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3.2.1.2 dac dosing

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

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

NCI-CTCAE Grade Treatment Modification for Pembrolizumab  
- Once the pembrolizumab infusion rate has been decreased by 50% or interrupted due to an infusion-related reaction, it must remain decreased for all subsequent infusions. - If the subject has a second infusion-related reaction Grade ≥ 2 on the slower infusion rate, the infusion should be stopped and the subject should be removed from study treatment. - If hypersensitivity reaction occurs, the subject must be treated according to the best available medical practice. IV=intravenous, NCI-CTCAE=National Cancer Institute-Common Terminology Criteria for Adverse Event, NSAIDs=nonsteroidal anti-inflammatory drugs.  
3.2.3 Additional Modifications for Subjects with Grade 2 Infusion-Related Reactions If, in the event of a Grade 2 infusion-related reaction that does not improve or worsens after implementation of the modifications indicated in **Table 8** (including reducing the infusion rate by 50%), the investigator may consider treatment with corticosteroids and the infusion should be stopped for that day. At the next cycle, the investigator may consider the addition of H2blocker antihistamines (e.g., famotidine), in addition to the mandatory premedication, for select subjects. However, prophylactic steroids are NOT permitted.
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3.2.4 severe hypersensitivity reactions and

A. Symptoms Impaired airway Decreased oxygen saturation (< 92%) Confusion Lethargy Hypotension Pale/clammy skin Cyanosis  
B. Management 1. Epinephrine injection and dexamethasone infusion; 2. Patient should be placed on monitor immediately; 3. Alert intensive care unit (ICU) for possible transfer if required.
For prophylaxis of flu-like symptoms, 25 mg indomethacin or comparable NSAID dose (e.g., ibuprofen 600 mg, naproxen sodium 500 mg) may be administered 2 hours before and 8 hours after the start of each dose of pembrolizumab IV infusion. Alternative treatments for fever (e.g., paracetamol) may be given to subjects at the discretion of the investigator. For Grade 1 Symptoms: (Mild reaction; infusion interruption not indicated; intervention not indicated): Remain at bedside and monitor subject until recovery from symptoms. The following prophylactic pre-medications are recommended for future infusions: diphenhydramine 50 mg (or equivalent) and/or paracetamol 325 to 1000 mg (acetaminophen) at least 30 minutes before additional pembrolizumab administrations. For Grade 2 Symptoms: (Moderate reaction requires therapy or infusion interruption but responds promptly to symptomatic treatment [e.g., antihistamines, non-steroidal antiinflammatory drugs, narcotics, corticosteroids, IV fluids]; prophylactic medications indicated for 24 hours): Stop the pembrolizumab infusion, begin an IV infusion of normal saline, and treat the subject with diphenhydramine 50 mg IV (or equivalent) and/or paracetamol 325 to 1000 mg (acetaminophen); remain at bedside and monitor subject until resolution of symptoms to Grade 1 or less. Resume infusion at 50% rate.
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3.2.3 ccr self -administered study

All oral self-administered investigational agents will be properly accounted for, handled, and disposed in accordance with existing federal regulations and principles of Good Clinical Practice. All oral study drugs will be recorded in the patient diary found in **Appendix 2**. This will be used as a memory aide for subjects. The clinical research team will maintain the primary source record. Subjects should be asked to bring the diary as well as unused study agent and empty containers with them at each study visit. If a subject goes off study while at home, the research nurse will ensure and document the return of the unused oral investigational agents from the participant. Unused investigational study agents will be disposed and destroyed per CC pharmacy SOPs.
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3.3 dosing delays and modifications

This study is intended to evaluate the safety and potential efficacy of oral DAC-THU and pembrolizumab as epigenetic immunotherapy in patients with NSCLC, EsC and MPM. The proposed oral DAC dose of ~0.17 mg/kg to be combined with THU ~10 mg/kg administered 60 minutes before oral DAC, is based on non-human primate studies and the Phase 1 clinical trial in SCD which had an identical pharmacologic objective of DAC distribution through tissues with high CDA expression (intestines, liver etc.), and the wide concentration-time profile desired for non-cytotoxic DNMT1-depletion. This dose of DAC administered orally is 10-20% of the FDAapproved dosages administered intravenously and is < 50% of the dose of oral decitabine administered together with another CDA-inhibitor (a THU-analogue) in clinical trials by Astex/Otsuka. Although it is anticipated that this therapy will be well tolerated at the revised doses, myelosuppression resulting from reprogramming of hematopoietic stem cell differentiation may become dose limiting (50). If dose limiting myelosuppression is observed during the first 2 cycles of Course 1, DAC-THU will be held until toxicities have resolved to Grade 2 or less, and DACTHU therapy will recommence at the next lower dose, or at the SAME dose if deemed appropriate by the PI, as indicated in the dose escalation schema (**Table 7**). Only one dose reduction will be allowed.
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3.3.1 general guidance for dose

3.3.1.1 Patients may continue to receive therapy provided the following criteria are met on Day 1 of each cycle:
Non-hematologic toxicity recovered to < Grade 2 (or tolerable Grade 2 or baseline).  
No evidence of progressive disease.  
3.3.1.2 In the event of an adverse event at least possibly related to the investigational agent, the dose of the investigational agent should be adjusted according to the parameters articulated above and guidelines listed in the Dose Delays / Dose Modifications tables shown below (**Table 9** and **Table 10**). If an adverse event is not covered in such tables, doses may be held at the discretion of the investigator for the subject's safety.  
3.3.1.3 If patients fail to recover to CTCAE Grade 0-1, tolerable Grade 2 (or within 1 grade of starting values for pre-existing laboratory abnormalities), or baseline from a treatmentrelated adverse event attributable to DAC-THU within 42 days (2 cycles), they cannot receive additional DAC-THU. Patients may continue pembrolizumab monotherapy at the investigator discretion provided that discontinuation criteria for pembrolizumab toxicity have not been met.  
3.3.2 Treatment Delays and Modifications for Medical Needs
Brief interruptions and delays up to 3 weeks may be required due to patients experiencing complications of their disease or other medical illness not attributable to disease progression, or protocol therapy. A patient that interrupts therapy for more than 3 weeks will be taken off treatment.
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3.3.4 dosage modification and management

Since inhibition of PD-L1 stimulates the immune system, irAEs may occur. Treatment of irAEs is mainly dependent upon severity (NCI-CTCAE grade):
Grade 1 to 2: treat symptomatically or with moderate dose steroids, more frequent monitoring.  
Grade 1 to 2 (persistent): manage similar to high grade AE (Grade 3 to 4).  
Grade 3 to 4: treat with high dose corticosteroids.
Treatment of irAEs should follow accepted guidelines set forth in **Table 10**.
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3.3.5 dose modification of dac-thu

