 
 
 
 
 
Record prior 
anti-cancer 
therapies 
X 
 
 
 
 
 
 
Physical 
examination, 
performance 
status, vital 
signs, weight 
X 
X 
 
 
 
X 
 
ECOG 
performance 
status 
X 
X 
 
 
 
X 
 
Hematology1 
X 
X 
X 
X 
 
X 
 
Serum 
chemistry 2 
X 
X 
X 
X 
 
X 
 
Reticulocyte 
count 
X 
X 
 
 
 
 
 
Pregnancy 
testing3 
X 
 
 
 
 
 
 
Record adverse 
events4 
 
X 
 
X 
 
X 
 


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Record 
concomitant 
medications/ 
procedures 
 
X 
 
 
 
X 
 
Bone marrow 
aspirate and 
biopsy and 
peripheral blood 
for disease 
assessment and 
correlative 
studies5 
X 
 
 
 
X (of first 
cycle, 
and 
then every 2 
cycles [C3, 5, 
etc]) 
X 
 
TSH 
X 
 
 
X 
 
 
 
Survival 
Follow-up6 
 
 
 
 
 
 
X 
Cycle1: Entinostat 8mg po on D1, 8 and 15, no Pembrolizumab (MK-3475) 
DL1: Entinostat 8mg on D1, 8 of 21-day cycles, Pembrolizumab (MK-3475) 200mg IV over 30 
mins on D1 of 21-day cycles 
DL2: Entinostat 8mg on D1, 8, 15 of 21-day cycles, Pembrolizumab (MK-3475) 200mg IV over 
30 mins on D1 of 21-day cycles 
DL-1: 8mg on D1 of 21-day cycles, Pembrolizumab (MK-3475) 200mg IV over 30 mins on D1 
of 21-day cycles 
- An unscheduled visit can occur at any time during the study. Source must be maintained for 
these unscheduled visits. The date for the visit and any data generated must be recorded on the 
appropriate CRF. Source documents for these unscheduled visits must also be maintained. In 
cases of aplasia, CBCs will be performed weekly and Bone marrow aspirates and biopsies will 
be performed every 3 months until recovery. 
1: Hematology assessment includes complete blood count and differential blood count. 
2: Chemistry assessment includes serum sodium, potassium, chloride, carbon dioxide, serum 
urea nitrogen, creatinine, calcium, glucose, alkaline phosphatase, AST, ALT, total bilirubin, total 
protein, albumin. Blood samples for LFTs must be collected and analyzed at local or central labs 
within 3 days prior to dosing. LFT results must be reviewed by the principal investigator (or 
designee) to meet dosing criteria specifications: d3 x ULN for AST, ALT and d2 x ULN for 
Total bilirubin unless liver metastases are present in which case LFT d5 x ULN for AST, ALT 
and Total bilirubin d3.0 x ULN) prior to dosing. If, during the course of treatment abnormal LFT 
values are detected, entinostat and Pembrolizumab (MK-3475) will not be administered. The 
subject should be evaluated and managed referring to the protocol, as clinically appropriate. 
In addition, evaluation for pituitary function must be made prior to each infusion either by 
clinical history, TSH or both. 
3: Pregnancy tests for females of childbearing potential. A female of childbearing potential is a 
sexually mature female who: 1) has not undergone a hysterectomy or bilateral oophorectomy; or 
2) has not been naturally postmenopausal for at least 24 consecutive months (i.e., has had menses 
at any time in the preceding 24 consecutive months). 
4: An additional safety assessment will be done before each maintenance dose. 
5: Bone marrow assessment will be performed in patients at baseline (before cycle 1), after 
cycles 1 (entinostat alone) and then every 2 cycles of entinostat and Pembrolizumab (MK-3475) 
dose. At baseline, bone marrows will be sent for analysis of aspirate, biopsy, flow cytometry, 
iron stain, correlative studies, cytogenetics and FISH. FISH panel will include probes to identify 
5q-, 7q-, trisomy 8, 20q-. Follow-up bone marrows will be analyzed for aspirate, biopsy, and 
flow cytometry. Cytogenetics will be sent after cycle 2, 4 and subsequent bone marrow studies. 


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Iron stains and FISH will be repeated at discretion of investigator. 
6: Survival follow-up should be obtained monthly from time of study drug discontinuation or 
disease progression, whichever occurs first, for 6 months. Survival follow-up will be collected 
via telephone calls, patient medical records, or clinic visits. 
 
11. MEASUREMENT OF EFFECT 
 
Although response is not the primary endpoint of this trial, patients will be assessed for objective 
response by the standard criteria of modified IWG-2006 guidelines (Appendix A1 and 
Appendix A2). Responses in AML patients will be assessed using IWG 2003 criteria 
(Appendix A3). In addition to a baseline bone marrow biopsy and scan, bone marrow biopsy 
will be performed at baseline, after cycles 2 and 4, and before each maintenance dose. 
 
11.1 
Definitions 
 
Evaluable for toxicity. All patients will be evaluable for toxicity from the time of their first 
treatment with Entinostat in combination with Pembrolizumab (MK-3475) 
 
Evaluable for objective response. Only those patients who have measurable disease present 
at baseline, have received at least one cycle of therapy, and have had their disease re- 
evaluated will be considered evaluable for response. These patients will have their response 
classified according to the definitions stated below. (Note: Patients who exhibit objective 
disease progression prior to the end of cycle 1 will also be considered evaluable.) 
 
11.2 
Response criteria 
 
Myelodysplastic syndrome (MDS) 
Responses for MDS patients will be defined as in the modified IWG-2006 criteria 
(Appendix A1 and Appendix A1). These response criteria do not apply to AML as these 
patients are required to be in full remission by eligibility criteria. Only relapse and survival 
as measured by PFS and overall survival will be measured to assess response for AML 
patients. The OS and PFS will be measured for both AML and MDS as described in 
Appendix A1 and Appendix A2. 
Duration of 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). 
 
Duration of stable disease: Stable disease is measured from the start of the treatment until 
the criteria for progression are met. 
 
Chronic Myelomonocytic Leukemia (CMML) 
The definitions below are consistent with those detailed by Cheson, et al for MDS61 except 
that, as discussed earlier, for disease progression based on BM blast percentage, the patient 
must exceed 10% absolute BM blast percentage in addition to increasing above 50% of BM 
blast percentage at baseline to count as disease progression. 


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Complete response is defined by bone marrow showing <5% myeloblasts with normal 
trilineage maturation without dysplasia. When erythroid precursors constitute <50% of 
marrow cells, the blast percentage is based on total nucleated cells; otherwise, the blast 
percentage is based on non-erythroid compartment. Peripheral blood criteria consist of 
Hemoglobin >11 gm/dl, ANC > 1 x 109/L, Platelets > 100 x 109/L, absence of peripheral 
blood blasts, all in the absence of transfusions and growth factors. 
Partial response is defined by all of the above criteria with the exception of bone marrow 
blasts decreasing by >50% from pretreatment levels and/or achievement of a less advanced 
MDS FAB classification. 
Stable Disease is defined by failure to achieve PR but without progression for at least 2 
months. 
Progression is defined according to initial blast percentages: 
For patients with <5% blasts: >50% increase in blasts to >10% 
For patients with 5-10% blasts: >50% increase in blasts to >10% 
For patients with >10-20% blasts: >50% increase in blasts to >20% 
For patients with >20-30% blasts: >50% increase in blasts to >30% 
In addition, there must be >1 of the following criteria: >50% decrease from maximum 
response levels in granulocytes or platelets, a decrease in hemoglobin concentration by at 
least 2 gm/dl, or becoming transfusion dependent. 
 
11.3 
Other response parameters 
 
Progression-free survival (PFS) and overall survival (OS) will be recorded from start of study 
to progression or death (PFS), or time from start of study to death (OS). 
 
 
12. STUDY OVERSIGHT AND DATA REPORTING / REGULATORY 
REQUIREMENTS 
 
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0 
(Adverse Events: List and Reporting Requirements). 
 
12.1 
Study Oversight 
 
This protocol is monitored at several levels, as described in this section. The Protocol Principal 
Investigator is responsible for monitoring the conduct and progress of the clinical trial, including 
the ongoing review of accrual, patient-specific clinical and laboratory data, and routine and 
serious adverse events; reporting of expedited adverse events; and accumulation of reported 
adverse events from other trials testing the same drug(s). The Protocol Principal Investigator and 
statistician have access to the data at all times through the CTMS web-based reporting portal. 
 
For the Phase 1 portion of this study, all decisions regarding dose escalation/expansion/de- 
escalation require sign-off by the Protocol Principal Investigator through the CTMS/IWRS. In 
addition, for the Phase 1 portion, the Protocol Principal Investigator will have at least monthly, 


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or more frequently, conference calls with the Study Investigators and the CTEP Medical 
Officer(s) to review accrual, progress, and adverse events and unanticipated problems. 
 
All Study Investigators at participating sites who register/enroll patients on a given protocol are 
responsible for timely submission of data via Medidata Rave and timely reporting of adverse 
events for that particular study. This includes timely review of data collected on the electronic 
CRFs submitted via Medidata Rave. 
All studies are also reviewed in accordance with the enrolling institution’s data safety monitoring 
plan. 
There are two safety/stopping rules included in this clinical trial: 
Stopping rule 1: Delayed immune related toxicity from dose escalation phase. If 4 or more 
patients in any dose level develop delayed protocol defined immune related grade 3/4 events, we 
will halt further therapy on that dose level 
Stopping rule 2: Excess toxicity in the expansion cohort. We will cease enrollment in the 
expansion phase at the dose determined from the dose escalation part of the study if we observe 
two or more DLTs among the first six evaluable patients enrolled in the expansion cohort. An 
evaluable patient is any patient who received a study drug. If the true toxicity rate is 50% or 
greater at the MTD then this rule will stop the trial with probability at least .89. Similarly, if the 
true toxicity rate is 33% at the MTD then this stopping rule will end the trial with probability .65. 
In that event, a new expansion cohort will start at the dose level immediately below the dose 
level that was expanded. If the dose level being expanded is dose level -1, then the study will be 
terminated. 
 
12.2 
Data Reporting 
 
Data collection for this study will be done exclusively through Medidata Rave. Access to the 
trial in Rave is granted through the iMedidata application to all persons with the appropriate 
roles assigned in the Regulatory Support System (RSS). To access Rave via iMedidata, the site 
user must have an active CTEP IAM account (check at < https://ctepcore.nci.nih.gov/iam >) and 
the appropriate Rave role (Rave CRA, Read-Only, CRA (Lab Admin, SLA or Site Investigator) 
on either the LPO or participating organization roster at the enrolling site. To the hold Rave CRA 
role or CRA Lab Admin role, the user must hold a minimum of an AP registration type. To hold 
the Rave Site Investigator role, the individual must be registered as an NPIVR or IVR. 
Associates can hold read-only roles in Rave. 
 
Upon initial site registration approval for the study in RSS, all persons with Rave roles assigned 
on the appropriate roster will be sent a study invitation e-mail from iMedidata. To accept the 
invitation, site users must log into the Select Login (https://login.imedidata.com/selectlogin) 
using their CTEP-IAM user name and password, and click on the “accept” link in the upper 
right-corner of the iMedidata page. Please note, site users will not be able to access the study in 
Rave until all required Medidata and study specific trainings are completed. Trainings will be in 
the form of electronic learnings (eLearnings), and can be accessed by clicking on the link in the 
upper right pane of the iMedidata screen. 


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Users that have not previously activated their iMedidata/Rave account at the time of initial site 
registration approval for the study in RSS will also receive a separate invitation from iMedidata 
to activate their account. Account activation instructions are located on the CTSU website, Rave 
tab under the Rave resource materials (Medidata Account Activation and Study Invitation 
Acceptance). Additional information on iMedidata/Rave is available on the CTSU members’ 
website under the Rave tab or by contacting the CTSU Help Desk at 1-888-823-5923 or by e- 
mail at ctsucontact@westat.com. 
 
12.2.1 Method 
 
CTMS Routine Monitoring: 
This study will be monitored by the Clinical Trials Monitoring Service (CTMS). Data 
will be submitted to CTMS at least once every two weeks via Medidata Rave (or other 
modality if approved by CTEP). Information on CTMS reporting is available at 
http://www.theradex.com/clinicalTechnologies/?National-Cancer-Institute-NCI-11. On- 
site audits will be conducted on an 18-36 month basis as part of routine cancer center site 
visits. More frequent audits may be conducted if warranted by accrual or due to concerns 
regarding data quality or timely submission. For CTMS monitored studies, after users 
have activated their accounts, please contact the Theradex Help Desk at (609) 799-7580 
or by email at CTMSSupport@theradex.com for additional support with Rave and 
completion of CRFs. 
 
 
12.2.2 Responsibility for Data Submission 
 
For ETCTN trials, it is the responsibility of the PI(s) at the site to ensure that all 
investigators at the ETCTN Sites understand the procedures for data submission for each 
ETCTN protocol and that protocol specified data are submitted accurately and in a timely 
manner to the CTMS via the electronic data capture system, Medidata Rave. 
 
Data are to be submitted via Medidata Rave to CTMS on a real-time basis, but no less 
than once every 2 weeks. The timeliness of data submissions and timeliness in resolving 
data queries will be tracked by CTMS. Metrics for timeliness will be followed and 
assessed on a quarterly basis. For the purpose of Institutional Performance Monitoring, 
data will be considered delinquent if it is greater than 4 weeks past due. 
 