Table 9 lists standard laboratory values that should trigger dose modifications of the investigational drug.  
| Table 9: Trigger Values that Result in Dose Modification General Adverse Events Action Non-hematological, Grade 1 or 2  Continue DAC-THU therapy at full dose prescribed.   Apply maximum supportive care recommendations.  If prolonged duration of Grade 2 adverse event (≥ 7 days)  is affecting quality of life, start the next treatment cycle at  DL-1.  If event persists and continues to affect quality of life for  ≥ 7 more days following initial dose reduction,  discontinue DAC-THU. Grade 3 ALT and AST  Hold DAC-THU therapy until recovery to Grade  1.   If ALT and AST resolve to Grade  1 within 10 days,  maintain current dose level.  If ALT and AST do NOT resolve to Grade  1 within 10  days, dose reduce 1 level.  If Grade 3 ALT or AST occurs after dose reduction,  discontinue DAC-THU.(excluding hemorrhage)   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| Version Date: 08/01/2022 General Adverse Events                      | Action                                                                                                                                                                                                                                                                                        |                                                                                                                                                                                                                                                          |    |            |       |       |      |            |
|----------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----|------------|-------|-------|------|------------|
| Grade 4 ALT and AST                                                  |                                                                                                                                                                                                                                                                                              | Hold DAC-THU therapy until recovery to Grade  1.                                                                                                                                                                                                        |    |            |       |       |      |            |
|                                                                     | Resume DAC-THU at DL-1.                                                                                                                                                                                                                                                                       |                                                                                                                                                                                                                                                          |    |            |       |       |      |            |
|                                                                     | If                                                                                                                                                                                                                                                                                            | Grade                                                                                                                                                                                                                                                    | 4  | toxicities | occur | after | dose | reduction, |
| discontinue DAC-THU.                                                 |                                                                                                                                                                                                                                                                                               |                                                                                                                                                                                                                                                          |    |            |       |       |      |            |
| Non-hematological, Grade 3 or 4                                      |                                                                                                                                                                                                                                                                                              | Apply maximum supportive care recommendations. Hold  DAC-THU until recovery to Grade  1 and resume DACTHU at the next lower dose level.                                                                                                                                                                                                                                                          |    |            |       |       |      |            |
|                                                                     | If symptoms continue to persist at Grade 3 or 4, following  dose reduction, discontinue drug.                                                                                                                                                                                                 |                                                                                                                                                                                                                                                          |    |            |       |       |      |            |
| QT prolongation, Grade 3 or 4                                        |                                                                                                                                                                                                                                                                                              | Cardiology consultation will be obtained to determine  whether any cardiac functional assessment is warranted or  if therapy should be delayed or discontinued. When  warranted, ECGs will subsequently be reviewed by the  NCI consulting cardiologist. |    |            |       |       |      |            |
|                                                                     | If therapy is continued, dose reduce 1 level for Grade 4  events.                                                                                                                                                                                                                             |                                                                                                                                                                                                                                                          |    |            |       |       |      |            |
|                                                                     | If Grade 3 or 4 toxicities occur after dose reduction,  discontinue DAC-THU.                                                                                                                                                                                                                  |                                                                                                                                                                                                                                                          |    |            |       |       |      |            |
| (excluding ALT, AST, hemorrhage  and QT prolongation) Thrombocytosis |                                                                                                                                                                                                                                                                                              | If platelet count > 500/ mcL but < 1.2M/mcL, continue  DAC-THU at current dose. Begin ASA 325mg PO daily.                                                                                                                                                |    |            |       |       |      |            |
|                                                                     | If platelet count 1.2M /mcL or more, hold DAC-THU until  platelet count < 1.2 M/mcL, begin ASA 325 mg PO daily  and resume DAC-THU at DL-1 if within the first two  Cycles of Course 1; or at SAME dose or DL-1 per PI  discretion in subsequent cycles.                                      |                                                                                                                                                                                                                                                          |    |            |       |       |      |            |
| Hemoglobin                                                           |                                                                                                                                                                                                                                                                                              | Hgb > 18 g/dL: hold DAC-THU until Hgb WNL. Resume  DAC-THU at DL-1.                                                                                                                                                                                      |    |            |       |       |      |            |
| Hematological Adverse Events Grades 1 and 2 events                   |                                                                                                                                                                                                                                                                                              | Continue DAC-THU therapy at full dose prescribed.  Apply maximum supportive care recommendations.                                                                                                                                                        |    |            |       |       |      |            |
| Grade 3 events                                                       |                                                                                                                                                                                                                                                                                              | Apply supportive care.                                                                                                                                                                                                                                   |    |            |       |       |      |            |
|                                                                     | If toxicities occur in the first two cycles of therapy and  resolve to Grade 1 or less, in 10 days, resume DAC-THU  at current dose.                                                                                                                                                          |                                                                                                                                                                                                                                                          |    |            |       |       |      |            |
|                                                                     | If toxicities occur in the first two cycles of therapy and  resolve to Grade 1 or less > 10 days following drug hold,  resume DAC-THU at next lower dose.                                                                                                                                     |                                                                                                                                                                                                                                                          |    |            |       |       |      |            |
|                                                                     | If toxicities occur in subsequent cycles and resolve to  Grade 1 or less in 21 days following drug hold, resume  DAC-THU at SAME dose. If toxicities recur within two  cycles of resuming DAC-THU, hold until toxicities  resolve to Grade 1 or less, and resume DAC-THU at next  lower dose. |                                                                                                                                                                                                                                                          |    |            |       |       |      |            |
|                                                                     | If toxicities occur in subsequent cycles and resolve to 63                                                                                                                                                                                                                                    |                                                                                                                                                                                                                                                          |    |            |       |       |      |            |  
| General Adverse Events   | Action Grade 1 or less > 21 days following drug hold, resume  DAC-THU at next lower dose.   If toxicities recur within two cycles after maximal dose  reduction, discontinue DAC-THU.                                                                                                                                                            |                                                |
|--------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------|
| Grade 4 events           |                                                                                                                                                                                                                                                                                                                                                  | Apply maximum supportive care recommendations. |
|                         | Hold DAC-THU therapy until patient meets hematologic  criteria for retreatment and resume DAC-THU at next  lower dose.                                                                                                                                                                                                                            |                                                |
|                         | If recurrence of hematological adverse event after drug  hold/ interruptions is observed with treatment at DL-1, in  the setting of maximum supportive care measures applied,  hold drug once again until patient meets hematologic  criteria for retreatment, further dose reduce DAC-THU if  possible or discontinue DAC-THU per PI discretion. |                                                |
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3.3.6 dose delay of pembrolizumab