Data from Medidata Rave and CTEP-AERS is reviewed by the CTMS on an ongoing 
basis as data is received. Queries will be issued by CTMS directly within Rave. The 
queries will appear on the Task Summary Tab within Rave for the CRA at the ETCTN to 
resolve. Monthly web-based reports are posted for review by the Drug Monitors in the 
IDB, CTEP. Onsite audits will be conducted by the CTMS to ensure compliance with 
regulatory requirements, GCP, and NCI policies and procedures with the overarching 
goal of ensuring the integrity of data generated from NCI-sponsored clinical trials, as 
described in the ETCTN Program Guidelines, which may be found on the CTEP 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/adverse_events.htm 


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) and CTSU websites. 
 
An End of Study CRF is to be completed by the PI, and is to include a summary of study 
endpoints not otherwise captured in the database, such as (for phase 1 trials) the 
recommended phase 2 dose (RP2D), and a description of any dose-limiting toxicities 
(DLTs). CTMS will utilize a core set of eCRFs that are Cancer Data Standards Registry 
and Repository (caDSR) compliant (http://cbiit.nci.nih.gov/ncip/biomedical-informatics- 
resources/interoperability-and-semantics/metadata-and-models). Customized eCRFs will 
be included when appropriate to meet unique study requirements. The PI is encouraged 
to review the eCRFs, working closely with CTMS to ensure prospectively that all 
required items are appropriately captured in the eCRFs prior to study activation. CTMS 
will prepare the eCRFs with built-in edit checks to the extent possible to promote data 
integrity. 
 
CDUS data submissions for ETCTN trials activated after March 1, 2014, will be carried 
out by the CTMS contractor, Theradex. CDUS submissions are performed by Theradex 
on a monthly basis. The trial’s lead institution is responsible for timely submission to 
CTMS via Rave, as above. 
 
Further information on data submission procedures can be found in the ETCTN Program 
Guidelines 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/adverse_events.htm 
). 
 
 
12.3 
Collaborative Agreements Language 
 
The agent(s) supplied by CTEP, DCTD, NCI used in this protocol is/are provided to the NCI 
under a Collaborative Agreement (CRADA, CTA, CSA) between the Pharmaceutical 
Company(ies) (hereinafter referred to as “Collaborator(s)”) and the NCI Division of Cancer 
Treatment and Diagnosis. Therefore, the following obligations/guidelines, in addition to the 
provisions in the “Intellectual Property Option to Collaborator” 
(http://ctep.cancer.gov/industryCollaborations2/intellectual_property.htm) contained within the 
terms of award, apply to the use of the Agent(s) in this study: 
 
1. Agent(s) may not be used for any purpose outside the scope of this protocol, nor can 
Agent(s) be transferred or licensed to any party not participating in the clinical study. 
Collaborator(s) data for Agent(s) are confidential and proprietary to Collaborator(s) and shall 
be maintained as such by the investigators. The protocol documents for studies utilizing 
Agents contain confidential information and should not be shared or distributed without the 
permission of the NCI. If a copy of this protocol is requested by a patient or patient’s family 
member participating on the study, the individual should sign a confidentiality agreement. A 
suitable model agreement can be downloaded from: http://ctep.cancer.gov. 
 
2. For a clinical protocol where there is an investigational Agent used in combination with 


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(an)other Agent(s), each the subject of different Collaborative Agreements, the access to and 
use of data by each Collaborator shall be as follows (data pertaining to such combination use 
shall hereinafter be referred to as "Multi-Party Data”): 
 
a. NCI will provide all Collaborators with prior written notice regarding the existence and 
nature of any agreements governing their collaboration with NCI, the design of the 
proposed combination protocol, and the existence of any obligations that would tend to 
restrict NCI's participation in the proposed combination protocol. 
 
b. Each Collaborator shall agree to permit use of the Multi-Party Data from the clinical trial 
by any other Collaborator solely to the extent necessary to allow said other Collaborator 
to develop, obtain regulatory approval or commercialize its own Agent. 
 
c. Any Collaborator having the right to use the Multi-Party Data from these trials must 
agree in writing prior to the commencement of the trials that it will use the Multi-Party 
Data solely for development, regulatory approval, and commercialization of its own 
Agent. 
 
3. Clinical Trial Data and Results and Raw Data developed under a Collaborative Agreement 
will be made available to Collaborator(s), the NCI, and the FDA, as appropriate and unless 
additional disclosure is required by law or court order as described in the IP Option to 
Collaborator (http://ctep.cancer.gov/industryCollaborations2/intellectual_property.htm). 
Additionally, all Clinical Data and Results and Raw Data will be collected, used and 
disclosed consistent with all applicable federal statutes and regulations for the protection of 
human subjects, including, if applicable, the Standards for Privacy of Individually 
Identifiable Health Information set forth in 45 C.F.R. Part 164. 
 
4. When a Collaborator wishes to initiate a data request, the request should first be sent to the 
NCI, who will then notify the appropriate investigators (Group Chair for Cooperative Group 
studies, or PI for other studies) of Collaborator's wish to contact them. 
 
5. Any data provided to Collaborator(s) for Phase 3 studies must be in accordance with the 
guidelines and policies of the responsible Data Monitoring Committee (DMC), if there is a 
DMC for this clinical trial. 
 
6. Any manuscripts reporting the results of this clinical trial must be provided to CTEP by the 
Group office for Cooperative Group studies or by the principal investigator for non- 
Cooperative Group studies for immediate delivery to Collaborator(s) for advisory review and 
comment prior to submission for publication. Collaborator(s) will have 30 days from the 
date of receipt for review. Collaborator shall have the right to request that publication be 
delayed for up to an additional 30 days in order to ensure that Collaborator’s confidential and 
proprietary data, in addition to Collaborator(s)’s intellectual property rights, are protected. 
Copies of abstracts must be provided to CTEP for forwarding to Collaborator(s) for courtesy 
review as soon as possible and preferably at least three (3) days prior to submission, but in 
any case, prior to presentation at the meeting or publication in the proceedings. Press 
releases and other media presentations must also be forwarded to CTEP prior to release. 


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Copies of any manuscript, abstract and/or press release/ media presentation should be sent to: 
Email: ncicteppubs@mail.nih.gov 
The Regulatory Affairs Branch will then distribute them to Collaborator(s). No publication, 
manuscript or other form of public disclosure shall contain any of Collaborator’s confidential/ 
proprietary information. 
 
 
13. STATISTICAL CONSIDERATIONS 
 
13.1 
Study Design/Endpoints 
This will be a phase 1b study [schema]. The proposed dosing of ENT has been previously 
tested in MDS and showed to be well-tolerated. While the combination of ENT with the anti- 
PD1, Pembrolizumab (MK-3475) has not been studied clinically in MDS patients, it is 
unlikely that this combination would have overlapping toxicities. 
 
PART 1: ENT has been previously evaluated as a single agent therapy in patients with MDS 
at these doses and has been well tolerated. The ENT monotherapy run-in part (cycle 1) will 
allow to study the changes in myeloid derived suppressor cells (MDSCs) at pre- and post- 
ENT treatment (the end of cycle 1) to dissect the effects of ENT monotherapy from those of 
the combined ENT/anti-PD1 therapy. 
 
We will perform ‘3+3’ classical dose escalation design to determine dose limiting toxicity 
(DLT). The first 3 patients will receive ENT at dose level 1 (DL1: 8mg on D1, 8 of 21-day 
cycles). Pembrolizumab (MK-3475) will be added starting day 1 cycle 2 at the approved dose 
of 200mg intravenously [IV] over 30 minutes on D1 of 21-day cycles) for 4 cycles. A safety 
committee that includes the study chair, the principal investigator from any study site that has 
enrolled a patient on the specific DL, as well as two sub-investigators from Yale will review 
safety data after enrollment of every 3 patients during the dose escalation phase. The 
committee will have the discretion to decide to enroll 3 more patients in the specific dose 
level even if no DLT occurred in the first 3 treated patients in that dose level if there are 
toxicity/safety concerns that did not qualify as DLT (maximum of 6 evaluable patients to be 
enrolled at any dose level). The committee can also decide to enroll patients at a lower dose 
level after the first 3 or after the first 6 patients enrolled in any dose level even if no or one 
DLT occurred at that dose level if there are toxicity/safety concerns that did not qualify as 
DLT. The following regarding enrollment in different dose levels will be considered 
guidance for the safety committee but can be modified in accordance of above: If no patient 
of the 3 patients treated with combined therapy at DL1 develops DLT, then dose level 2 
(DL2) will be tested. For instance, DL2 will be with ENT at 8mg on D1, 8 and 15 and MK- 
3475 (pembrolizumab) at 200mg IV on D1 of 21-day cycles. If 1 patient out of the 3 patients 
has DLT at DL1, we will expand cohort to 3 more patients and if 1 patient of the 6 patients 
has DLT (i.e. only 0/3 or 1/6), we will test next dose (DL2). If no patient of the 3 patients 
treated with combined therapy at DL2 develop DLT, then DL2 will be used for part 2 
(expansion cohort). If 1 patient out of the 3 patients has DLT at DL2, we will expand cohort 
to 3 more patients and if 1 patient of the 6 patients has DLT (i.e only 0/3 or 1/6), dose level 2 


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(DL2) will be used for part 2 (expansion cohort). If 2 or more patients of the 6 patients have 
DLT, DL1 will be used for expansion cohort. 
An additional dose level (DL -1) in which day 8 of ENT will be omitted (i.e. ENT at 8mg on 
D1, and Pembrolizumab (MK-3475) at 200mg IV on D1 of 21-day cycles) will be used if 2 
or more patients develop DLT during the first 2 cycles of combined therapy at DL1. If 0 or 1 
patients of the 6 patients treated with combined therapy at DL-1 develop DLT then DL-1 will 
be used in the second part of the study. If 2 or more 6 patients enrolled in DL -1 develop 
DLT, then the study will be terminated. 
Toxicities will be tabulated and graded according to the Common Terminology Criteria for 
Adverse Events Version 5 (CTCAE-5). DLT will be assessed after the first 2 cycles of 
combined therapy. Responses will be as defined by IWG-2006 criteria (CR, PR, or HI) and 
assessed at the end of cycle 1, the combined first 2 cycles, and every 2 cycles afterwards. 
Patients who achieve an objective response or maintain a stable disease (SD) status after the 
first 4 combination cycles will continue receiving both drugs at the same schedule till 
progression up to 1 year of combined therapy. Participants who do not complete the first 2 
cycles of combined therapy without experiencing a DLT during dose escalation (as DLT period 
is defined in the first 2 combined therapy cycles) will also be replaced. To be DLT-evaluable, 
the patient must complete the first two cycles of combined therapy without experiencing a 
DLT. Any patient who is dose reduced during the entinostat run-in first cycle due to entinostat- 
related toxicity will be analyzed on the dose level that corresponds to the reduced dose level 
delivered during the first cycle of combined therapy. 
 
PART 2: After the safe dose of the combination is established, 15 patients will be treated 
with ENT+ Pembrolizumab (MK-3475) combination at that same dose and schedule. 
Assessment of response will occur after 4 combination cycles and similar to part 1, those 
who achieve an objective response or SD will continue to receive the therapy till progression. 
Patients who progress or respond and subsequently lose response will be withdrawn from 
study. BM biopsy/aspirate and PB samples will be done at baseline, after first cycle of ENT 
monotherapy, every 2 cycles of combined therapy, and at time of progression or study 
discontinuation. Formal safety assessment will be performed after the first 6 patients are 
enrolled and each have received at least 5 cycles (i.e. 1 ENT monotherapy cycle and 4 cycles 
of combined therapy (DL1, DL2) for each patient) to assess for any unexpected autoimmune 
or other toxicity. 
 
Primary Endpoint(s): MTD will be defined by the occurrence of DLT among 2 dose 
schedules (see section 5.2) 
Definitions of DLT: see section 5.2 
 
 
The following early stopping rule for excess toxicity in the expansion cohort will be used: 
We will cease enrollment in the expansion phase at the dose determined from dose escalation 
part of the study if we observe two or more DLTs among the first six evaluable patients 
enrolled in the expansion cohort. An evaluable patient is any patient who received a study 
drug. If the true toxicity rate is 50% or greater at the MTD then this rule will stop the trial 
with probability at least .89. Similarly, if the true toxicity rate is 33% at the MTD then this 
stopping rule will end the trial with probability .65. In that event, a new expansion cohort will 


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start at the dose level immediately below the dose level that was expanded. If the dose level 
being expanded is dose level -1, then the study will be terminated. 
 
Secondary Endpoint(s): Overall response rate (CR, PR, and HI) as defined by the modified 
International working group 2006 (IWG-2006) criteria for CR, PR, HI (Appendix A1 and 
Appendix A2) with at least 15% to be continued 
 
Exploratory Endpoint(s): 
x Median response duration for responders, median time of progression to AML, median 
overall survival (OS) and 1- and 2-year OS rate 
x Assessment of the dynamic quantitative change in proportion of MDSCs in BM with 
combined therapy (target of reducing to <10%) and correlation with any observed clinical 
responses 
 
13.2 
Sample Size/Accrual Rate 
 
We expect to accrue 1-2 patients with MDS per month once the study is open. 
Currently there is no therapy that has been shown to prolong survival in MDS in the post- 
DNMTi setting and that the expected best response in patients receiving supportive care only in 
post-DNMTi setting is negligible (1% or less). It is generally accepteGWKDWD255LQWKH
post-DNMTi is clinically meaningful and would be worth further evaluation in larger studies. 
The total number of patients enrolled in the study will range from 21 to 27. The null 
hypothesis response rate of 1% (only supportive care) will be rejected if we observe 2 or more 
favorable responses. These criterion has significance level between .02 and .03 using a one-sided 
binomial test. This criterion has power between .84 and .93 for testing an alternative response 
rate of 15% with the combination therapy. Therefore, we will treat a total of 21 patients in the 
combination arm (6 in the first part of the study and 15 in the second part of the study). 
 
DOMESTIC PLANNED ENROLLMENT REPORT 
 
Racial Categories 
Ethnic Categories 
 
Total 
Not Hispanic or Latino 
Hispanic or Latino 
Female 
Male 
Female 
Male 
American Indian/ 
Alaska Native 
1 
1 
1 
1 
4 
Asian 
1 
1 
0 
0 
2 
Native Hawaiian or 
Other Pacific 
Islander 
1 
0 
0 
0 
1 
Black or African 
American 
1 
1 
1 
1 
4 
White 
4 
4 
1 
1 
10 
More Than One 
Race 
0 
0 
0 
0 
 
Total 
8 
7 
3 
3 
21 
 
13.3 
Stratification Factors 
 
There are no planned stratifications for this protocol. 
 
13.4 
Analysis of Secondary Endpoints 
Overall response rate: defined by the International working group 2006 (IWG-2006) criteria 
for CR, PR, HI (Appendix A1 and Appendix A2). We will also record PFS and OS for the 


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MDS. These endpoints will be assessed after cycle 2 and 4 of induction therapy, and every 3 
months during follow-up. Bone marrow biopsies will be done at baseline, after 1 cycle and 
then every 2 cycles, at end of study, and as clinically indicated (e.g. suspicion of progression, 
decreasing blood counts). We aim to obtain preliminary efficacy data of entinostat in 
combination with Pembrolizumab (MK-3475) in terms of CR, PR, HI as defined by IWG- 
2006 and PFS, OS. Rates of CR, PR and HI will be summarized separately by cohort and 
reported with an exact 95% confidence interval. Median PFS and OS will also be reported 
with a 95% confidence interval. 
The quantitative change in MDSCs (% MDSCs by flow cytometry, QIF score by AQUA) 
during treatment with the ENT/anti-PD1 combined therapy will be estimated using mixed 
effects models to take into account the within-patient correlation. Likelihood ratio tests will 
be performed to confirm if random intercepts and slopes are necessary in the model. The 
fixed effect for change in MDSCs over time will be evaluated for significance. The 
variability in the rate of change in MDSCs across patients will also be examined. The 
association between the clinical outcome and a meaningful reduction in MDSCs, which will 
be defined after a review of the data, will be assessed with the chi-square test. The quantity of 
MDSCs at baseline and during treatment as continuous variables can also be compared 
between responding and non-responding patients using a t-test or Mann-Whitney U-Test, if 
more appropriate. 
 
13.5 
Reporting and Exclusions 
 
13.5.1  Evaluation of toxicity: All patients will be evaluable for toxicity from the time of their 
first treatment with entinostat/ Pembrolizumab (MK-3475). 
 
13.5.2 Evaluation of response: All patients included in the study will be assessed for response 
to treatment, even if there are major protocol treatment deviations or if they are 
ineligible. Each patient will be assigned one of the following categories: 1) complete 
response, 2) partial response, 3) stable disease, 4) progressive disease, 5) early death 
from malignant disease, 6) early death from toxicity, 7) early death because of other 
cause, or 9) unknown (not assessable, insufficient data). [Note: By arbitrary 
convention, category 9 usually designates the “unknown” status of any type of data in a 
clinical database.] 
 
All of the patients who met the eligibility criteria (with the possible exception of those 
who received no study medication) should be included in the main analysis of the 
response rate. Patients in response categories 4-9 should be considered to have a 
treatment failure (disease progression). Thus, an incorrect treatment schedule or drug 
administration does not result in exclusion from the analysis of the response rate. Precise 
definitions for categories 4-9 will be protocol specific. 
 
All conclusions should be based on all eligible patients. Sub-analyses may then be 
performed on the basis of a subset of patients, excluding those for whom major 
protocol deviations have been identified (e.g., early death due to other reasons, early 
discontinuation of treatment, major protocol violations, etc.). However, these sub- 
analyses may not serve as the basis for drawing conclusions concerning treatment 


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efficacy, and the reasons for excluding patients from the analysis should be clearly 
reported. The 95% confidence intervals should also be provided. 
 
 
14. STUDY STATUS UPDATES AND STUDY CLOSURE 
 
14.1 
Definitions of Study Status Changes 
 
14.1.1 Temporarily Closed to Accrual 
 
The study status is Temporarily Closed to Accrual when no patient slots are currently 
available, but there is the possibility that the trial will re-open for accrual (patient slots 
become available). Sites are not permitted to accrue additional patients until CTEP is 
notified of Re-Activation. 
 
Study status will need to be changed to Temporarily Closed to Accrual when any of the 
following criteria are met: 
 
 
x Sites are notified by CTEP (via Request for Rapid Amendment [RRA]) of 
changes in the risk/benefit ratio that necessitate changes to the patient Informed 
Consent document. Requested changes will be specified in the RRA and must be 
reviewed by the study’s IRB. 
x CTEP and the lead investigator agree that unacceptable toxicities necessitate a 
discussion to change the dosing/regimen. 
x A protocol-defined benchmark has been achieved (such as an interim analysis 
before proceeding to the next stage). 
x Investigators encounter any of the stopping criteria described in Section 5.3. 
 
14.1.2 Closed to Accrual 
 
The study status is (permanently) Closed to Accrual when no more patient enrollment 
slots are available, and at least one patient is still actively receiving the study treatment. 
Sites are no longer permitted to enroll additional patients. 
 
Patient slots are no longer available when the following criteria are met: 
 
x The pre-specified number of evaluable patients has been successfully enrolled, 
treated, and evaluated. 
x The study treatment has failed to meet the pre-specified efficacy goal at the stage 
1 interim analysis. 
x CTEP and the investigators agree that unacceptable toxicities preclude further 
enrollment. 
x Investigators encounter any of the stopping criteria described in Section 5.3. 


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14.1.3 Closed to Accrual and Treatment 
 
The study status is Closed to Accrual and Treatment when no more patient enrollment 
slots are available and no patients are currently receiving the study treatment. Patients 
may still be enrolled on the protocol only for the purposes of follow-up. 
 
Patient accrual and treatment will be permanently halted when any of the following 
criteria are met: 
 
x Enrollment was previously closed (study status of “Closed to Accrual”), and no 
patients are receiving the study treatment. 
x CTEP and the investigators agree that unacceptable toxicities preclude further 
enrollment. In this case, CTEP and the investigators must collaborate to alter the 
regimen or to halt the study treatment altogether as soon as it can be safely done 
for patients currently receiving treatment. 
 
CTEP and Theradex must be notified when patients are no longer receiving treatment 
[i.e., when the last patient(s) to be receiving treatment is/are no longer receiving the study 
regimen for any reason]. 
 
14.1.4 Closed to Follow-Up 
 
The study is considered Closed to Follow-Up when all protocol-defined follow-up 
procedures have been completed for all patients who have not been removed from the 
study for other reasons. That is, there are no outstanding follow-up procedures to be 
performed as mandated by the protocol. 
 
CTEP does not need to be notified of a status change to “Closed to Follow Up.” 
 
14.1.5 Complete 
 
Study is considered Complete if it has been at least thirty (30) days since the last patient 
follow-up evaluation. 
 
A citation to a final study report (manuscript, meeting abstract, etc.) is required with the 
submission of the Protocol Status Update Form to CTEP PIO. 
 
 
14.2 
Responsibility for Filing Protocol Status Update Forms 
 
CTEP must be notified of all study status changes in Section 14.1 (except for Closed to Follow- 
Up) by the Corresponding Organization via Protocol Status Update Form, available from the 
CTEP website at http://ctep.cancer.gov/protocolDevelopment/default.htm#amendments. 


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Theradex must be notified as soon as all patients are off treatment (i.e., when study status 
changes to Closed to Accrual and Treatment). Theradex will produce a report within 90 days of 
this notification. 


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REFERENCES 
1. 
Fenaux P, Mufti GJ, Hellstrom-Lindberg E, et al: Efficacy of azacitidine 
compared with that of conventional care regimens in the treatment of higher-risk 
myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol 10:223-32, 
2009 
2. 
Prebet T, Gore SD, Esterni B, et al: Outcome of high-risk myelodysplastic 
syndrome after azacitidine treatment failure. J Clin Oncol 29:3322-7, 2011 
3. 
Zeidan AM, Kharfan-Dabaja MA, Komrokji RS: Beyond hypomethylating 
agents failure in patients with myelodysplastic syndromes. Curr Opin Hematol 21:123-30, 2014 
4. 
Klimek VM, Fircanis S, Maslak P, et al: Tolerability, pharmacodynamics, 
and pharmacokinetics studies of depsipeptide (romidepsin) in patients with acute myelogenous 
leukemia or advanced myelodysplastic syndromes. Clin Cancer Res 14:826-32, 2008 
5. 
Odenike OM, Alkan S, Sher D, et al: Histone deacetylase inhibitor 
romidepsin has differential activity in core binding factor acute myeloid leukemia. Clin Cancer 
Res 14:7095-101, 2008 
6. 
Pardoll DM: The blockade of immune checkpoints in cancer 
immunotherapy. Nat Rev Cancer 12:252-64, 2012 
7. 
Egen JG, Kuhns MS, Allison JP: CTLA-4: new insights into its biological 
function and use in tumor immunotherapy. Nat Immunol 3:611-8, 2002 
8. 
Herbst RS, Soria JC, Kowanetz M, et al: Predictive correlates of response 
to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 515:563-7, 2014 
9. 
Robert C, Ribas A, Wolchok JD, et al: Anti-programmed-death-receptor-1 
treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised 
dose-comparison cohort of a phase 1 trial. Lancet 384:1109-17, 2014 
10. 
Powles T, Eder JP, Fine GD, et al: MPDL3280A (anti-PD-L1) treatment 
leads to clinical activity in metastatic bladder cancer. Nature 515:558-62, 2014 
11. 
Ansell SM, Lesokhin AM, Borrello I, et al: PD-1 blockade with 
nivolumab in relapsed or refractory Hodgkin's lymphoma. N Engl J Med 372:311-9, 2015 
12. 
Kondo A, Yamashita T, Tamura H, et al: Interferon-gamma and tumor 
necrosis factor-alpha induce an immunoinhibitory molecule, B7-H1, via nuclear factor-kappaB 
activation in blasts in myelodysplastic syndromes. Blood 116:1124-31, 2010 
13. 
Yang H, Bueso-Ramos C, DiNardo C, et al: Expression of PD-L1, PD-L2, 
PD-1 and CTLA4 in myelodysplastic syndromes is enhanced by treatment with hypomethylating 
agents. Leukemia 28:1280-8, 2014 
14. 
Chen X, Eksioglu EA, Zhou J, et al: Induction of myelodysplasia by 
myeloid-derived suppressor cells. J Clin Invest 123:4595-611, 2013 
15. 
Kim K, Skora AD, Li Z, et al: Eradication of metastatic mouse cancers 
resistant to immune checkpoint blockade by suppression of myeloid-derived cells. Proc Natl 
Acad Sci U S A 111:11774-9, 2014 
16. 
Ma X: Epidemiology of myelodysplastic syndromes. Am J Med 125:S2-5, 
2012 
17. 
Sekeres MA: Epidemiology, natural history, and practice patterns of 
patients with myelodysplastic syndromes in 2010. J Natl Compr Canc Netw 9:57-63, 2011 
18. 
Goldberg SL, Chen E, Corral M, et al: Incidence and clinical 
complications of myelodysplastic syndromes among United States Medicare beneficiaries. J Clin 
Oncol 28:2847-52, 2010 