No dose modification for pembrolizumab is allowed. Because of the potential for clinically meaningful pembrolizumab-related AEs requiring early recognition and prompt intervention, management algorithms have been developed for suspected AEs of selected categories. Dose delay criteria apply for all drug-related adverse events (regardless of whether or not the event is attributed to pembrolizumab). If AE is felt to be immune related and attributable to pembrolizumab, the patient can continue DAC-THU while pembrolizumab is held. Dose delay criteria apply for all drug-related AEs. Pembrolizumab must be delayed until treatment can resume. 3.3.6.1 Pembrolizumab Administration Should be Delayed for the Following:  
| Gastrointestinal irAEs                                        |                                          |                                                                                           |
|---------------------------------------------------------------|------------------------------------------|-------------------------------------------------------------------------------------------|
| Severity of Diarrhea/Colitis (NCI-CTCAE v5)                   | Initial Management                       | Follow-up Management                                                                      |
| Grade 1                                                       | Continue Pembrolizumab                   | Close monitoring for worsening  symptoms Educate subject to report worsening  immediately |
| Diarrhea: < 4 stools/day over  Baseline Colitis: asymptomatic | Symptomatic treatment (e.g.  loperamide) | If worsens: Treat as Grade 2, 3 or 4.                                                     |  
| Gastrointestinal irAEs                                                                                                                                                                                                                            |                                                                                                                                                                                                                                                                                                    |                                                                                                                                                                                                                                                                                                                                                    |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Severity of Diarrhea/Colitis (NCI-CTCAE v5)                                                                                                                                                                                                       | Initial Management                                                                                                                                                                                                                                                                                 | Follow-up Management                                                                                                                                                                                                                                                                                                                               |
| Grade 2                                                                                                                                                                                                                                           | Withhold Pembrolizumab                                                                                                                                                                                                                                                                             | If improves to Grade ≤ 1:                                                                                                                                                                                                                                                                                                                          |
| Diarrhea: 4 to 6 stools per day over  Baseline; IV fluids indicated < 24  hours; not interfering with ADL Colitis: abdominal pain; blood in  stool                                                                                                | Symptomatic treatment                                                                                                                                                                                                                                                                              | Resume Pembrolizumab therapy If persists > 5-7 days or recurs: Treat as Grade 3 or 4.                                                                                                                                                                                                                                                              |
| Grade 3 to 4                                                                                                                                                                                                                                      | Withhold Pembrolizumab for Grade 3.                                                                                                                                                                                                                                                                | If improves:                                                                                                                                                                                                                                                                                                                                       |
| Diarrhea (Grade 3): ≥ 7 stools per  day over Baseline; incontinence; IV  fluids ≥ 24 h; interfering with ADL Colitis (Grade 3): severe abdominal  pain, medical intervention indicated,  peritoneal signs Grade 4: life-threatening,  perforation | Permanently discontinue  Pembrolizumab for Grade 4 or  recurrent Grade 3. 1.0 to 2.0 mg/kg/day prednisone IV or  equivalent Add prophylactic antibiotics for  opportunistic infections  Consider lower endoscopy                                                                                   | Continue steroids until Grade ≤ 1,  then taper over at least 1 month;  resume Pembrolizumab therapy  following steroids taper (for initial  Grade 3).  If worsens, persists > 3 to 5 days, or  recurs after improvement: Add infliximab 5mg/kg (if no  contraindication). Note: infliximab  should not be used in cases of  perforation or sepsis. |
| Dermatological irAEs                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                    |                                                                                                                                                                                                                                                                                                                                                    |
| Grade of Rash                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                                                                    |                                                                                                                                                                                                                                                                                                                                                    |
| (NCI-CTCAE v5)                                                                                                                                                                                                                                    | Initial Management                                                                                                                                                                                                                                                                                 | Follow-up Management                                                                                                                                                                                                                                                                                                                               |
| Grade 1 to 2                                                                                                                                                                                                                                      | Continue Pembrolizumab  Symptomatic therapy (for example,  antihistamines, topical steroids)                                                                                                                                                                                                       | If persists > 1 to 2 weeks or recurs:                                                                                                                                                                                                                                                                                                              |
| Covering ≤ 30% body surface area                                                                                                                                                                                                                  | Withhold Pembrolizumab therapy  Consider skin biopsy  Consider 0.5-1.0 mg/kg/day  prednisone or equivalent. Once  improving, taper steroids over at least  1 month, consider prophylactic  antibiotics for opportunistic  infections, and resume  Pembrolizumab therapy following  steroids taper. |                                                                                                                                                                                                                                                                                                                                                    |  
| Dermatological irAEs                                                                 |                                                                                                                                                                                                                  |                                                                                                                       |
|--------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------|
| Grade of Rash                                                                        |                                                                                                                                                                                                                  |                                                                                                                       |
| (NCI-CTCAE v5)                                                                       | Initial Management                                                                                                                                                                                               | Follow-up Management                                                                                                  |
| If worsens:  Treat as Grade 3 to 4.                                                  |                                                                                                                                                                                                                  |                                                                                                                       |
| Grade 3 to 4                                                                         | Withhold Pembrolizumab for Grade 3.                                                                                                                                                                              | If improves to Grade ≤ 1:                                                                                             |
| Grade 3: Covering > 30% body  surface area;  Grade 4: Life threatening  consequences | Permanently discontinue for Grade 4  or recurrent Grade 3.  Consider skin biopsy  Dermatology consult  1.0 to 2.0 mg/kg/day prednisone or  equivalent Add prophylactic antibiotics for  opportunistic infections | Taper steroids over at least 1 month;  resume Pembrolizumab therapy  following steroids taper (for initial  Grade 3). |  
| Pulmonary irAEs                     |                                                                                                                                                                                                                                             |                                                                                                                                                                                                         |
|-------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade of Pneumonitis (NCI-CTCAE v5) | Initial Management                                                                                                                                                                                                                          | Follow-up Management                                                                                                                                                                                    |
| Grade 1                             | Consider withholding Pembrolizumab  therapy  Monitor for symptoms every 2 to 3  days  Consider Pulmonary and Infectious  Disease consults                                                                                                   | Re-assess at least every 3 weeks                                                                                                                                                                        |
| Radiographic changes only           | If worsens:  Treat as Grade 2 or Grade 3 to 4.                                                                                                                                                                                              |                                                                                                                                                                                                         |
| Grade 2                             | Withhold Pembrolizumab therapy                                                                                                                                                                                                              | Re-assess every 1 to 3 days                                                                                                                                                                             |
| Mild to moderate new symptoms       | Pulmonary and Infectious Disease  consults Monitor symptoms daily; consider  hospitalization  1.0 to 2.0 mg/kg/day prednisone or  equivalent Add prophylactic antibiotics for  opportunistic infections  Consider bronchoscopy, lung biopsy | If improves:  When symptoms return to Grade ≤ 1,  taper steroids over at least 1 month,  and then resume Pembrolizumab  therapy following steroids taper  If not improving after 2 weeks or  worsening: |  
| Pulmonary irAEs                                                                   |                                                                                                                                                                                                                                                         |                                                                                                                                    |
|-----------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------|
| Grade of Pneumonitis (NCI-CTCAE v5)                                               | Initial Management                                                                                                                                                                                                                                      | Follow-up Management                                                                                                               |
| Treat as Grade 3 to 4.                                                            |                                                                                                                                                                                                                                                         |                                                                                                                                    |
| Grade 3 to 4                                                                      | Permanently discontinue  Pembrolizumab therapy. Hospitalize. Pulmonary and Infectious Disease  consults. 1.0 to 2.0 mg/kg/day prednisone or  equivalent  Add prophylactic antibiotics for  opportunistic infections  Consider bronchoscopy, lung biopsy | If improves to Grade ≤ 1:                                                                                                          |
| Grade 3: Severe new symptoms;  New/worsening hypoxia; Grade 4: Life-threatening   | Taper steroids over at least 1 month  If not improving after 48 hours or  worsening:  Add additional immunosuppression  (for example, infliximab,  cyclophosphamide, IV  immunoglobulin, or mycophenolate  mofetil)                                     |                                                                                                                                    |
| Hepatic irAEs                                                                     |                                                                                                                                                                                                                                                         |                                                                                                                                    |
| Grade of Liver Test Elevation (NCI-CTCAE v5)                                      | Initial Management                                                                                                                                                                                                                                      | Follow-up Management                                                                                                               |
| Grade 1                                                                           | Continue Pembrolizumab therapy                                                                                                                                                                                                                          | Continue liver function monitoring                                                                                                 |
| Grade 1 AST or ALT > ULN to 3.0  x ULN and/or Total bilirubin >  ULN to 1.5 x ULN | If worsens:   Treat as Grade 2 or 3 to 4.                                                                                                                                                                                                               |                                                                                                                                    |
| Grade 2                                                                           | Withhold Pembrolizumab therapy                                                                                                                                                                                                                          | If returns to Grade ≤ 1:                                                                                                           |
| AST or ALT > 3.0 to ≤ 5 x ULN  and/or total bilirubin > 1.5 to ≤ 3 x  ULN         | Increase frequency of monitoring to  every 3 days.                                                                                                                                                                                                      | Resume routine monitoring; resume  Pembrolizumab therapy. If elevation persists > 5 to 7 days or  worsens:  Treat as Grade 3 to 4. |
| Grade 3 to 4                                                                      | Permanently discontinue  Pembrolizumab therapy  Increase frequency of monitoring to  every 1 to 2 days  1.0 to 2.0 mg/kg/day prednisone or  equivalent                                                                                                  | If returns to Grade ≤ 1:                                                                                                           |
| AST or ALT > 5 x ULN and/or total  bilirubin > 3 x ULN                            | Taper steroids over at least 1 month  If does not improve in > 3 to 5 days,  worsens or rebounds:                                                                                                                                                       |                                                                                                                                    |  
| Hepatic irAEs                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |                                                                                                                                                                |                                    |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------|
| Grade of Liver Test Elevation (NCI-CTCAE v5)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Initial Management                                                                                                                                             | Follow-up Management               |
| Add prophylactic antibiotics for  opportunistic infections  Consult gastroenterologist/  hepatologist Consider obtaining MRI/CT scan of  liver and liver biopsy if clinically  warranted                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | Add mycophenolate mofetil 1 gram  (g) twice daily  If no response within an additional 3  to 5 days, consider other  immunosuppressants per local  guidelines. |                                    |
| Renal irAEs                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |                                                                                                                                                                |                                    |
| Grade of Creatinine Increased  (NCI-CTCAE v5)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | Initial Management                                                                                                                                             | Follow-up Management               |
| Grade 1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      | Continue Pembrolizumab therapy                                                                                                                                 | Continue renal function monitoring |
| Creatinine increased > ULN to 1.5  x ULN                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     | If worsens: Treat as Grade 2 to 3 or 4.                                                                                                                        |                                    |
| Grade 2 to 3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Withhold Pembrolizumab therapy                                                                                                                                 | If returns to Grade ≤ 1:           |
| Creatinine increased > 1.5 and ≤ 6 x  ULN Increase frequency of monitoring to every  3 days and consider Nephrology consult 1.0 to 2.0 mg/kg/day prednisone or  equivalent. Add prophylactic antibiotics for  opportunistic infections Consider renal biopsy Follow Nephrology recommendations  if consult obtained; alternatively,  taper steroids over at least 1 month,  and resume Pembrolizumab therapy  following steroids taper. If worsens:  Treat as Grade 4. Grade 4 Permanently discontinue Pembrolizumab  therapy Monitor creatinine daily 1.0 to 2.0 mg/kg/day prednisone or  equivalent. Add prophylactic antibiotics for  opportunistic infections Consider renal biopsy Nephrology consult If returns to Grade ≤ 1: Creatinine increased > 6 x ULN Follow Nephrology  recommendations; alternatively, taper  steroids over at least 1 month. |                                                                                                                                                                |                                    |  
| Cardiac irAEs                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |                                                                                                                |                                                                                                  |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------|
| Myocarditis                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | Initial Management                                                                                             | Follow-up Management                                                                             |
| New onset of cardiac signs or  symptoms and / or new laboratory  cardiac biomarker elevations (e.g.,  troponin, CK-MB, BNP) or cardiac  imaging abnormalities suggestive  of myocarditis.                                                                                                                                                                                                                                                                                                                                                                                                                    | Withhold Pembrolizumab therapy.                                                                                | If symptoms improve and  immune-mediated etiology is ruled  out, re-start Pembrolizumab therapy. |
| Hospitalize. In the presence of life threatening cardiac  decompensation, consider transfer to a  facility experienced in advanced heart  failure and arrhythmia management. Cardiology consult to establish etiology  and rule-out immune-mediated  myocarditis.  Guideline based supportive treatment as  per cardiology consult. * Consider myocardial biopsy if  recommended per cardiology consult. If symptoms do not  improve/worsen, viral myocarditis  is excluded, and immune-mediated  etiology is suspected or confirmed  following cardiology consult,  manage as immune-mediated  myocarditis. |                                                                                                                |                                                                                                  |
| Immune-mediated myocarditis                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | Permanently discontinue                                                                                        | Once improving, taper steroids over  at least 1 month.                                           |
| Pembrolizumab. Guideline based supportive treatment as  appropriate as per cardiology consult. * 1.0 to 2.0 mg/kg/day prednisone or  equivalent. Add prophylactic antibiotics for  opportunistic infections.                                                                                                                                                                                                                                                                                                                                                                                                 | If no improvement or worsening,  consider additional  immunosuppressants (e.g.  azathioprine, cyclosporine A). |                                                                                                  |
| *Local guidelines, or e.g., ESC or AHA guidelines  ESC guidelines website: https://www.escardio.org/Guidelines/Clinical-Practice-Guidelines AHA guidelines website:  http://professional.heart.org/professional/GuidelinesStatements/searchresults.jsp?q=&y=&t=1001                                                                                                                                                                                                                                                                                                                                          |                                                                                                                |                                                                                                  |  
| Endocrine irAEs                                                                                                                                                                                                                                               |                                |                                                                                                  |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------|--------------------------------------------------------------------------------------------------|
| Endocrine Disorder                                                                                                                                                                                                                                            | Initial Management             | Follow-up Management                                                                             |
| Grade 1 or Grade 2  endocrinopathies  (hypothyroidism,  hyperthyroidism, adrenal  insufficiency, type I diabetes  mellitus)                                                                                                                                   | Continue Pembrolizumab therapy | Continue hormone  replacement/suppression and  monitoring of endocrine function as  appropriate. |
| Endocrinology consult if needed Start thyroid hormone replacement  therapy (for hypothyroidism),  anti-thyroid treatment (for  hyperthyroidism), corticosteroids (for  adrenal insufficiency) or insulin (for  Type I diabetes mellitus) as  appropriate.  69 |                                |                                                                                                  |  
| Endocrine irAEs                                                                                                                                                                                                                                                          |                                                                                                                                                                    |                                                                                                                                                                                                                                                                        |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Endocrine Disorder                                                                                                                                                                                                                                                       | Initial Management                                                                                                                                                 | Follow-up Management                                                                                                                                                                                                                                                   |
| Rule-out secondary endocrinopathies  (i.e., hypopituitarism / hypophysitis)                                                                                                                                                                                              |                                                                                                                                                                    |                                                                                                                                                                                                                                                                        |
| Grade 3 or Grade 4  endocrinopathies  (hypothyroidism,  hyperthyroidism, adrenal  insufficiency, type I diabetes  mellitus)                                                                                                                                              | Withhold Pembrolizumab therapy                                                                                                                                     | Resume Pembrolizumab once  symptoms and/or laboratory tests  improve to Grade ≤ 1 (with or  without hormone  replacement/suppression).                                                                                                                                 |
| Consider hospitalization Endocrinology consult Start thyroid hormone replacement  therapy (for hypothyroidism),  anti-thyroid treatment (for  hyperthyroidism), corticosteroids (for  adrenal insufficiency) or insulin (for  type I diabetes mellitus) as  appropriate. | Continue hormone  replacement/suppression and  monitoring of endocrine function as  appropriate.                                                                   |                                                                                                                                                                                                                                                                        |
| Rule-out secondary endocrinopathies  (i.e. hypopituitarism / hypophysitis)                                                                                                                                                                                               |                                                                                                                                                                    |                                                                                                                                                                                                                                                                        |
| Hypopituitarism/Hypophysitis                                                                                                                                                                                                                                             | If secondary thyroid and/or adrenal                                                                                                                                |                                                                                                                                                                                                                                                                        |
| (secondary endocrinopathies)                                                                                                                                                                                                                                             | insufficiency is confirmed (i.e.  subnormal serum FT4 with  inappropriately low TSH and/or low  serum cortisol with inappropriately  low ACTH):                    | Resume Pembrolizumab once  symptoms and hormone tests  improve to Grade ≤ 1 (with or  without hormone replacement). In addition, for hypophysitis with  abnormal MRI, resume  Pembrolizumab only once shrinkage  of the pituitary gland on MRI/CT  scan is documented. |
|                                                                                                                                                                                                                                                                         | Refer to endocrinologist for  dynamic testing as indicated and  measurement of other hormones  (FSH, LH, GH/IGF-1, PRL,  testosterone in men, estrogens in  women) | Continue hormone  replacement/suppression therapy as  appropriate.                                                                                                                                                                                                     |
|                                                                                                                                                                                                                                                                         | Hormone  replacement/suppressive therapy  as appropriate                                                                                                           |                                                                                                                                                                                                                                                                        |
|                                                                                                                                                                                                                                                                         | Perform pituitary MRI and visual  field examination as indicated                                                                                                   |                                                                                                                                                                                                                                                                        |
| If hypophysitis confirmed:  Continue Pembrolizumab if mild  symptoms with normal MRI.  Repeat the MRI in 1 month                                                                                                                                                        |                                                                                                                                                                    |                                                                                                                                                                                                                                                                        |  
| Endocrine irAEs                                                                                                                            |                                                                                                                                              |                                                                                                                              |
|--------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------|
| Endocrine Disorder                                                                                                                         | Initial Management                                                                                                                           | Follow-up Management                                                                                                         |
|                                                                                                                                           | Withhold Pembrolizumab if  moderate, severe or lifethreatening symptoms of  hypophysitis and/or abnormal  MRI. Consider hospitalization.  Initiate corticosteroids (1 to 2  mg/kg/day prednisone or  equivalent) followed by  corticosteroids taper during at  least 1 month.                                                                                                                                              |                                                                                                                              |
|                                                                                                                                           | Add prophylactic antibiotics for  opportunistic infections.                                                                                  |                                                                                                                              |
| Other irAEs (not described above)                                                                                                          |                                                                                                                                              |                                                                                                                              |
| Grade of other irAEs                                                                                                                       | Initial Management                                                                                                                           | Follow-up Management                                                                                                         |
| (NCI-CTCAE v5)                                                                                                                             |                                                                                                                                              |                                                                                                                              |
| Grade 2 or Grade 3 clinical signs  or symptoms suggestive of a  potential irAE                                                             | Withhold Pembrolizumab therapy                                                                                                               | If irAE is ruled out, manage as                                                                                              |
| pending clinical investigation                                                                                                             | appropriate according to the  diagnosis and consider re-starting  Pembrolizumab therapy If irAE is confirmed, treat as Grade 2  or 3 irAE.   |                                                                                                                              |
| Grade 2 irAE or first occurrence                                                                                                           | Withhold Pembrolizumab therapy                                                                                                               |                                                                                                                              |
| of Grade 3 irAE                                                                                                                            | 1.0 to 2.0 mg/kg/day prednisone or  equivalent  Add prophylactic antibiotics for  opportunistic infections  Specialty consult as appropriate | If improves to Grade ≤ 1:  Taper steroids over at least 1 month  and resume Pembrolizumab therapy  following steroids taper. |
| Recurrence of same Grade 3                                                                                                                 | Permanently discontinue                                                                                                                      |                                                                                                                              |
| irAEs                                                                                                                                      | Pembrolizumab therapy                                                                                                                        |                                                                                                                              |
| 1.0 to 2.0 mg/kg/day prednisone or equivalent Add prophylactic antibiotics for  opportunistic infections  Specialty consult as appropriate | If improves to Grade ≤ 1:  Taper steroids over at least 1 month.                                                                             |                                                                                                                              |
| Grade 4                                                                                                                                    | Permanently discontinue  Pembrolizumab therapy                                                                                               |                                                                                                                              |
| 1.0 to 2.0 mg/kg/day                                                                                                                       | If improves to Grade ≤ 1:  Taper steroids over at least 1 month                                                                              |                                                                                                                              |  
| Other irAEs (not described above)                                                                                                                                   |                                                                                                                                                   |                      |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------|----------------------|
| Grade of other irAEs                                                                                                                                                | Initial Management                                                                                                                                | Follow-up Management |
| (NCI-CTCAE v5)                                                                                                                                                      | prednisone or equivalent and/or other  immunosuppressant as needed Add prophylactic antibiotics for  opportunistic infections  Specialty consult. |                      |
| Requirement for 10 mg per day  or greater prednisone or  equivalent for more than 12  weeks for reasons other than  hormonal replacement for  adrenal insufficiency | Permanently discontinue  Pembrolizumab therapy Specialty consult                                                                                  |                      |
| Persistent Grade 2 or 3 irAE  lasting 12 weeks or longer                                                                                                            |                                                                                                                                                   |                      |  
ADL=activities of daily living, ALT=alanine aminotransferase, AST=aspartate aminotransferase, irAE=immune-related adverse event, IV=intravenous, LLN=lower limit of normal, MRI=magnetic resonance imaging, NCI-CTCAE=National Cancer Institute-Common Terminology Criteria for Adverse Event, NSAIDs=nonsteroidal anti-inflammatory drugs, T4=thyroxine, TSH=thyroid-stimulating hormone, ULN=upper limit of normal, IVIG=intravenous immunoglobulins, CPK=creatine phosphokinase.  
Note: Subjects who require delay of pembrolizumab should be re-evaluated closely as clinically indicated and resume pembrolizumab dosing when re-treatment criteria are met.
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3.3.6.2 criteria to resume pembrolizumab