99 
                NCI Protocol #:10009  
                Version Date: 09/04/2020 
 
 
19. 
Zeidan AM, Komrokji RS: There's risk, and then there's risk: The latest 
clinical prognostic risk stratification models in myelodysplastic syndromes. Curr Hematol Malig 
Rep 8:351-60, 2013 
20. 
Greenberg P, Cox C, LeBeau MM, et al: International scoring system for 
evaluating prognosis in myelodysplastic syndromes. Blood 89:2079-88, 1997 
21. 
Sekeres MA, Maciejewski JP, List AF, et al: Perceptions of disease state, 
treatment outcomes, and prognosis among patients with myelodysplastic syndromes: results from 
an internet-based survey. Oncologist 16:904-11, 2011 
22. 
Gore SD, Jiemjit A, Silverman LB, et al: Combined 
Methyltransferase/Histone deacetylase inhibition with 5Azacitidine and MS-275 in patients with 
MDS, CMMoL and AML: Clinical response, Histone Acetylation and DNA damage. Blood 
108:156a-157a, 2006 
23. 
Prebet T, Sun Z, Figueroa ME, et al: Prolonged administration of 
azacitidine with or without entinostat for myelodysplastic syndrome and acute myeloid leukemia 
with myelodysplasia-related changes: results of the US Leukemia Intergroup trial E1905. J Clin 
Oncol 32:1242-8, 2014 
24. 
Okazaki T, Chikuma S, Iwai Y, et al: A rheostat for immune responses: 
the unique properties of PD-1 and their advantages for clinical application. Nat Immunol 
14:1212-8, 2013 
25. 
Garon EB, Rizvi NA, Hui R, et al: Pembrolizumab for the treatment of 
non-small-cell lung cancer. N Engl J Med 372:2018-28, 2015 
26. 
Seiwert TY, Burtness B, Mehra R, et al: Safety and clinical activity of 
pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and 
neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial. Lancet Oncol 17:956-65, 
2016 
27. 
Zeidan AM, Linhares Y, Gore SD: Current therapy of myelodysplastic 
syndromes. Blood Rev 27:243-59, 2013 
28. 
Zeng H, Wu DP, Ouyang J, et al: [Expressions of costimulatory molecules 
on CD3(+)CD4(+) T cells in myelodysplastic syndrome]. Zhongguo Shi Yan Xue Ye Xue Za 
Zhi 16:1082-5, 2008 
29. 
Zou W, Chen L: Inhibitory B7-family molecules in the tumour 
microenvironment. Nat Rev Immunol 8:467-77, 2008 
30. 
Dong H, Strome SE, Salomao DR, et al: Tumor-associated B7-H1 
promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 8:793-800, 2002 
31. 
Pardoll DM: Immunology beats cancer: a blueprint for successful 
translation. Nat Immunol 13:1129-32, 2012 
32. 
Thompson RH, Dong H, Kwon ED: Implications of B7-H1 expression in 
clear cell carcinoma of the kidney for prognostication and therapy. Clin Cancer Res 13:709s- 
715s, 2007 
33. 
Nomi T, Sho M, Akahori T, et al: Clinical significance and therapeutic 
potential of the programmed death-1 ligand/programmed death-1 pathway in human pancreatic 
cancer. Clin Cancer Res 13:2151-7, 2007 
34. 
Gao Q, Wang XY, Qiu SJ, et al: Overexpression of PD-L1 significantly 
associates with tumor aggressiveness and postoperative recurrence in human hepatocellular 
carcinoma. Clin Cancer Res 15:971-9, 2009 
35. 
Hamanishi J, Mandai M, Iwasaki M, et al: Programmed cell death 1 ligand 


100 
                NCI Protocol #:10009  
                Version Date: 09/04/2020 
 
 
1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer. 
Proc Natl Acad Sci U S A 104:3360-5, 2007 
36. 
Hodi FS, Dranoff G: The biologic importance of tumor-infiltrating 
lymphocytes. J Cutan Pathol 37 Suppl 1:48-53, 2010 
37. 
Robert C, Thomas L, Bondarenko I, et al: Ipilimumab plus dacarbazine for 
previously untreated metastatic melanoma. N Engl J Med 364:2517-26, 2011 
38. 
Ribas A, Tumeh PC: Cancer therapy: Tumours switch to resist. Nature 
490:347-8, 2012 
39. 
Ribas A, Chesney JA, Gordon MS, et al: Safety profile and 
pharmacokinetic analyses of the anti-CTLA4 antibody tremelimumab administered as a one hour 
infusion. J Transl Med 10:236, 2012 
40. 
Topalian SL, Hodi FS, Brahmer JR, et al: Safety, activity, and immune 
correlates of anti-PD-1 antibody in cancer. N Engl J Med 366:2443-54, 2012 
41. 
Hamid O, Robert C, Daud A, et al: Safety and tumor responses with 
lambrolizumab (anti-PD-1) in melanoma. N Engl J Med 369:134-44, 2013 
42. 
Hamid O, Schmidt H, Nissan A, et al: A prospective phase II trial 
exploring the association between tumor microenvironment biomarkers and clinical activity of 
ipilimumab in advanced melanoma. J Transl Med 9:204, 2011 
43. 
Hess-Stumpp H, Bracker TU, Henderson D, et al: MS-275, a potent orally 
available inhibitor of histone deacetylases--the development of an anticancer agent. Int J 
Biochem Cell Biol 39:1388-405, 2007 
44. 
Shah P, Gau Y, Sabnis G: Histone deacetylase inhibitor entinostat reverses 
epithelial to mesenchymal transition of breast cancer cells by reversing the repression of E- 
cadherin. Breast Cancer Res Treat 143:99-111, 2014 
45. 
Juergens RA, Wrangle J, Vendetti FP, et al: Combination epigenetic 
therapy has efficacy in patients with refractory advanced non-small cell lung cancer. Cancer 
Discov 1:598-607, 2011 
46. 
Hauschild A, Trefzer U, Garbe C, et al: Multicenter phase II trial of the 
histone deacetylase inhibitor pyridylmethyl-N-{4-[(2-aminophenyl)-carbamoyl]-benzyl}- 
carbamate in pretreated metastatic melanoma. Melanoma Res 18:274-8, 2008 
47. 
Acharya MR, Karp JE, Sausville EA, et al: Factors affecting the 
pharmacokinetic profile of MS-275, a novel histone deacetylase inhibitor, in patients with 
cancer. Invest New Drugs 24:367-75, 2006 
48. 
Gabrilovich DI, Bronte V, Chen SH, et al: The terminology issue for 
myeloid-derived suppressor cells. Cancer Res 67:425; author reply 426, 2007 
49. 
Gabrilovich DI, Nagaraj S: Myeloid-derived suppressor cells as regulators 
of the immune system. Nat Rev Immunol 9:162-74, 2009 
50. 
Gabrilovich DI, Ostrand-Rosenberg S, Bronte V: Coordinated regulation 
of myeloid cells by tumours. Nat Rev Immunol 12:253-68, 2012 
51. 
Ostrand-Rosenberg S, Sinha P: Myeloid-derived suppressor cells: linking 
inflammation and cancer. J Immunol 182:4499-506, 2009 
52. 
Rodriguez PC, Zea AH, Culotta KS, et al: Regulation of T cell receptor 
CD3zeta chain expression by L-arginine. J Biol Chem 277:21123-9, 2002 
53. 
Gojo I, Jiemjit A, Trepel JB, et al: Phase 1 and pharmacologic study of 
MS-275, a histone deacetylase inhibitor, in adults with refractory and relapsed acute leukemias. 
Blood 109:2781-90, 2007 


101 
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54. 
Ryan QC, Headlee D, Acharya M, et al: Phase I and pharmacokinetic 
study of MS-275, a histone deacetylase inhibitor, in patients with advanced and refractory solid 
tumors or lymphoma. J Clin Oncol 23:3912-22, 2005 
55. 
Gore L, Rothenberg ML, O'Bryant CL, et al: A phase I and 
pharmacokinetic study of the oral histone deacetylase inhibitor, MS-275, in patients with 
refractory solid tumors and lymphomas. Clin Cancer Res 14:4517-25, 2008 
56. 
Batlevi CL, Kasamon Y, Bociek RG, et al: ENGAGE- 501: phase II study 
of entinostat (SNDX-275) in relapsed and refractory Hodgkin lymphoma. Haematologica 
101:968-75, 2016 
57. 
Denise A. Yardley SFJ, Peter Ordentlich, Carol J Braun, Helen S Pentikis, 
Jeannette Lynne Hasapidis, Miranda Rees, Howard A. Burris: A phase 1 study to assess the food 
effect on the pharmacokinetics (PK) of entinostat. J Clin Oncol 32:5s, 2014 (suppl; abstr 591), 
2014 
58. 
Wood ML, Slater DN: Cutaneous manifestations of the hypereosinophilic 
syndrome treated with sodium cromoglycate. Clin Exp Dermatol 9:139-42, 1984 
59. 
Schooley RT, Flaum MA, Gralnick HR, et al: A clinicopathologic 
correlation of the idiopathic hypereosinophilic syndrome. II. Clinical manifestations. Blood 
58:1021-6, 1981 
60. 
Flaum MA, Schooley RT, Fauci AS, et al: A clinicopathologic correlation 
of the idiopathic hypereosinophilic syndrome. I. Hematologic manifestations. Blood 58:1012-20, 
1981 
61. 
Cheson BD, Greenberg PL, Bennett JM, et al: Clinical application and 
proposal for modification of the International Working Group (IWG) response criteria in 
myelodysplasia. Blood 108:419-25, 2006 
62. 
Gajewski TF, Meng Y, Blank C, et al: Immune resistance orchestrated by 
the tumor microenvironment. Immunol Rev 213:131-45, 2006 
63. 
Nagaraj S, Gabrilovich DI: Myeloid-derived suppressor cells in human 
cancer. Cancer J 16:348-53, 2010 
64. 
Johansson CC, Mougiakakos D, Trocme E, et al: Expression and 
prognostic significance of iNOS in uveal melanoma. Int J Cancer 126:2682-9, 2010 
65. 
Diaz-Montero CM, Salem ML, Nishimura MI, et al: Increased circulating 
myeloid-derived suppressor cells correlate with clinical cancer stage, metastatic tumor burden, 
and doxorubicin-cyclophosphamide chemotherapy. Cancer Immunol Immunother 58:49-59, 
2009 
66. 
Kusmartsev S, Gabrilovich DI: Role of immature myeloid cells in 
mechanisms of immune evasion in cancer. Cancer Immunol Immunother 55:237-45, 2006 
67. 
Youn JI, Kumar V, Collazo M, et al: Epigenetic silencing of 
retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer. Nat Immunol 
14:211-20, 2013 
68. 
Zeidan AM, Zeidner JF, Duffield A, et al: Stabilization of 
Myelodysplastic Syndromes (MDS) Following Hypomethylating Agent (HMAs) Failure Using 
the Immune Checkpoint Inhibitor Ipilimumab: A Phase I Trial. Blood 126, 2015 
69. 
Sallusto F, Lenig D, Forster R, et al: Two subsets of memory T 
lymphocytes with distinct homing potentials and effector functions. Nature 401:708-12, 1999 
70. 
Twyman-Saint Victor C, Rech AJ, Maity A, et al: Radiation and dual 
checkpoint blockade activate non-redundant immune mechanisms in cancer. Nature 520:373-7, 


102 
                NCI Protocol #:10009  
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2015 
71. 
Almand B, Clark JI, Nikitina E, et al: Increased production of immature 
myeloid cells in cancer patients: a mechanism of immunosuppression in cancer. J Immunol 
166:678-89, 2001 
72. 
Mirza N, Fishman M, Fricke I, et al: All-trans-retinoic acid improves 
differentiation of myeloid cells and immune response in cancer patients. Cancer Res 66:9299- 
307, 2006 
73. 
Daud AI, Mirza N, Lenox B, et al: Phenotypic and functional analysis of 
dendritic cells and clinical outcome in patients with high-risk melanoma treated with adjuvant 
granulocyte macrophage colony-stimulating factor. J Clin Oncol 26:3235-41, 2008 
74. 
Kusmartsev S, Su Z, Heiser A, et al: Reversal of myeloid cell-mediated 
immunosuppression in patients with metastatic renal cell carcinoma. Clin Cancer Res 14:8270-8, 
2008 
75. 
Liu CY, Wang YM, Wang CL, et al: Population alterations of L-arginase- 
and inducible nitric oxide synthase-expressed CD11b+/CD14(-)/CD15+/CD33+ myeloid-derived 
suppressor cells and CD8+ T lymphocytes in patients with advanced-stage non-small cell lung 
cancer. J Cancer Res Clin Oncol 136:35-45, 2010 
76. 
Hoechst B, Ormandy LA, Ballmaier M, et al: A new population of 
myeloid-derived suppressor cells in hepatocellular carcinoma patients induces 
CD4(+)CD25(+)Foxp3(+) T cells. Gastroenterology 135:234-43, 2008 
77. 
Filipazzi P, Valenti R, Huber V, et al: Identification of a new subset of 
myeloid suppressor cells in peripheral blood of melanoma patients with modulation by a 
granulocyte-macrophage colony-stimulation factor-based antitumor vaccine. J Clin Oncol 
25:2546-53, 2007 
78. 
Poschke I, Mougiakakos D, Hansson J, et al: Immature 
immunosuppressive CD14+HLA-DR-/low cells in melanoma patients are Stat3hi and 
overexpress CD80, CD83, and DC-sign. Cancer Res 70:4335-45, 2010 
79. 
Vuk-Pavlovic S, Bulur PA, Lin Y, et al: Immunosuppressive CD14+HLA- 
DRlow/- monocytes in prostate cancer. Prostate 70:443-55, 2010 
80. 
van Cruijsen H, van der Veldt AA, Vroling L, et al: Sunitinib-induced 
myeloid lineage redistribution in renal cell cancer patients: CD1c+ dendritic cell frequency 
predicts progression-free survival. Clin Cancer Res 14:5884-92, 2008 
81. 
Kotsakis A, Harasymczuk M, Schilling B, et al: Myeloid-derived 
suppressor cell measurements in fresh and cryopreserved blood samples. J Immunol Methods 
381:14-22, 2012 
82. 
Camp RL, Chung GG, Rimm DL: Automated subcellular localization and 
quantification of protein expression in tissue microarrays. Nat Med 8:1323-7, 2002 
83. 
Schalper KA, Velcheti V, Carvajal D, et al: In situ tumor PD-L1 mRNA 
expression is associated with increased TILs and better outcome in breast carcinomas. Clin 
Cancer Res 20:2773-82, 2014 
84. 
Brown JR, Wimberly H, Lannin DR, et al: Multiplexed quantitative 
analysis of CD3, CD8, and CD20 predicts response to neoadjuvant chemotherapy in breast 
cancer. Clin Cancer Res 20:5995-6005, 2014 
85. 
Bordeaux J, Welsh A, Agarwal S, et al: Antibody validation. 
Biotechniques 48:197-209, 2010 
86. 
Rodriguez PC, Quiceno DG, Ochoa AC: L-arginine availability regulates 


103 
                NCI Protocol #:10009  
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T-lymphocyte cell-cycle progression. Blood 109:1568-73, 2007 
87. 
Ostrand-Rosenberg S: Myeloid-derived suppressor cells: more 
mechanisms for inhibiting antitumor immunity. Cancer Immunol Immunother 59:1593-600, 
2010 
88. 
Youn JI, Nagaraj S, Collazo M, et al: Subsets of myeloid-derived 
suppressor cells in tumor-bearing mice. J Immunol 181:5791-802, 2008 
89. 
Corzo CA, Cotter MJ, Cheng P, et al: Mechanism regulating reactive 
oxygen species in tumor-induced myeloid-derived suppressor cells. J Immunol 182:5693-701, 
2009 
90. 
Kitano S, Postow MA, Ziegler CG, et al: Computational algorithm-driven 
evaluation of monocytic myeloid-derived suppressor cell frequency for prediction of clinical 
outcomes. Cancer Immunol Res 2:812-21, 2014 
91. 
Bendall SC, Simonds EF, Qiu P, et al: Single-cell mass cytometry of 
differential immune and drug responses across a human hematopoietic continuum. Science 
332:687-96, 2011 


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APPENDIX A1 
MODIFIED INTERNATIONAL WORKING GROUP (IWG)-2006 
RESPONSE CRITERIA FOR ALTERING NATURAL HISTORY OF MDS 
 
Category 
Response criteria (responses must last at least 4 week) 
 
Complete 
remission 
%RQHPDUURZP\HOREODVWVZLWKQRUPDOPDWXUDWLRQRIDOOFHOOOLQHV* 
 
Persistent dysplasia will be noted*† 
Peripheral blood‡ 
+JEJG/
3ODWHOHWV[9/L 
1HXWURSKLOV[9/L† 
Blasts 0% 
 
Partial 
remission 
All CR criteria if abnormal before treatment except: 
 
%RQHPDUURZEODVWVGHFUHDVHGE\RYHUSUHWUHDWPHQWEXWVWLOO!
Cellularity and morphology not relevant 
 
Marrow CR† 
%RQHPDUURZP\HOREODVWVDQGGHFUHDVHE\RYHUSUHWUHDWPHQW† 
Peripheral blood: if HI responses, they will be noted in addition to marrow CR† 
Stable disease Failure to achieve at least PR, but no evidence of progression for > 8 wks 
Failure 
Death during treatment or disease progression characterized by worsening of 
cytopenias, increase in percentage of bone marrow blasts, or progression to a 
more advanced MDS FAB subtype than pretreatment 
 
 
 
 
Relapse after 
CR or PR 
At least 1 of the following: 
 
Return to pretreatment bone marrow blast percentage 
DeFUHPHQWRIIURPPD[LPXPUHPLssion/response levels inIgranulocytes 
or platelets 
Reduction in Hgb FRQFHQWUDWLRQE\JG/RUWUDQVIXVLRQGHSHQGHQFH 
 
Cytogenetic 
response 
 
 
 
Disease 
progression 
Complete 
 
Disappearance of the chromosomal abnormality without appearance of new ones 
Partial 
At least 50% reduction of the chromosomal abnormality 
For patients with: 


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/HVVWKDQEODVWVLQFUHDVHLQEODVWVWR!EODVWV
5%-EODVWVLQFUHDVHWR!EODVWV 
10%-EODVWVLQFUHDVHWR! blasts 
20%-30% EODVWVLQFUHDVHWR! blasts 
 
Any of the following: 
At least 50% decrement from maximum remission/response in granulocytes or 
platelets 
5HGXFWLRQLQ+JEE\JG/
Transfusion dependency 
 
Survival 
Endpoints: 
Overall: death from any cause 
Event free: failure or death from any cause 
PFS: disease progression or death from MDS 
DFS: time to relapse 
Cause-specific death: death related to MDS 
 
To convert hemoglobin from grams per deciliter to grams per liter, multiply grams per deciliter 
by 10. 
MDS indicates myelodysplastic syndromes; Hgb, hemoglobin; CR, complete remission; HI, 
hematologic improvement; PR, partial remission; FAB, French-American-British; AML, acute 
myeloid leukemia; PFS, progression-free survival; DFS, disease-free survival. 
* Dysplastic changes should consider the normal range of dysplastic changes (modification). 
† Modification to IWG response criteria. 
‡ In some circumstances, protocol therapy may require the initiation of further treatment (eg, 
consolidation, maintenance) before the 4-week period. Such patients can be included in the 
response category into which they fit at the time the therapy is started. Transient cytopenias 
during repeated chemotherapy courses should not be considered as interrupting durability of 
response, as long as they recover to the improved counts of the previous course. 


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APPENDIX A2 
MODIFIED INTERNATIONAL WORKING GROUP (IWG)-2006 
RESPONSE CRITERIA FOR HEMATOLOGIC IMPROVEMENT 
 
 
Hematologic 
improvement* 
Erythroid response 
(pretreatment, < 11 
g/dL) 
 
 
 
 
 
Platelet response 
(pretreatment, 
< 100 x 109/L) 
 
 
Neutrophil response 
(pretreatment, 
< 1.0 x 109/L) 
Response criteria (responses must last at least 8 wk)† 
 
+JELQFUHDVHE\JG/ 
 
 
Relevant reduction of units of RBC transfusions by an absolute number of 
at least 4 RBC transfusions/8 wk compared with the pretreatment 
transfusion number in the previous 8 wk. Only RBC transfusions given for 
D+JERIJG/SUHWUHDWPHQWZLOOFRXQWLQ the RBC transfusion 
response evaluation† 
 
$EVROXWHLQFUHDVHRI[9/L for patients starting with > 20x 109/L 
platelets 
 
Increase from < 20 x 109/L to > 20 x 109/L and by at least 100%† 
At least 100% increase and an absolute increase > 0.5 x 109/L† 
 
Progression or 
relapse after HI‡ 
At least 1 of the following: 
 
At least 50% decrement from maximum response levels in granulocytes or 
platelets 
5HGXFWLRQLQ+JEE\JG/
Transfusion dependence 
 
Deletions to the IWG response criteria are not shown. 
To convert hemoglobin levels from grams per deciliter to grams per liter, multiply grams per 
deciliter by 10. 
Hgb indicates hemoglobin; RBC: red blood cell; HI: hematologic improvement. 
* 3UHWUHDWPHQWFRXQWVDYHUDJHVRIDWOHDVWPHDVXUHPHQWVQRWLQIOXHQFHGE\WUDQVIXVLRQV
week apart (modification). 
† Modification to IWG response criteria. 
‡ In the absence of another explanation, such as acute infection, repeated courses of 
chemotherapy (modification), gastrointestinal bleeding, hemolysis, and so forth. It is 
recommended that the 2 kinds of erythroid and platelet responses be reported overall as well as 
by the individual response pattern. 


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APPENDIX A3 
INTERNATIONAL WORKING GROUP ACUTE 
MYELOID LEUKEMIA RESPONSE CRITERIA 
 
 
 
Response 
Criterion 
 
Time of 
Assessment 
 
Neutrophils 
(μL) 
 
Platelets 
(μL) 
Bone 
Marrow 
Blasts 
(%) 
 
Other 
Early treatment 
assessment 
7-10 days after 
therapy 
 
NA 
 
NA 
 
< 5 
 
Morphologic 
leukemia-free 
State 
 
Varies by protocol 
 
NA 
 
NA 
 
< 5 
 
Flow cytometry EMD 
Morphologic CR 
Varies by protocol 
> 1,000 
> 100,000 
< 5 
Transfusion EMD 
 
Cytogenetic CR 
 
Varies by protocol 
 
> 1,000 
 
> 100,000 
 
< 5 
Cytogenetics—normal, 
EMD 
 
Molecular CR 
 
Varies by protocol 
 
> 1,000 
 
> 100,000 
 
< 5 
Molecular—negative, 
EMD 
 
Partial Remission 
 
Varies by protocol 
 
> 1,000 
 
> 100,000 
> 50 or 
decrease 
to 5-25 
 
Blasts < 5% if Auer rod 
positive 
Table 11: Hematologic Response According to IWG Criteria for AML 
 
AML = acute myelogenous leukemia; CR = complete remission; EMD = extramedullary disease; 
onal Working Group; NA = not applicable (Cheson, 2003). 


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APPENDIX B 
INTERNATIONAL PROGNOSTIC SCORING SYSTEM (IPSS) 
FOR MYELODYSPLASTIC SYNDROME 
 
SCORE VALUES 
PROGNOSTIC VARIABLE 
0 
0.5 
1.0 
1.5 
2.0 
Marrow Blasts (%)† 
<5 
5-10 
– 
11-20 
21-30 
Karyotype* 
Good 
Intermediate Poor 
 
 
Cytopenias** 
0/1 
2/3 
 
 
 
†Patients with 21-30% blasts are considered as MDS or AML (WHO) 
*Cytogenetics: Good = normal, -<DORQHGHOTDORQHGHOTDORQH3RRU FRPSOH[
abnormalities) or chromosome 7 anomalies; Intermediate = other abnormalities 
**Cytopenias: neutrophil count<1800/μl, platelets <100,000, Hb <10 g/dL. 
 
 
Risk category 
(% IPSS pop.) 
Overall score 
Median survival 
(yr) 
25% 
AML 
progression (yr) 
LOW (33) 
0 
5.7 
9.4 
INT-1 (38) 
0.5-1.0 
3.5 
3.3 
INT-2 (22) 
1.5-2.0 
1.1 
1.1 
HIGH (7) 
>2.5 
0.4 
0.2 


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


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APPENDIX D 
PROTOCOL FOR MONONUCLEAR CELLS ISOLATION 
(FICOLL HYPAQUE): 
 
1. Resuspend bone marrow aspirates with PBS (1:1) 
 
2. Gently layer the diluted suspension over 5 ml of F/H in a 15 ml conical tube. You can load up 
to 10 mL however limiting this to 9 ml or less reduced the chances of contacting the cap. 
 
3. Spin at 400g (1200rpm) for 30 minutes at Room Temp (22-24C). Do not set the brake on the 
centrifuge. 
 
4. Carefully remove the tube from the centrifuge. Using a glass pipette carefully remove the 
interface (white) containing the MNC. This should take less than 2 ml per 15 ml conical (1 ml if 
you are careful not to disturb the layer). 
 
5. Transfer the cells to a fresh 15 ml conical tube and add PBS up to 10 ml (at least a 1:4 
dilution). Mix well then spin at 400g (1200rpm) for 10 minutes at Room Temp (22-24C). 
 
6. Carefully aspirate the supernatant and resuspend the cells in 10 ml of PBS. Mix well then spin 
at 400g (1200rpm) for 10 minutes at Room Temp (22-24C). 
 
7. Carefully aspirate the supernatant and resuspend the cells in 10 ml of PBS. Mix well then spin 
at 400g (1200rpm) for 10 minutes at Room Temp (22-24C). 
 
8. Resuspend the cell in a small volume of PBS or culture medium and count after mixing well. 
 
9. To cryopreserve the cells (minimum of 4 vials not to exceed 2x107/vial). 
 
a. The final concentration will usually be 40% Human Serum, 10% DMSO: 
1. The easiest method is to dilute the cells to a twice the final concentration in PBS and to 
add an equivalent volume of cells (500 ul) to a 2X freezing medium (80% Serum, 20% DMSO) 
prechilled to 4C. 
2. Transfer right away to a labeled cryovial and use a controlled rate device to drop the 
temperature to -80C prior to transfer to Liquid Nitrogen. The rate is usually ideal at -1C per 
minute for PBMC. 
 
Important considerations: 
 
1. All work should be done using standard BSL2 procedures (blood and body fluid precautions). 
2. Maintain a clean workspace and use a containment laminar airflow hood when possible. 
3. Minimize the chance of contamination. 
4. Work quickly but methodically. 
5. Keep tubes closed as much as possible and work quickly. 
6. Change gloves frequently and maintain situational awareness of contact surfaces. 


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APPENDIX E 
PATIENT DRUG INFORMATION HANDOUT AND WALLET 
CARD 
 
Information for Patients, Their Caregivers, and Non-Study Healthcare Team on Possible 
Interactions with Other Drugs and Herbal Supplements 
 
The patient  
is enrolled on a clinical trial using the 
experimental study drug, entinostat and Pembrolizumab (MK-3475). This clinical trial is 
sponsored by the National Cancer Institute. This form is addressed to the patient, but includes 
important information for others who care for this patient. 
 