Subjects may resume treatment with study drug when the drug-related AE(s) resolve to Grade ≤ 1 or baseline value, with the following exceptions:
Subjects may resume treatment in the presence of Grade 2 fatigue.  
Subjects who have not experienced a Grade 3 drug-related skin AE may resume treatment in the presence of Grade 2 skin toxicity.  
Subjects with baseline Grade 1 AST/ALT or total bilirubin who require dose delays for reasons other than a 2-grade shift in AST/ALT or total bilirubin may resume treatment in the presence of Grade 2 AST/ALT OR total bilirubin.  
Subjects with combined Grade 2 AST/ALT AND total bilirubin values meeting discontinuation parameters should have treatment permanently discontinued.  
Drug-related pulmonary toxicity, diarrhea, or colitis, must have resolved to baseline before treatment is resumed. Subjects with persistent Grade 1 pneumonitis after completion of a steroid taper over at least 1 month may be eligible for retreatment.  
Drug-related endocrinopathies adequately controlled with only physiologic hormone replacement may resume treatment.  
If the criteria to resume treatment are met, the subject should restart treatment at the next scheduled time point per protocol. However, if the treatment is delayed past the next scheduled time point per protocol, the next scheduled time point will be delayed until dosing resumes. If treatment is delayed or interrupted for > 6 weeks, the subject must be permanently discontinued from study therapy unless there is evidence (stable disease or disease regression) of clinical benefit.
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3.4 on study protocol evaluation

Note: Baseline assessments do not need to be repeated if performed during screening in the designated time frame. Any Time Prior to Initiation of Treatment:
a. HLA class I and class II analysis of PBMC.  
b. Tumor biopsy if fresh sample from previous biopsy is not available. Note: Samples collected on 06C0014 or any other NCI protocol in which the patient is co-enrolled can be used.
Within 10 Days Prior to Commencing DAC-THU/Pembrolizumab Therapy:
a. Physical examination including assessment of vital signs and ECOG status.  
b. Chemistries i. Acute Care Panel - Sodium (Na), Potassium (K), Chloride (Cl), Total CO2
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN),
ii. Mineral Panel - Albumin, Calcium total, Magnesium total (Mg),
Phosphorus iii. Hepatic Panel - Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin
iv. LD, Total Protein, Total CK, Uric Acid c. Fetal hemoglobin d. CBC, differential, platelet count, PT, PTT  
e. T, B, NK cell subsets f. Urinalysis and culture if indicated g. Pulmonary Function Test h. Arterial Blood Gas on room air (to be drawn if pulse oximetry <90% on room air). i. Research Labs (refer to **Appendix 4** for details).
Within 48 Hours Prior to Initiation of DAC-THU/Pembrolizumab Therapy:
a. Women of child-bearing potential will have a urine or serum hCG pregnancy test.
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3.4.2 treatment evaluation

Twice Weekly During First Course of Therapy, and Weekly Thereafter Unless Clinically Indicated:
a. CBC with differential  
b. Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LD, Total Protein, Total CK, Uric Acid c. PT/PTT
Note: The patient may have lab work obtained through his or her local physician.  
Every Three Weeks During Therapy:
a. T, B, NK cell subsets
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performed after each course

a. Physical examination including assessment of vital signs and ECOG status.  
b. Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg), Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LD, Total Protein, Total CK, Uric Acid c. CBC, differential, platelet count, PT, PTT d. Thyroid Function Tests e. T, B, NK cell subsets f. Urinalysis and culture if indicated
g. Biopsy of index lesion (only on first treatment evaluation).  
3.4.3 Research Evaluations (Refer to Appendix 4, Unless Otherwise Indicated Below)  
Pharmacokinetic (PK) Studies Research Labs Radiological Assessments (refer to **Appendix 3**)
Tumor Biopsies (see Appendix 3)
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3.4.4 follow-up evaluations

Patients who are removed from treatment due to disease progression or unacceptable, dose-limiting toxicities or who voluntarily withdraw from treatment will be monitored weekly until treatment related toxicities have resolved to baseline at which time they will be removed from study. Toxicity Evaluation:  
o Laboratory evaluation: CBC, Chemistries (Sodium, potassium, chloride, bicarbonate, creatinine, glucose, BUN, albumin, total calcium, ionized calcium, magnesium, phosphorus, alkaline phosphatase, ALT/SGPT, AST/SGOT, total
bilirubin, direct bilirubin, lactate dehydrogenase, total protein, creatine kinase, uric acid), PT/PTT
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3.5 costs and compensation 3.5.1

NIH does not bill health insurance companies or participants for any research or related clinical care that participants receive at the NIH Clinical Center. If some tests and procedures are performed outside the NIH Clinical Center, participants may have to pay for these costs if they are not covered by their insurance company. Medicines that are not part of the study treatment will not be provided or paid for by the NIH Clinical Center.
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3.5.3 reimbursement

The NCI will cover the costs of some expenses associated with protocol participation. Some of these costs may be paid directly by the NIH and some may be reimbursed to the participant/guardian as appropriate. The amount and form of these payments are determined by the NCI Travel and Lodging Reimbursement Policy.
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3.6 criteria for removal from

Prior to removal from protocol, effort must be made to have all subjects complete a safety visit approximately 30 days after the last dose of study therapy.
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3.6.1 criteria for removal from

Patient refusal of further treatments  
It is deemed in the best interest of the patient A patient who develops a concurrent serious medical condition that might preclude or contraindicate administration of DAC-THU/pembrolizumab therapy will be removed from treatment A patient who becomes pregnant will be immediately taken off therapy Progressive disease Failure to meet criteria for additional treatment as defined in Sections **3.3.2** and **3.3.6**
Delay of treatment for three weeks Note: Patients will be followed until toxicities related to experimental therapy resolve to baseline. 3.6.2 Off-Study Criteria
Voluntary withdrawal for any reason, or noncompliance with protocol requirements  
Completion of the follow up period (see Section **3.4.4**)
Progressive disease unless the patient is being followed for a serious adverse event related to the research. The event must resolve to baseline prior to removing from the study If the investigator determines that it is in the best interest of the patient to discontinue follow up Lost to follow up Death PI decision to close the study
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3.6.3 lost to follow-up

A participant will be considered lost to follow-up if he or she fails to return for 3 scheduled visits and is unable to be contacted by the study site staff. The following actions must be taken if a participant fails to return to the clinic for a required study visit:  
The site will attempt to contact the participant and reschedule the missed visit within 5 business days and counsel the participant on the importance of maintaining the assigned visit schedule and ascertain if the participant wishes to and/or should continue in the study.  
Before a participant is deemed lost to follow-up, the investigator or designee will make every effort to regain contact with the participant (where possible, 3 telephone calls/video calls and, if necessary, an IRB approved certified letter to the participant's last known mailing address or local equivalent methods). These contact attempts should be documented in the participant's medical record or study file.  
Should the participant continue to be unreachable, he or she will be considered to have withdrawn from the study with a primary reason of lost to follow-up.
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4 concomitant medications/measures 4.1 hematologic

Blood product support should be provided to maintain platelets > 20,000 cells/mcl, Hgb > 8.0 gm/dl and as clinically indicated. Growth factor support is not permitted with exception of administration of filgrastim. Filgrastim will not be administered prophylactically, but may be administered if a patient experiences neutropenic sepsis, or if clinically indicated.
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4.2 antidiarrheals

Antidiarrheal agents will be prescribed using standard clinical practice guidelines at the preference of the investigator.
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4.3 electrolyte replacement

Electrolyte replacement will be provided to maintain serum levels within normal limits.
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4.4 proton pump inhibitors

Given the uncertainties regarding effects of proton pump inhibitors on absorption of DAC-THU, proton pump inhibitors will not be used except on days when DAC-THU is not administered.
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5 correlative studies 5.1 biospecimen

Detailed plasma pharmacokinetic (PK) sampling of DAC and THU will be collected as outlined in **Appendix 4**. PK analysis will be performed at the NCI, Genitourinary Malignancies Branch (GMB). Samples will be stored in the Thoracic Epigenetics Lab (TEL) and batched and later transferred to the Figg Lab on dry ice for analysis.
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5.1.2 studies on tumor tissue

Portions of biopsy materials will be sent for frozen section or permanent section confirmation of malignancy and percent viable tumor cells. Tissue will be processed for focused gene, endogenous retroviral (ERV) and microRNA expressions, and DNA methylation signatures using quantitative RT-PCR, nanostring, pyrosequencing and digital droplet PCR techniques. Blood from EDTA and red top tubes (see **Appendix 4**) will be used to isolate plasma circulating tumor DNA and serum for focused methylation analysis and immune response to treatment. Remaining tissue samples will be snap frozen in liquid nitrogen for subsequent isolation of RNA and DNA for qRT-PCR and nanostring experiments. If sufficient tissue is available, another portion will be imbedded in paraffin for subsequent immunostaining experiments, focusing on expression of genes focusing on those proteins encoded by genes that have been identified to be clearly activated by epigenetic therapy. If sufficient materials are present, additional more comprehensive analyses including multiplex IHC analysis of tumor microenvironment may be performed with the focus of materials from patients treated at the MTD. All of the analyses, which are predicated on acquisition of sufficient materials, will be performed in the Thoracic Epigenetics Lab under direction of the PI or submitted to sequencing cores at the NCI.
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5.1.3 immune subset analyses

Peripheral blood mononuclear cells (PBMC) will be assessed using multiparameter flow cytometry for immune subsets including but not necessarily limited to Tregs, MDSC, effector and exhausted CD4+ or CD8+ T-cells, and CD14+ monocytes. Assessment will include functional markers, i.e. PD-1, PD-L1, Tim-3, CTLA-4, HLA-DR and/or CD40. Samples will be drawn (as indicated in Appendix 4) and picked up by the Trepel Lab (see Section **5.2.4**) for analysis. The Trepel Lab will prepare the samples for staining, stain and run the samples by multiparametric flow cytometry (MACSQuant, Miltenyi Biotec, Bergisch Gladbach, DE), and analyze the data using FlowJo (FlowJo LLC, Ashland, OR) software.
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5.1.4 circulating tumor cells

Peripheral blood will be collected to correlate changes in circulating tumor cells with clinical response. CTCs will be assessed using ferrofluidic enrichment and multi-parameter flow cytometric detection. Samples will be drawn (as indicated in **Appendix 4**) and picked up by the Trepel Lab (Section **5.2.4**) for analysis. The Trepel Lab will process the samples, stain and run one tube immediately. The other tube will be processed, 2D barcoded and viably stored for potential future analysis. The samples will be analyzed by multiparametric flow cytometry (MACSQuant, Miltenyi Biotec, Bergisch Gladbach, DE) and FlowJo (FloJo LLC, Ashland, OR) software.
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5.2 sample storage tracking and

Samples will be ordered and tracked through Clinical Trial Data Management System. Should a CRIS screen not be available, the CRIS downtime procedures will be followed. Samples will not be sent outside NIH without appropriate approvals and/or agreements, if required.
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5.2.1 samples managed by dr.