These are the things that you as a healthcare provider need to know: 
 
x Entinostat was found to induce CYP 1A2, CYP 2C6, and CYP 2B8 as well as UGT 1A4. 
x Entinostat was found to be a substrate for P-gp and BCRP transporters, but did not inhibit 
either of these transport proteins. 
 
To the patient: Take this paper with you to your medical appointments and keep the 
attached information card in your wallet. 
 
Entinostat may interact with other drugs which can cause side effects. For this reason, it is very 
important to tell your study doctors of any medicines you are taking before you enroll onto this 
clinical trial. It is also very important to tell your doctors if you stop taking any regular 
medicines, or if you start taking a new medicine while you take part in this study. When you talk 
about your current medications with your doctors, include medicine you buy without a 
prescription (over-the-counter remedy), or any herbal supplements such as St. John’s Wort. It is 
helpful to bring your medication bottles or an updated medication list with you. 
 
Many health care providers can write prescriptions. You must tell all of your health care 
providers (doctors, physician assistants, nurse practitioners, pharmacists) you are taking part in a 
clinical trial. 
 
These are the things that you and they need to know: 
 
Entinostat must be used very carefully with other medicines that use CYP 1A2, CYP 2C6, and 
CYP 2B8, UGT 1A4 and P-gp as well as BCRP transporters. Before you enroll onto the clinical 
trial, your study doctor will work with your regular health care providers to review any 
medicines and herbal supplements that are considered strong inducers/inhibitors or substrates of 
CYP 1A2, CYP 2C6, and CYP 2B8 as well as UGT 1A4, and P-gp and BCRP transporters 
 
x Please be very careful! Over-the-counter drugs (including herbal supplements) may 
contain ingredients that could interact with your study drug. Speak to your doctors or 
pharmacist to determine if there could be any side effects. 
 
x Your regular health care provider should check a frequently updated medical reference or 
call your study doctor before prescribing any new medicine or discontinuing any 


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medicine. Your study doctor’s name is 
  
and he or she can be contacted at 
 
  
. 


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Entinostat interacts with a specific liver enzyme called CYP 2B6, 3A4, 
1A2, CYP 2C6, and CYP 2B8 as well as UGT 1A4, gp and BCRP 
transport protein and must be used very carefully with other 
medicines that interact with CYP 2B6, 3A4, 1A2, CYP 2C6, 2B8 as well 
as UGT 1A4, gp and BCRP transport protein. 
¾ Before you enroll onto the clinical trial, your study doctor will 
work with your regular health care providers to review any 
medicines and herbal supplements that are considered “strong 
inducers/inhibitors or substrates of CYP 2B6, 3A4, 1A2, CYP 2C6, 
2B8 as well as UGT 1A4, gp and BCRP transportprotein.” 
¾ Before prescribing new medicines, your regular health care 
providers should go to a frequently-updated medical referencefor 
a list of drugs to avoid, or contact your studydoctor.
¾ Your study doctor’s nameis
and can be contacted at
. 
STUDY DRUG INFORMATION WALLET CARD
You are enrolled on a clinical trial using the experimental study 
drug Entinostat and Pembrolizumab (MK-3475). This clinical trial is 
sponsored by the NCI. Entinostat may interact with drugs that are 
processed by your liver, or use certain transport proteins in your 
body. Because of this, it is very important to:
¾ Tell your doctors if you stop taking any medicines or if youstart 
taking any new medicines.
¾ Tellall of your health care providers (doctors, physician assistants, 
nurse practitioners, or pharmacists) that you are taking part in a 
clinical trial.
¾ Check with your doctor or pharmacist whenever you need touse 
an over-the-counter medicine or herbalsupplement.


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APPENDIX F 
TRANSPORTER MDR1/PGP TRANSPORTER MDR1/PGP 
 
Amiodarone 
S/Inhib 
Methadone 
Inhib 
Amitriptyline 
Inhib 
Mibefradil 
Inhib 
Amprenavir 
Induc 
Midazolam 
Inhib 
Astemizole 
Inhib 
Mifepristone 
Inhib 
Atorvastatin 
S/Inhib 
Nelfinavir 
S/Induc 
Boceprevir 
S/Inhib 
Nicardipine 
S/Inhib 
Bromocriptine 
Inhib 
Nifedipine 
Inhib 
Carvedilol 
Inhib 
Ofloxacin 
Inhib 
Chlorpromazine 
Inhib 
Pentazocine 
Inhib 
Clarithromycin 
Inhib 
Prazosin 
Induc 
Clotrimazole 
Induc 
Prochlorperazine 
Inhib 
Cyclosporine 
S/Inhib 
Progesterone 
Inhib/Induc 
Desipramine 
Inhib 
Propafenone 
Inhib 
Dexamethasone 
S/Induc 
Propranolol 
S/Inhib 
Dexverapamil 
Inhib 
Quercetin 
Induc 
Diltiazem 
S/Inhib 
Quinidine 
S/Inhib 
Dipyridamole 
Inhib 
Quinine 
Inhib 
Disulfiram 
Inhib 
Retinoic acid 
Induc 
Doxepin 
Inhib 
Reserpine 
inhib 
Erythromycin 
S/Inhib 
Rifampin 
S/Induc 
Fluphenazine 
Inhib 
Ritonavir 
S/inhib 
Glibenclamide 
Inhib 
Saquinavir 
S/Inhib 
Haloperidol 
Inhib 
Simvastatin 
S/Inhib 
Hydrocortisone 
S/Inhib 
St. John’s Wort 
Induc 
Imipramine 
Inhib 
Tacrolimus 
S/Inhib 


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Indinavir 
S/Induc 
Tamoxifen 
Inhib 
Itraconazole 
S/Inhib 
Telaprevir 
S/Inhib 
Ketoconazole 
Inhib 
Temsirolimus 
S/Inhib 
Lidocaine 
S/Inhib 
Testosterone 
Inhib 
Lovastatin 
S/Inhib 
Trimipramine 
Inhib 
Maprotiline 
Inhib 
Valspodar 
Inhib 
Mefloquine 
Inhib 
Verapamil 
S/Inhib 
Meperidine 
Inhib 
 
 
Inhib = Inhibition; Induc = Induction; S=substrate 


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APPENDIX G 
EXAMPLES OF SENSITIVE IN VIVO CYP SUBSTRATES AND 
CYP SUBSTRATES WITH NARROW THERAPEUTIC RANGE1 
 
CYP Enzymes 
Sensitive substrates2 
Substrates with 
narrow therapeutic range3 
CYP1A2 
Alosetron, caffeine, 
duloxetine, melatonin, 
ramelteon, 
tacrine, tizanidine 
Theophylline, tizanidine 
CYP2B64 
Bupropion, efavirenz 
 
CYP2C8 
Repaglinide5 
Paclitaxel 
CYP2C9 
Celecoxib 
Warfarin, phenytoin 
CYP2C19 
Lansoprazole, omeprazole, S- 
mephenytoin 
S-mephenytoin 
CYP3A6 
Alfentanil, aprepitant, 
budesonide, buspirone, 
conivaptan, darifenacin, 
darunavir, dasatinib, 
dronedarone, eletriptan, 
eplerenone, everolimus, 
felodipine, indinavir, 
fluticasone, lopinavir, 
lovastatin, 
lurasidone, maraviroc, 
midazolam, nisoldipine, 
quetiapine, saquinavir, 
sildenafil, simvastatin, 
sirolimus, tolvaptan, 
tipranavir, triazolam, 
vardenafil 
Alfentanil, astemizole7, 
cisapride7, cyclosporine, 
dihydroergotamine, 
ergotamine, fentanyl, 
pimozide, quinidine, 
sirolimus, tacrolimus, 
terfenadine7 
CYP2D6 
Atomoxetine, desipramine, 
dextromethorphan, 
metoprolol, 
nebivolol, perphenazine, 
tolterodine, 
venlafaxine 
Thioridazine 
1 Note that this is not an exhaustive list. For an updated list, see the following link: Drug Development and Drug 
Interactions. 
2 Sensitive CYP substrates refers to drugs whose plasma AUC values have been shown to increase 5-fold or higher 
when co-administered with a known CYP inhibitor. 
3 CYP substrates with narrow therapeutic range refers to drugs whose exposure-response relationship indicates that small 
increases in their exposure levels by the concomitant use of CYP inhibitors may lead to serious safety 
concerns (e.g., Torsades de Pointes). 
4 The AUC of these substrates were not increased by 5-fold or more with a CYP2B6 inhibitor, but they represent the 
most sensitive substrates studied with available inhibitors evaluated to date. 
5 Repaglinide is also a substrate for OATP1B1, and it is only suitable as a CYP2C8 substrate if the inhibition of 
OATP1B1 by the investigational drug has been ruled out. 
6 Because a number of CYP3A substrates (e.g., darunavir, maraviroc) are also substrates of P-gp, the observed 
increase in exposure could be due to inhibition of both CYP3A and P-gp. 
7 Withdrawn from the United States market because of safety reasons. 


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Also refer to the following link for the classification of substrates: 
Classification of Substrates: http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugIntera 
ctionsLabeling/ucm093664.htm#4) 


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APPENDIX H 
GASTRIC ACID REDUCING DRUGS 
 
 
Proton Pump Inhibitors 
• Omeprazole (Prilosec, Zegerid) 
• Lansoprazole (Prevacid) 
• Rabeprazole (AcipHex) 
• Pantoprazole (Protonix) 
• Esomeprazole (Nexium) 
H2 Inhibitors 
• Cimetidine (Tagamet) 
• Ranitidine (Zantac) 
• Famotidine (Pepcid) 
• Nizatidine (Axid) 
Antacids 
• Alka-Seltzer 
• Alka-2, Surpass Gum, Titralac, Tums 
• Milk of Magnesia 
• ALternaGEL, Amphojel 
• Gaviscon, Gelusil, Maalox, Mylanta, Rolaids 
• Pepto-Bismol 
 
Also refer to the following link for examples of gastric acid reducing drugs: 
Gastric acid reducing drugs: http://www.everydayhealth.com/ulcer/ulcer-treatment.aspx 


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APPENDIX I 
BIOASSAY TEMPLATES 
 
If the protocol includes any integral biomarker studies using in situ hybridization (ISH), 
immunohistochemistry (IHC), and/or DNA-based mutation assays, you may fill out the 
appropriate template (found at 
http://www.cancerdiagnosis.nci.nih.gov/diagnostics/templates.htm) and attach to this protocol 
submission as separate Appendices. 
 
If the laboratory or laboratories performing the studies has an alternatively-formatted document 
that supplies the same level of information regarding validation, materials and methods, etc., it 
may be used instead of the templates. 
 
 
N/A since the correlative biomarker of MDSCs measurements are exploratory 


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APPENDIX J 
CORRELATIVE STUDY PROPOSAL 
 
BACKGROUND and RATIONALE: Besides allogeneic stem cell transplantation, the DNA 
methyltransferase inhibitor (DMNTi), azacitidine (AZA) is the only treatment proven to prolong 
survival in higher risk myelodysplastic syndrome (HR-MDS). Nonetheless, HR-MDS patients 
treated with AZA have a median survival advantage of 9.5 months (24.5 vs 15 months median 
OS)1 and the objective response rate is only 50% with most patients eventually relapsing within 2 
years1. Failure of DNMTi therapy occurs in the majority of treated patients and carries a dismal 
prognosis with a median survival of less than 6 months2. To date, no drug or combination of drugs 
have improved survival of MDS patients in the post-DNMTi failure setting in randomized trials, 
and there is currently no standard of care for those patients. Therefore, improving outcomes of 
MDS patients after failure of DNMTi remains a top clinical and research priority3. 
Recent advances in tumor immunology have facilitated understanding the role of immune escape 
as an important mechanism of cancer progression and drug resistance62. Recent data suggest that 
myeloid-derived suppressor cells (MDSCs) contribute to immune escape in some cancers63-65. 
MDSCs have been shown to exert a suppressive role against T cells in tumor microenvironment 
and lead to cancer progression in tumor-bearing mice models and cancer patients66. Murine 
MDSCs have been well defined by a combination of surface markers and its correlation with in 
vitro and in vivo suppressive functions49. However, much less has been reported on their human 
counterparts. An important recent study published in the Journal of Clinical Investigation also 
demonstrated the importance of MDSCs which were significantly increased in bone marrows (BM) 
of MDS patients (~35% vs <5% in non-MDS BM controls) and played an important role in 
ineffective hematopoiesis in MDS14. Thus, reprograming MDSCs to normal myeloid lineage cells 
may be important to prevent ineffective hematopoiesis and avoid immune escape. 
Epigenetic silencing of retinoblastoma gene appears to regulate the differentiation of myeloid cells 
to MDSCs in cancers and histone deacetylase inhibitors (HDACi) prevent this pathologic 
differentiation67. Another recent study demonstrated that MDSCs induce resistance to immune 
checkpoint blockade (anti-CTLA4 and anti-PD1) in colon cancer bearing mice15. Importantly,the 
MDSCs were decreased by the use of the epigenetic modulator, entinostat (ENT), leading to 
rejection of tumors when combined with immune checkpoint blockade15. Thus, it will be important 
to investigate whether HDAC inhibitors can decrease the quantity of MDSCs and impact the course 
of disease. 
As single agents, several HDACi were safe and well-tolerated but exhibited only modest clinical 
activities in patients with MDS4,5. Therefore, the interest to develop these agents further has 
focused on combination-based strategies, usually with DNMTi. To date, no HDACi-DNMTi 
combination has improved survival of MDS patients in randomized studies when used in the 
frontline or the post-DNMTi failure setting. These observations suggest that while HDACi therapy 
has activity in MDS, DNMTi might not be the best choice for combination-based regimen with 
HDACi. Exploration of alternative agents to combine with HDACi in the post-DNMTi failure 
setting might be a more rational approach. 
Another important mechanism which cancers employ commonly to evade the immune system 
involve the exploitation of T-cell inhibitory pathways involving inhibitory molecules such as the 
programed death-1 (PD-1) and cytotoxic T-lymphocyte associated antigen-4 (CTLA-4)6,7. Immune 
checkpoint blockade with antibodies directed against PD-1, its ligand (PD-L1), and CTLA-4 has 
emerged as a novel promising approach to reverse immune evasion, and has resulted in a 
significant and durable clinical activity in some patients with advanced solid malignancies 


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especially metastatic melanoma, lung cancer, bladder cancer, and Hodgkin lymphoma8-11. 
Compared to expanding clinical applications of immune checkpoint inhibitors in solid tumors, the 
mechanisms of immune evasion and their therapeutic targeting in myeloid malignancies, including 
MDS, have not been fully elucidated. Several studies suggest that interaction between PD-1 and 
PD-L1 may induce immune evasion in MDS12 and moreover the upregulation of these molecules 
could be associated with resistance to DNMTi therapy in MDS13. 
 