Please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred). For sample pickup, page 102-11964. For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 240-760-6190 (main clinical pharmacology lab number). For questions regarding sample processing, send request to NCIBloodcore@mail.nih.gov. 5.2.1.2 Sample Data Collection All samples sent to the Blood Processing Core (BPC) will be barcoded, with data entered and stored in the Labmatrix utilized by the BPC. This is a secure program, with access to Labmatrix limited to defined Figg lab personnel, who are issued individual user accounts. Installation of Labmatrix is limited to computers specified by Dr. Figg. These computers all have a password restricted login screen. Labmatrix creates a unique barcode ID for every sample and sample box, which cannot be traced back to patients without Labmatrix access. The data recorded for each sample includes the patient ID, name, trial name/protocol number, time drawn, cycle time point, dose, material type, as well as box and freezer location. Patient demographics associated with the clinical center patient number are provided in the system. For each sample, there are notes associated with the processing method (delay in sample processing, storage conditions on the ward, etc.).
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5.2.1.3 sample storage and destruction

Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -80C according to stability requirements. These freezers are located onsite in the BPC and offsite at NCI Frederick Central Repository Services in Frederick, MD. Visitors to the laboratory are required to be accompanied by laboratory staff at all times. Access to stored clinical samples is restricted. Samples will be stored until requested by a researcher named on the protocol. All requests are monitored and tracked in Labmatrix. All researchers are required to sign a form stating that the samples are only to be used for research purposes associated with this trial (as per the IRB approved protocol) and that any unused samples must be returned to the BPC. It is the responsibility of the NCI Principal Investigator to ensure that the samples requested are being used in a manner consistent with IRB approval. Following completion of this study, samples will remain in storage as detailed above. Access to these samples will only be granted following IRB approval of an additional protocol, granting the rights to use the material. If, at any time, a patient withdraws from the study and does not wish for their existing samples to be utilized, the individual must provide a written request. Following receipt of this request, the samples will be destroyed (or returned to the patient, if so requested). The PI will record any loss or unanticipated destruction of samples as a deviation. Reporting will be per the requirements of Section **7.2**. Sample bar-codes are linked to patient demographics and limited clinical information. This information will only be provided to investigators listed on this protocol, via registered use of Labmatrix. It is critical that the sample remains linked to patient information such as race, age, dates of diagnosis and death, and histological information about the tumor, in order to correlate genotype with these variables.
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5.2.2 procedures for storage of

Tissues designated for clinical diagnostics are transported to the Laboratory of Pathology (LP) where they are examined grossly and relevant portions fixed, embedded in paraffin and sectioned and stained for diagnostic interpretation. Unutilized excess tissue that is not embedded in paraffin is stored in formalin for up to three months, in accordance with College of American Pathologists/Joint Commission on Accreditation of Healthcare Organizations (CAP/JCAHO) guidelines, and then discarded. Following completion of the diagnostic workup, the slides and tissue blocks are stored indefinitely in the LP's clinical archives. All specimens are catalogued and retrieved utilizing the clinical laboratory information systems, in accordance with CAP/JCAHO regulations. The use of any stored specimens for research purposes is only allowed when the appropriate IRB approval has been obtained. In some cases, this approval has been obtained via the original protocol on which the patient was enrolled.
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5.2.3 thoracic epigenetics laboratory

This study will be conducted within the Thoracic Surgery Branch, NCI. Sample collection and initial processing will be performed in the Thoracic Epigenetics Laboratory (TEL). Samples will be stored in designated monitored freezers (at least -20°C). All samples obtained on this study will be tracked using Labmatrix. Pharmacokinetic samples will be analyzed in the Figg Lab, GMB. Samples will be identified and tracked using unique identifiers linked to each subject's unique patient number (study number). Codes linking personal identifiable information to the unique identifier will be stored in secure, computer servers with limited coded access or locked file cabinets in the Thoracic Surgery Branch, with access limited to the PI or study coordinator. Gene expression, microRNA, and DNA methylation signatures, as well as methylation status of circulating tumor DNA and serologic responses to treatment will be analyzed in Dr. David Schrump's laboratory.
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5.2.4 trepel laboratory

Contact the Trepel Lab by email (Dr. Jane Neckers (Trepel): trepel@helix.nih.gov; Min-Jung Lee: leemin@mail.nih.gov; Akira Yuno: akira.yuno@nih.gov and Sunmin Lee: lees@pop.nci.nih.gov) when the patient is scheduled and by phone as soon as the blood is drawn at 240-760-6330. A lab member will come to pick up the blood. Please keep blood at ambient temperature. Members of the lab will enter the samples into a secure password protected patient's sample tracking database (Translational Pharmacodynamics Research Group Patient Sample Management System) and process the samples. Sample barcodes are linked to patient demographics and limited clinical information. This information will only be provided to investigators listed on this protocol. It is critical that the sample remains coded and linked to patient information such as race, age, dates of diagnosis and death, and histological information about the tumor, in order to correlate with these variables.  
Blood samples will be stored initially in the Trepel Lab in the Magnuson Clinical Center. If, at any time, a subject withdraws from the study and does not wish for their existing samples to be utilized, the individual must provide a written request. Following receipt of this request, the samples will be destroyed (or returned to the patient, if so requested). When a patient withdraws consent the participant's data will be excluded from future distributions, but data that have already been distributed for approved research use will not be able to be retrieved. The PI will record any loss or unanticipated destruction of samples as a deviation (refer to Section **5.2.5**). Reporting will be per the requirements of Section 7.2.
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5.2.5 sample destruction end of

All specimens obtained in the protocol are used as defined in the protocol. Any specimens that are remaining at the completion of the protocol will be stored in the conditions described below. The study will remain open so long as sample or data analysis continues. Following completion of this study, if the subject has co-enrolled on 06C0014, samples will be transferred to the Tissue Procurement protocol and remain in storage in the Blood Processing Core (BPC) as detailed within 06C0014. Access to these samples will only be granted following IRB approval of an additional protocol, granting the rights to use the material. If the subject has not co-enrolled on the Tissue Procurement protocol, the samples will be destroyed. If the patient withdraws consent the participants data will be excluded from future distributions, but data that have already been distributed for approved research use will not be able to be retrieved. The PI will record any loss or unanticipated destruction of samples as a deviation. Reporting will be per the requirements of Section 7.2.
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6 data collection and evaluation

For the purposes of the research sample analyses and correlation with clinical outcomes, demographic information, histology, operative and peri-operative interventions, pathologic findings, laboratory and imaging parameters (performed as part of routine or protocol specified patient care) may be collected on this study. The PI will be responsible for overseeing entry of data into a 21 CFR Part 11-compliant data capture system provided by the NCI CCR and ensuring data accuracy, consistency and timeliness. The principal investigator, associate investigators/research nurses and/or a contracted data manager will assist with the data management efforts. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant. End of Study Procedures: Data will be stored according to HHS, FDA regulations, and NIH Intramural Records Retention Schedule as applicable. Loss or Destruction of Data: Should we become aware that a major breach in our plan to protect subject confidentiality and trial data has occurred, this will be reported expeditiously per requirements in Section **7.2.1**.
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6.1.1 routine data collection

Following registration, all adverse events will be described in the source documents, reviewed by the designated research nurse, and captured. All adverse events, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until return to baseline or stabilization of event. Document AEs from the first study intervention, Study Day 1, through 30 days after the last administration of study therapy. Beyond 30 days after the last intervention, only adverse events which are serious and related to the study intervention need to be recorded.
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6.1.2 exclusions to routine data

6.1.2.1 Adverse Events The following events will be captured only in the source documents and will not be recorded:
- All Grade 1 events. - Grade 2 events that are not related to the treatment. - Grade 1, 2, and 3 Lab values drawn outside of the protocol specified time points that are not assessed as clinically significant by the PI or his designee and are not associated with an adverse event. - Events related to vascular access devices (occlusion, thrombi, hospitalizations for insertion or removal).  
Note: Events that result in a hospitalization for convenience will not be reported. 6.1.2.2 Concomitant Medications/Measures All concomitant medications and measures will be captured in the source documents. Only those medications that the patient is taking at baseline on a routine basis or medications that cause an AE will be captured (e.g., onetime medications, PRN medications, supportive medications, electrolyte replacement and medications given to treat adverse events will not be captured).
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6.2 data sharing plans 6.2.1

I will share human data generated in this research for future research as follows:  
Coded, linked data in an NIH-funded or approved public repository.  
Coded, linked data in BTRIS (automatic for activities in the Clinical Center).  
Identified or coded, linked data with approved outside collaborators under appropriate agreements.
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how and where will the

Data will be shared through:  
An NIH-funded or approved public repository, clinicaltrials.gov, dbGaP. BTRIS (automatic for activities in the Clinical Center).  
Publication and/or public presentations.
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when will the data be

Before publication.  
At the time of publication or shortly thereafter.
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6.2.2 genomic data sharing plan

Unlinked genomic data will be deposited in public genomic databases such as dbGaP in compliance with the NIH Genomic Data Sharing Policy.
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6.3 response criteria

For the purposes of this study, patients will be re-evaluated for response every 10 weeks (± 1 week). In addition to a baseline scan, confirmatory scans should also be obtained not less than 4 weeks following initial documentation of objective response. Objective response and progression will be evaluated in this study using the new international criteria proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) [(Version 1.1)(109)]. Changes in the largest diameter (unidimensional measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph nodes are used in the RECIST criteria. Note: Lesions are either measurable or non-measurable using the criteria provided below. The term "evaluable" in reference to measurability will not be used because it does not provide additional meaning or accuracy.
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6.3.1 confirmatory measurement/duration of response

- Confirmation To be assigned a status of PR or CR, changes in tumor measurements must be confirmed by repeat assessments that should be performed at least 4 weeks after the criteria for response are first met.  
- Duration of Overall Response The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented.
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6.3.2 definitions

Evaluable for toxicity: All patients will be evaluable for toxicity from the time of their first treatment with DAC/THU/pembrolizumab. Dose limiting toxicities will be assessed during the first 3 weeks of DAC-THU/pembrolizumab therapy. Evaluable for objective response: Patients who have measurable disease present at baseline, have received at least 9 weeks of DAC-THU/pembrolizumab therapy (one full course), and have had their disease re-evaluated will be considered evaluable for response provided: (1) the patient demonstrates progressive disease or death while on protocol therapy; (2) the tumor is not removed surgically prior to the time complete response or partial response is confirmed; or (3) the patient demonstrates a complete or partial response as confirmed according to protocol criteria. These patients will have their response classified according to the definitions stated below. [Note:
Patients who electively terminate therapy before completing 9 weeks of DACTHU/pembrolizumab therapy and do not expire within 14 days from start of treatment will be replaced. Patients who experience disease progression after 3 weeks of drug therapy but before completing the minimum of 9 weeks of drug therapy will be taken off-treatment and will only be evaluable for toxicity (not evaluable for response)]. Evaluable Non-Target Disease Response: Patients who have lesions present at baseline that are evaluable but do not meet the definitions of measurable disease, have received at least 8 weeks of DAC-THU/pembrolizumab therapy, and have had their disease re-evaluated will be considered evaluable for non-target disease. The response assessment is based on the presence, absence, or unequivocal progression of the lesions.
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6.3.3 disease parameters 6.3.3.1 measurable

Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as:
By chest x-ray: > 20 mm;  
By CT scan:
o Scan slice thickness 5 mm or under as >10 mm o Scan slice thickness > 5 mm: double the slice thickness With calipers on clinical exam: >10 mm.
All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters). 6.3.3.2 Malignant Lymph Nodes To be considered pathologically enlarged and measurable, a lymph node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). At baseline and in follow-up, only the short axis will be measured and followed.
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6.3.3.3 non-measurable disease

All other lesions (or sites of disease), including small lesions (longest diameter < 10 mm or pathological lymph nodes with ≥ 10 to < 15 mm short axis), are considered non-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by CT or MRI), are considered as non-measurable. Note: Cystic lesions that meet the criteria for radiographically defined simple cysts should not be considered as malignant lesions (neither measurable nor non-measurable) since they are, by definition, simple cysts. 'Cystic lesions' thought to represent cystic metastases can be considered as measurable lesions, if they meet the definition of measurability described above. However, if non-cystic lesions are present in the same patient, these are preferred for selection as target lesions.
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6.3.3.4 target lesions

All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, should be identified as **target lesions** and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. It may be the case that, on occasion, the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected. A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease. Progressive disease by RECIST criteria (109) noted after the first re-staging scan may represent disease that was not detected on the pre-study scan, and a confirmatory scan will be required at the next scheduled re-staging evaluation unless clinically not indicated. If confirmed, progression should be dated by the initial time when the lesions are first detected. If progressive disease by RECIST criteria is seen after Cycle 3, but not confirmed on subsequent restaging scan, the scans from after Cycle 3 would serve as the baseline scan to evaluate for disease progression (110).
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6.3.3.5 non-target lesions

All other lesions (or sites of disease) including any measurable lesions over and above the 5 target lesions should be identified as **non-target lesions** and should also be recorded at baseline. Measurements of these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each should be noted throughout follow-up.
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6.3.4 methods for evaluation of

All measurements should be taken and recorded in metric notation using a ruler or calipers. All baseline evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 weeks before the beginning of the treatment. The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be imaged but are assessable by clinical exam.
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6.3.4.2 metastatic bone lesions

Disease progression is considered if a minimum of two new lesions is observed on bone scan. New lesions seen by the end of Cycle 3 (with the first re-staging bone scan) may represent disease that was not detected on the pre-study scan, and a confirmatory scan will be required at the next scheduled re-staging bone scan unless clinically not indicated. If confirmed, progression should be dated by the initial time when the lesions are first detected. If new lesions are seen after Cycle 3, but no additional lesions are seen on confirmatory scans, the scans from post-Cycle 3 would serve as the baseline scan to evaluate for disease progression (109).
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6.3.4.3 clinical lesions

Clinical lesions will only be considered measurable when they are superficial (e.g., skin nodules and palpable lymph nodes) and ≥ 10 mm diameter as assessed using calipers (e.g., skin nodules). In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended.
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6.3.4.4 methods of measurement

Chest X-ray - Lesions on chest x-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung. However, CT is preferable.  
CT and MRI - CT and MRI are the best currently available and reproducible methods to measure target lesions selected for response assessment. For this study helical Multi-detector CT will be performed with cuts of 5 mm in slice thickness for chest, abdomen and pelvis lesions and 2-3 mm thickness for head and neck lesions. 6.3.4.5 Additional Response Evaluation Using Volumetric Analysis In addition, the utility of volumetric tumor measurement in patients with measurable disease will be prospectively evaluated and compared to 1D and 2D measurements.
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6.3.5 response criteria for radiographic

6.3.5.1 Measuring of Soft Tissue Disease a. Evaluation of Target Lesions
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complete response (cr)

Disappearance of all target lesions. Any pathological lymph nodes (whether target or nontarget) must have reduction in short axis to < 10 mm.
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partial response (pr)

At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum diameters.
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progressive disease (pd)

At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions). Note: For this study, determination of PD will not be made prior to the Day 60 evaluation.
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stable disease (sd)

Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.
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b. evaluation of non-target lesions

Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (< 10 mm short axis). Note: Tumor markers alone cannot be used to assess response. If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response.
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non-cr/non-pd (stable disease sd)

Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits.
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progressive disease (pd)

Appearance of one or more new lesions and/or *unequivocal progression* of existing nontarget lesions. *Unequivocal progression* should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase. Although a clear progression of "non-target" lesions only is exceptional, the opinion of the treating physician should prevail in such circumstances, and the progression status should be confirmed at a later time by the review panel (or Principal Investigator).
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c. evaluation of best overall