HYPOTHESIS: We hypothesize that the HDACi ENT can reduce the quantity and suppressive 
function of MDSCs in BM of MDS patients who fail to get responses from DNMTi. We also 
hypothesize that ENT with have a synergistic effect with the anti-PD-1 antibody pembrolizumab 
leading to meaningful clinical responses when used in combination. The objective of this proposal 
is to assess the in vivo effects of the combined therapy with PD1 blockade and HDACi on three 
exploratory immunologic endpoints through correlative and mechanistic studies of samples 
collected from BM of treated patients. 
 
SPECIFIC AIMS: 
 
Aim 1. Assess the dynamic changes in MDSCs (quantitative and qualitative) in relation to 
ENT/anti-PD1 combined therapy and its correlation with the clinical response. 
 
Aim 2. Evaluate PD-L1 expression in CD34+ blasts from human MDS BM and its correlation with 
the response to anti-PD1 therapy. 
 
Aim 3. Characterize functional dynamics of immune cell subsets in MDS BM following ENT and 
anti-PD1 treatment. 
 
Successful completion of these studies will describe the pembrolizumab-induced reprogramming 
in T-cell populations, assess whether these changes correlate with clinical events, provide critical 
insights into the future wider use of PD-1 blockade-based immunotherapy in patients with MDS 
and other myeloid malignancies, and demonstrate the significance of targeting MDSCs via 
HDACi. 
 
PRELIMINARY DATA/PRIOR STUDIES: 
The role of MDSCs in MDS and the preclinical data of HDACi to suppress MDSCs: 
Myelopoiesis is altered in cancer including the expansion of a subset of relatively immature and 
activated myeloid cells, now called MDSCs48. MDSCs have been reported to facilitate tumor 
metastasis and angiogenesis49-51. Recently, MDSCs were found to be significantly expanded in 
BM of patients with MDS and to play an important role in the pathogenesis of the disease 
especially in driving ineffective hematopoiesis14. The role of MDSCs in pathogenesis of MDS and 
the optimal strategy to target MDSCs remain under investigation. Kim and colleagues recently 
demonstrated that MDSCs can mediate resistance to immune checkpoint blockade therapy in a 
murine tumor model15. In this study, treatment with anti-PD-1 and anti-CTLA-4 inhibitor 
antibodies could not completely eliminate murine colon cancers or metastatic murine breast 
cancers15. Interestingly, when the animals were co-treated with epigenetic modifiers (i.e. 
ENT+AZA or ENT alone) in addition to the antibodies, the outcomes improved significantly with 
cure rates in excess of 80% of the tumor-bearing mice15. Importantly, AZA and ENT (alone or 


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together) in absence of the immune-checkpoint inhibitors also failed to eradicate the tumors. These 
findings suggested that the combination of the class 1 HDACi ENT with immune-checkpoint 
inhibition was sufficient to eradicate the primary tumors and their metastasis. Mechanistic studies 
showed that epigenetic modulators exerted their effect primarily by down-regulating MDSCs, 
suggesting that cancers which are resistant to immune checkpoint blockade can be potentially 
cured by targeting MDSCs15. Another study showed that epigenetic modification by HDAC-2 
regulates the differentiation of MDSCs in a murine cancer model and HDACi induces 
differentiation of MDSCs to normal macrophages and dendritic cells rather than expanding 
MDSCs67. Based on the significant role of MDSCs in pathogenesis and progression of MDS, the 
potentially important role of MDSCs in mediating resistance to immune checkpoint blockade 
therapy, and the preclinical data of the efficacy of the HDACi ENT in suppressing MDSCs, we 
will assess the quantitative and qualitative change of MDSCs in BM of MDS patients pre- and 
post- ENT/anti-PD1 therapy to achieve our primary goal of this study in specific aim 1. 
PD-L1 expression in MDS and its correlation with the response to anti-PD1 therapy: Kondo 
et al. demonstrated that PD-L1-positive MDS blasts have an intrinsic proliferative advantage and 
induce T-cell suppression through interactions with PD-1 receptors potentially contributing to 
disease progression12. They also showed in BM samples, blasts from HR-MDS patients expressed 
PD-L1 molecules more often compared with those from LR-MDS patients while the BM T-cells 
over-expressed PD-1 molecules12. The MD Anderson group studied mRNA in CD34+ cells from 
124 patients with MDS, acute myeloid leukemia (AML) and chronic myelomonocytic leukemia 
(CMML) and found that 34%, 15%, and 8% had aberrant up-regulation (စ2-fold) in PD-L1, PD- 
1 and CTLA4, respectively13. PD-L1 protein expression was observed in MDS CD34+ cells, 
whereas stromal and non-blast cellular elements were positive for PD-1. More importantly, in a 
subgroup of patients treated with DNMTi therapy, PD-L1, PD-1 and CTLA4 expression was 
increased. Furthermore, patients who exhibited resistance to DNMTi therapy showed higher 
relative up-regulation in gene expression than responding patients. Treatment of leukemia cells 
with decitabine resulted in a dose-dependent up-regulation of these genes and partial 
hypomethylation of PD-1 in leukemia cell lines and human samples13. These results suggest the 
PD-1/PD-L1 axis might mediate resistance to DNMTi therapy and provide rationale for using 
immune checkpoint blockade with an anti-PD1 antibody as a novel therapeutic approach to 
improve outcomes of HR-MDS patients. However, preliminary results from ongoing early phase 
clinical trials of ant-CTLA-4 and anti-PD1/PD-L1 agents in MDS patients suggest that immune 
checkpoint therapy as a monotherapy only leads to disease stabilization in some patients but very 
few responses68. This observation provides rationale for combining immune checkpoint therapy 
with other novel agents with distinct mechanisms of action to improve patient outcomes. 
Additionally, there is no single biomarker to predict the response to anti-PD1 therapy in cancers. 
Previous data indicate that the response rate of anti-PD-1 therapy is higher in PD-L1 expressing 
tumors40. On the other hand, recent two landmark studies suggest that the predicted response of 
anti-PD-L1 is associated with PD-L1 expression in tumor-infiltrating immune cells8,10. These 
studies have led us to test the question about the expression of PD-L1 in CD34+ blasts from human 
MDS BM and its correlation with the response to combined anti-PD1/ENT therapy in MDS in 
specific aim 2. 
Exploratory biomarkers to predict the response to anti-PD1 therapy in MDS: Compared to a 
murine model for linear T-cell differentiation, we have had to rely on cross-sectional assessment 
to define memory T-cell subsets. More than two markers (CD45RA, CCR7) enabled us to delineate 
memory T-cell compartments69. For instance, we simply divide T-cells into four subsets: naïve 


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(CD45RA+CCR7+), central memory (CD45RA-CCR7+), effector memory (CD45RA-CCR7-), 
late effector (CD45RA+CCR7-)69. The ratio of TCM (CD45RA-CCR7+) to TEMRA 
(CD45RA+CCR7-) and of TEM (CD45RA-CCR7-) to TEMRA (CD45RA+CCR7-) will be assessed 
in BM and peripheral blood using flow cytometry. More recent preclinical data suggest that the 
frequency of CD8+, and CD8+/regulatory T cells (Tregs) are two important predictive markers for 
anti-PD1 therapy70. Additionally, this study also demonstrated exhausted CD8+ T cells defined by 
the expression of PD-1 and Eomes are reinvigorated, which can be defined by increased Ki67 and 
GzmB, following anti-PD1 and anti-CTLA4 treatment in a murine model. In specific aim 3, we 
will determine whether known biomarkers predict the response to anti-PD1 therapy in MDS. 
 
METHODOLOGY OF CORRELATIVE STUDIES (EXPLORATORY BIOMARKERS): 
Specific Aim 1. Determine the numeric and functional alterations of MDSCs in relation to 
ENT/anti-PD1 therapy and their correlations with the clinical response 
Human MDSCs are defined by a cross-sectional assessment of well-known surface markers with 
functional phenotype. Initially, the expression of a common myeloid marker, CD33 without the 
expression of the lymphoid cell markers (CD3, CD19, CD56) defined human MDSCs63. 
Subsequent studies classified human MDSCs by HLA-DR-/Lineage-71, HLA-DR-/Lineage- 
/CD33+72-74, HLA-DR-/Lineage-/CD33+/CD11b+65, CD14-/CD15+/CD33+/CD11b+75, or HLA- 
DR-/CD1476-80. These heterogeneous definitions of human MDSCs cause a fundamental caveat 
especially in human correlative studies, suggesting that the most clinically relevant markers should 
be determined. Moreover, sample processing such as cryopreservation has been reported to affect 
some expression of surface markers and the suppressive function of human MDSCs81. Thus fresh 
BM aspirate will be assessed with flow cytometry to avoid any artifact from cryopreservation 
process. 
Historically, human BM core biopsy has been decalcified for immunohistochemistry (IHC). This 
additional manipulation may also affect the expression of surface markers of MDSCs. Thus, we 
will use surface markers that are relatively resistant to cryopreservation81 in formalin-fixed 
paraffin-embedded clot sections to avoid decalcification step to perform multiplex quantitative 
immunofluorescence (multiplex-QIF) while maintaining the immune architecture. We will also 
compare IHC/IF findings and its quantitative analyses by using automated quantitative analysis 
(AQUA) (described below) with flow cytometric data in BM aspirate. These procedures will be 
performed with a strong collaboration with Mina Xu at the Hematology tissue bank/Dr. Stephanie 
 Halene’s lab (Yale and at Dr. Tae Kon Kim laboratory at Vanderbilt University (Kim Lab).. 
We acknowledge the lack of understanding the baseline AQUA value of MDSCs in treatment naïve 
MDS patients, inter-patient or intra-patient variability of MDSCs at this time. While our study will 
provide important data, are collecting BM cells from treatment-naïve MDS patients (regardless of 
participation in the study) to show interpatient consistency of our biomarker the baseline value of 
MDSCs using QIF and flow cytometry. We will also use BM cells from one particular healthy 
donor as an inter-assay variability control. Intra-patient variability will be evaluated by the quantity 
of MDSCs in peripheral blood serially sampled from treatment naïve- MDS patients (before 
starting any treatment except transfusion) 
SA 1.1. Assess the quantitative change of MDSCs in BM of MDS patients in response to ENT/anti- 
PD1 treatment. 
Human MDSCs, defined as CD11b+/Lineage-ve (CD3, CD14, CD16, CD19, CD20, CD56)/HLA- 
DR-/CD33+ cells as in the prior landmark publication,14 will be enumerated in fresh BM aspirate 
of MDS patients by flow cytometry. All flow cytometric acquisition will be carried out using an 


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LSR II (BD) or FACS Calibur (BD) and acquired data will be analyzed using FlowJo software 
(TreeStar). 
In addition, formalin-fixed paraffin-embed clot sections of BM clot sections will be investigated 
by QIF using automated quantitative analysis (AQUA) as previously described 82. Briefly, 
AQUA® is a method to objectively and accurately measure biomarker expression within defined 
areas of interest and subcellular compartments based on co-localization with other biomarkers 
and DAPI (4',6-diamidino-2-phenylindole). Pixel intensity of the target of interest is measured 
within these compartments and divided by the compartment area resulting in a continuous 
scoring system directly proportional to the concentration of the biomarker of interest. 
Multiplexing different markers of immune cells and immune checkpoints allows identification 
and characterization of various cell types involved in immune response and in development of 
resistance to specific therapeutic approaches. Assays for multiplexing up to 4 different markers 
are established for QIF by AQUA and have been described previously83,84. 
The following multiplexing panel will be applied to characterize MDSCs and their macro/micro 
environment: 
Antibody 
CD33 
CD11b 
HLA-DR 
Company 
Abcam 
Novus 
Abcam 
Clone 
SP266 
CL1719 
MEM-267 
Species/Isotype 
Rabbit Monoclonal 
Mouse IgG1 
Mouse Monoclonal 
IgG2b 
Catalogue 
Number 
Ab199432 
NBP2-34490 
Ab26089 
 
For quality control purposes and standardization, each antibody undergoes rigorous validation 
using a previously described protocol 85. Only antibodies that validate for specificity and sensitivity 
are used for assessment of clinical specimens. Protein specific index TMAs consisting of 40 to 60 
cancer specimens and cell lines are run alongside each experiment for quality control and 
standardization purposes. This approach will allow to standardize the assays, control and normalize 
for batch effects. Assays will then be automated and standardized to optimize assay conditions and 
reproducibility. These studies will be performed at baseline, after the first cycle of ENT 
monotherapy, after every 2 cycles of combined therapy, and at progression or discontinuation from 
study. 
SA 1.2. Assess the spatial relationship between MDSCs and cell subsets in MDS BM 
Following the efforts of identification and quantification of MDSCs serial clot sections will be 
stained for CD11b, for CD34 and E-Cadherin in order to investigate the spatial relationships of 
MDSCs with myeloblasts and pro-erythroblasts. Further subclassification of MDSCs can be 
performed using the biomarkers CD14 and CD15 for differentiation of monocytic and granulocytic 
MDSCs, respectively. 
Further characterization of T cell subsets will be performed if sufficient tissue is available. The 
immune cell panel of CD3/CD4/CD8 will be analyzed as well as PD-1 and FOXP3 levels will be 
measured. 
In addition to protein quantification using the AQUA method of QIF, the slides will also be 
scanned on the Vectra platform, which is an automated quantitative pathology imaging system to 
be used with immunofluorescent or immunohistochemical stains. The Inform Software facilitates 
cell counting and characterization as opposed to the QIF AQUA system where scoring is based on 
measurement of protein concentration. 


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SA 1.3. Assess the change of functional characteristics of MDSCs in BM of MDS patients in 
response to ENT/anti-PD1 treatment 
The depletion of L-arginine inhibits T-cell proliferation through decreasing the expression of &'ȗ
chain52 and down-regulating the expression of cyclin D3 and cyclin-dependent kinase 486 leading 
to the arrest of T cells in G0-G1 of the cell cycle. The increased activity of arginase in MDSCs 
leads to enhanced L-arginine catabolism, which depletes L-arginine in tumor microenvironment. 
Now the assay to measure arginase activity is one of the classic methods to assess the function of 
MDSCs. Therefore, intracellular expression of arginase I in MDSCs of MDS patients will be 
assessed by multi-color flow cytometry using surface markers to define MDSCs following fixation 
and permeabilization compared87,88. 
We will also measure arginase activity of MDSCs in MDS patients. Briefly, sorted MDSCs from 
BM of MDS patients (sorted by FACS) will be lysed and the enzyme will be activated by heating 
for 10 mins at 56’C. Arginine hydrolysis will be conducted by incubating the lysate with 100μl of 
0.5M L-arginine at 37’C for 2 hrs. Urea concentration will be measured at 540nm after addition of 
ORIĮ-isonitrosopropiophenone, followed by heating at 95’C for 30mins. 
The ability to generate reactive oxygen species (ROS) has been reported to be one of the major 
mechanisms of MDSC-induced immune suppression in cancer patients89. Thus, we will use the 
ROS production following stimulation as a surrogate functional marker for MDSCs81. Briefly, 
MDS BM cells will be incubated at 37qC in medium in the presence of 10PM oxidation-sensitive 
dye, dichlororodihydrofluorescein diacetate (DCFDA) (Molecular Probes, CA) for 15 min. Cells 
will be stimulated by 50ng/ml phorbol 12-myristate-13-acetate (PMA) and 2PM ionomycin in the 
presence of DCFDA. Unstimulated and stimulated cells will be stained with antibodies staining 
surface markers to define MDSCs. 
These studies will be performed at baseline, after the first cycle of ENT monotherapy, after every 
2 cycles of combined therapy, and at progression or discontinuation from study. We expect the 
arginase I expression, the arginase activity and ROS generation of MDSCs to diminish following 
ENT treatment. 
SA 1.4. Evaluate the alteration of the suppressive function of MDSCs from BM of MDS patients 
following ENT/anti-PD1 treatment 
Functional properties of MDSC include suppression of antigen-stimulated T cell proliferation and 
IFN-Ȗ production51. To determine the functional alteration of MDSCs after ENT/anti-PD1 therapy, 
we will perform T-cell proliferation assay using thymidine incorporation and ELISA for IFN-Ȗ
detection. For T-cell suppression assay, MDSCs will be sorted from BM of MDS patients by 
Fluorescence-activated cell sorting (FACS). T-cells will be isolated by magnetic-activated cell 
sorting (MACS) using CD3 microbeads (Miltenyi Biotec) from autologous peripheral blood. T- 
cells (2x104) will be plated in 96-well flat bottom plates in triplicate in RPMI medium 
supplemented with 10% Fetal Bovine Serum. T-cells will be stimulated with IL-2 and anti- 
CD3/anti-CD28 coated beads (Invitrogen) at a 1:2 ratio of T cells to beads. MDSCs will beadded 
with T-cell cultures at ratios of 1:4. After 3 days of co-culture, supernatants will be harvested to 
measure IFN-ȖFRQFHQWUDWLRQ E\ (/,6$ 7K\PLGLQH LQFRUSRUDWLRQ DVVD\ ZLOO EH SHUIRUPHGWR
analyze T-cell proliferation and its suppression by MDSCs. These studies will be performed at 
baseline, after the first cycle of ENT monotherapy, after every 2 cycles of combined therapy, and 
at progression or discontinuation from study. We expect the suppressive activity of MDSCs on T- 
cell function to diminish following ENT treatment. 
Potential pitfall (SA1): The quantity of MDSCs in BM samples is hard to predict. In addition to 
QIF, we will also perform flow cytometric analyses using the same markers (CD11b, CD33, HLA- 


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DR) as well as CD14, and CD15 to define granulocytic MDSCs (CD11b+/CD33+/HLA-DR- 
/CD15+) and monocytic MDSCs (CD11b+/CD33+/HLA-DR-/CD14+) in fresh aspirate 
simultaneously obtained with BM core biopsy. 
 
We will prioritize patient BM aspirates if there is not enough to perform all of the assays described 
in SA1. Our first priority will be to measure the quantitative alteration of MDSCs pre- and post- 
anti-PD1 therapy/ENT by flow cytometry. We will measure the expression of arginase I, the 
generation of ROS (SA 1.3). We will then assess the arginase activity (SA 1.3) and the suppression 
assay (SA 1.4), and lastly measure arginase activity. Cryopreservation, decalcification and the type 
of collection tubes (heparin vs EDTA vs calcium phosphate) may affect the quantity or quality of 
MDSCs81,90. We will stain fresh cells for flow cytometry and functional assay and clot section of 
BM for QIF assay81. We will also use EDTA tubes as described before if we see any significant 
difference of the quantity or quality of MDSCs depending on the types of tube90. 
 
Specific Aim 2. Evaluate whether PD-L1 expression in CD34+ blasts from MDS BM 
correlates with the response to anti-PD1 therapy: Clot sections of BM will be fixed. PD-L1 
expression will be evaluated on MDS CD34+ blasts by IHC and QIF. Specimens will be classified 
as negative or positive if <5%,  of CD34+ cells are PD-L1 positive, respectively as previously 
described8,10,13. Flow cytometry will be carried out on the BM aspirate to analyze the percentage 
of PD-L1 expressing CD34+ blasts in BM of MDS patients using rabbit anti-PD-L1 monoclonal 
antibody (E1L3N, Cell Signaling). Specimens will be classified as FACS negative or positive if 
RI&'DUH3'-L1 positive, respectively. These studies will be performed at the 
beginning of trial and after the first cycle of ENT monotherapy, after every 2 cycles of combined 
therapy, and at progression or discontinuation from study 
 
Specific Aim 3. Characterize functional dynamics of immune cell subsets in MDS patients 
pre- and post- anti-PD1 therapy: Using multi-color flow cytometry, we will examine CD4+, 
CD8+ T-cell subpopulations (expressing CD45RA, CCR7, and CD27), Foxp3+ regulatory T cells 
(T-regs), and also natural killer (NK), NKT cells and dendritic cells (DC) with the following panel 
of mAbs: CD16 and CD56 (markers of NK cells and NKT cells), CD11c (a marker of myeloid 
DCs) and CD123 (a marker of plasmacytoid DCs) in BM aspirate and PBMCs of MDS patients 
obtained as above (pre- and post- anti-PD1). We will also analyze PD-1 expression and 
intracellular molecules such as Foxp3, Ki67, Eomes and Granzyme B (GzmB). All flow cytometric 
acquisition will be carried out using an LSR II (BD) or FACS Calibur (BD) and acquired data will 
be analyzed using FlowJo software (TreeStar). The changes in these subsets will be correlated with 
clinical responses. 
 
Potential pitfall and alternative methods (for SA2 & SA3): We will possibly have limited 
number of cells in BM aspirate, so we will prioritize patient samples if not enough to perform all 
the assays described in SA2, SA3. Our priority will be to carry out SA3 since SA2 will be assessed 
by IHC. Alternatively, we will analyze the immune profile combining a high-throughput technique 
that will allow us to study more than 20 markers in a single cell analysis using mass cytometry 
(CyTOF). These techniques will provide information about the architecture of the tumor 
microenvironment (TME) (i.e. BM) and the distribution of markers of interest detected with 
CyTOF. CyTOF-based single cell analysis is a new technology that combines flow cytometry with 
metal-conjugated antibodies detected by mass spectrometry permitting higher order multiplexing 
(up to 100 molecules) on a single cell analysis91. For this assay, the staining will be performed 


127 
                NCI Protocol #:10009  
                Version Date: 09/04/2020 
 
 
using MaxPar Human T Cell Phenotyping Panel Kit (CD11a, CD4, CD8a, CD16, CD25, CD45, 
CCR7, CD69, CD45RO, CD44, CD27, CD45RA, CD3, CD57, HLA-DR, and CD127) along with 
CD28, CD117, and CD95), modified MaxPar human AML phenotyping panel (added 
CD10,CD13, CD16 on top of CD19, CD117, CD11b, CD64, CD7, CD123, CD45, CD33, CD15, 
CD34, CD3, CD44, CD38, HLA-DR, CXCR4) for surface staining as per manufacturer’s protocol. 
Additionally, after fixation and permeabilization, cells will be stained with anti-human Ki-67- 
151Eu (B56; BD Pharmingen Ab conjugated with lanthanide MaxPar Europium Chloride 151Eu 
using the MaxPar X8 Ab labeling kit) for 30 min at room temperature. Cells will be acquired on 
CyTOF 2 instrument (DVS; Fluidigm Sciences). All data will be analyzed and graphs generated 
using the DVS Cytobank software (Cytobank). 
STATISTICAL CONSIDERATIONS and ANALYTICAL PLAN 
SPECIFIC AIM 1: 
The quantitative change in MDSCs (% MDSCs by flow cytometry, QIF score by AQUA) during 
treatment with the ENT/anti-PD1 combined therapy will be estimated using mixed effects models 
to take into account the within-patient correlation of measurements over time. Likelihood ratio 
tests will be performed to confirm if random intercepts and slopes are necessary in the model. The 
fixed effect for change in MDSCs over time will be evaluated for significance. The variability in 
the rate of change in MDSCs across patients will also be examined. The association between the 
clinical outcome and a meaningful reduction in MDSCs, which will be defined after a review of 
the data, will be assessed with the chi-square test. The quantity of MDSCs at baseline and during 
treatment as continuous variables can also be compared between responding and non-responding 
patients using a t-test or Mann-Whitney U-Test, if more appropriate. We will apply the same 
statistical analyses for the expression of arginase I and ROS and arginase activity of MDSCs (SA 
1.3) and suppressive activity of MDSCs by measuring T-cell proliferation & IFN-Ȗ production (SA 
1.4). 
SPECIFIC AIM 2: MDACC study demonstrated 20% of leukemic blasts express PD-L1 when 
positive is considered when more than 5% CD34+ blasts express PD-L1 by IHC while in normal 
CD34+ controls, PD-L1 is nearly 0%13. An association of clinical response with the expression of 
PD-L1 MDS CD34+ blasts will be assessed by a Pearson chi-square test on a 2x2 table of 
frequencies. The dependent variable will be defined as response (yes versus no), and flow 
cytometry/IHC categories (negative vs positive) will be the independent variables. We will also 
monitor the dynamic change of PD-L1 expression over the course of treatment and its correlation 
with clinical response. Longitudinal measurements of PD-L1 will be examined using mixed- 
effects modeling. 
SPECIFIC AIM 3: Statistical analyses of the frequency of CD8+, CD4+, Foxp3 Tregs, 
CD8+/Foxp3+ Tregs, TCM/TEMRA, TEM/TEMRA, the percentage of Ki67 and GzmB in PD-1+, 
Eomes+ CD8 T cells to compare changes over time from baseline to several time-points will be 
performed by using mixed effects modelling with a Benjamini-Hochberg correction to control for 
false discovery rates.