The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria.  
| For Patients with Measurable Disease (i.e., Target Disease) Target  NonTarget  Lesions Lesions New Lesions Overall                       | Best Overall Response                                                                                                |           |                                                    |                         |
|-----------------------|----------------------------------------------------------------------------------------------------------------------|-----------|----------------------------------------------------|-------------------------|
| Response              | when Confirmation is  Required*                                                                                      |           |                                                    |                         |
| CR                    | CR                                                                                                                   | No        | CR                                                 | > 4 wks. Confirmation** |
| CR                    | Non-CR                                                                                                               | No        | PR                                                 |                         |
| Non-PD                | > 4 wks. Confirmation**                                                                                              |           |                                                    |                         |
| CR                    | Not evaluated                                                                                                        | No        | PR                                                 |                         |
| PR                    | NonCR                                                                                                                      |           |                                                    |                         |
| Non-PD Not  evaluated | No                                                                                                                   | PR        |                                                    |                         |
| SD                    | NonCR                                                                                                                      |           |                                                    |                         |
| Non-PD Not  evaluated | No                                                                                                                   | SD        | Documented at least once  > 4 wks. from baseline** |                         |
| PD                    | Any                                                                                                                  | Yes or No | PD                                                 | No prior SD, PR or CR   |
| Any                   | PD***                                                                                                                | Yes or No | PD                                                 |                         |
| Any                   | Any                                                                                                                  | Yes       | PD                                                 |                         |
| *                     | See RECIST 1.1 manuscript for further details on what is evidence of a new lesion.                                   |           |                                                    |                         |
| **                    | Only for non-randomized trials with response as primary endpoint.                                                    |           |                                                    |                         |
| ***                   | In exceptional circumstances, unequivocal progression in non-target lesions may be  accepted as disease progression. |           |                                                    |                         |  
d. Patients with a global deterioration of health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be reported as "*symptomatic deterioration."* Every effort should be made to document the objective progression even after discontinuation of treatment.  
e. In some circumstances, it may be difficult to distinguish residual disease from normal tissue. When the evaluation of complete response depends on this determination, it is recommended that the residual lesions be investigated (fine needle aspirate/biopsy) before confirming the complete response status.
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6.4 toxicity criteria

The following adverse event management guidelines are intended to ensure the safety of each patient while on the study. The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for AE reporting. All appropriate treatment areas have access to a copy of the CTCAE version 5.0. A copy of the CTCAE version 5.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov).
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7 nih reporting requirements/data and

Please refer to definitions provided in Policy 801: Reporting Research Events found here.
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7.2 ohsrp office of compliance

Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 802 Non-Compliance Human Subjects Research found here. Note: Only IND Safety Reports that meet the definition of an unanticipated problem will need to be reported per these policies.
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7.2.2 irb requirements for pi

Please refer to the reporting requirements in Policy 801: Reporting Research Events found here.
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7.3 nci clinical director reporting

Problems expeditiously reported to the OHSRP in iRIS will also be reported to the NCI Clinical Director. A separate submission is not necessary as reports in iRIS will be available to the Clinical Director. In addition to those reports, all deaths that occur within 30 days after receiving a research intervention should be reported via email to the Clinical Director unless they are due to progressive disease. To report these deaths, please send an email describing the circumstances of the death to the Clinical Director/designee at NCICCRQA@mail.nih.gov within one business day of learning of the death.
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7.4 nih required data and

The clinical research team will meet weekly when patients are being actively treated on the trial to discuss each patient in detail. Decisions about dose level enrollment and dose escalation if applicable will be made based on the toxicity data from prior patients. All data will be collected in a timely manner and reviewed by the principal investigator or a clinical associate investigator in a timely manner. Events meeting requirements for expedited reporting as described in Section **7.2.1** will be submitted within the appropriate timelines. The principal investigator will review adverse event and response data on each patient to ensure safety and data accuracy. The principal investigator will personally conduct or supervise the investigation and provide appropriate delegation of responsibilities to other members of the research staff.
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8 sponsor safety reporting 8.1

Any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and which does not necessarily have a causal relationship with this treatment. An adverse event (AE) can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not related to the medicinal (investigational) product (ICH E6 (R2)).
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8.1.2 serious adverse event (sae)

An adverse event or suspected adverse reaction is considered serious if in the view of the investigator or the sponsor, it results in any of the following:
Death.  
A life-threatening adverse event (see Section **8.1.3**). Inpatient hospitalization or prolongation of existing hospitalization.  
o A hospitalization/admission that is pre-planned (i.e., elective or scheduled surgery arranged prior to the start of the study), a planned hospitalization for pre-existing condition, or a procedure required by the protocol, without a serious deterioration in health, is not considered a serious adverse event.  
o A hospitalization/admission that is solely driven by non-medical reasons (e.g.,
hospitalization for patient or subject convenience) is not considered a serious adverse event.  
o Emergency room visits or stays in observation units that do not result in admission to the hospital would not be considered a serious adverse event. The reason for seeking medical care should be evaluated for meeting one of the other serious criteria.  
Persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions.  
A congenital anomaly/birth defect. Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition.
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8.1.3 life-threatening

An adverse event or suspected adverse reaction is considered "life-threatening" if, in the view of either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a more severe form, might have caused death. (21CFR312.32)
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8.1.4 severity

The severity of each Adverse Event will be assessed utilizing the CTCAE version 5.0.
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8.1.5 relationship to study product

All AEs will have their relationship to study product assessed using the terms: related or not related.  
Related - There is a reasonable possibility that the study product caused the adverse event. Reasonable possibility means that there is evidence to suggest a causal relationship between the study product and the adverse event.  
Not Related - There is not a reasonable possibility that the administration of the study product caused the event.
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8.2 assessment of safety events

AE information collected will include event description, date of onset, assessment of severity and relationship to study product and alternate etiology (if not related to study product), date of resolution of the event, seriousness and outcome. The assessment of severity and relationship to the study product will be done only by those with the training and authority to make a diagnosis and listed on the Form FDA 1572 as the site principal investigator or sub-investigator. AEs occurring during the collection and reporting period will be documented appropriately regardless of relationship. AEs will be followed through resolution. SAEs will be:  
Assessed for severity and relationship to study product and alternate etiology (if not related to study product) by a licensed study physician listed on the Form FDA 1572 as the site principal investigator or sub-investigator.  
Recorded on the appropriate SAE report form, the medical record and captured in the clinical database.  
Followed through resolution by a licensed study physician listed on the Form FDA 1572 as the site principal investigator or sub-investigator.
For timeframe of recording adverse events, please refer to Section 6.1. All serious adverse events recorded from the time of first investigational product administration must be reported to the Sponsor with the exception of any listed in Section 8.4.
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8.3 reporting of serious adverse

Any AE that meets protocol-defined serious criteria or meets the definition of Adverse Event of Special Interest that require expedited reporting must be submitted immediately (within 24 hours of awareness) to OSRO Safety using the CCR SAE report form. Any exceptions to the expedited reporting requirements are found in Section 8.4. All SAE reporting must include the elements described in Section 8.2. SAE reports will be submitted to the Center for Cancer Research (CCR) at: OSROSafety@mail.nih.gov and to the CCR PI and study coordinator. CCR SAE report form and instructions can be found at: https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions Following the assessment of the SAE by OSRO, other supporting documentation of the event may be requested by the OSRO Safety and should be provided as soon as possible.
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8.4 waiver of expedited reporting

Death or Hospitalization that is deemed to be due to disease progression, and not attributable to the intervention will not be reported as an SAE. The event, and the assessment that it was caused by disease progression will be documented in the medical records. The causality assessment of Hospitalization will be re‐evaluated any time when new information is received. If the causality assessment changes from disease progression to related to the study intervention, SAE report will be sent to the Sponsor immediately **in an expedited manner according to Section 8.3**. If there is any uncertainty whether the intervention is a contributing factor to the event, the event should be reported as AE or SAE as appropriate.
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8.5 reporting pregnancy

All required pregnancy reports/follow-up to OSRO will be submitted to: OSROSafety@mail.nih.gov and to the CCR PI and study coordinator. Forms and instructions can be found here: https://ccrod.cancer.gov/confluence/display/CCRCRO/Forms+and+Instructions.
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8.5.1 maternal exposure

If a patient becomes pregnant during the course of the study, the study treatment should be discontinued immediately, and the pregnancy reported to the Sponsor no later than 24 hours of when the Investigator becomes aware of it. The Investigator should notify the Sponsor no later than 24 hours of when the outcome of the Pregnancy becomes known. Pregnancy itself is not regarded as an SAE. However, congenital abnormalities or birth defects and spontaneous miscarriages that meet serious criteria (Section **8.1.2**) should be reported as SAEs. The outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or congenital abnormality) should be followed up and documented.
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8.5.2 paternal exposure

Male patients should refrain from fathering a child or donating sperm during the study and for 60 days after the last dose of DAC-THU and pembrolizumab. Pregnancy of the patient's partner is not considered to be an AE. However, the outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or congenital abnormality) occurring from the date of the first dose until 60 days after the last dose should, if possible, be followed up and documented.
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8.6 regulatory reporting for studies

Following notification from the investigator, CCR, the IND sponsor, will report any suspected adverse reaction that is both serious and unexpected in expedited manner to the FDA in accordance to 21 CFR 31.2.32. CCR will report an AE as a suspected adverse reaction only if there is evidence to suggest a causal relationship between the study product and the adverse event. CCR will notify FDA and all participating investigators (i.e., all investigators to whom the Sponsor is providing drug under its INDs or under any investigator's IND) in an IND safety report of potential serious risks from clinical trials or any other source, as soon as possible, in accordance to 21 CFR Part 312.32. All serious events will be reported to the FDA at least annually in a summary format.
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8.7 sponsor protocol deviation reporting

A Protocol Deviation is defined as any non-compliance with the clinical trial Protocol, Manual of Operational Procedures (MOP) and other Sponsor approved study related documents, GCP, or protocol-specific procedural requirements on the part of the participant, the Investigator, or the study site staff inclusive of site personnel performing procedures or providing services in support of the clinical trial. It is the responsibility of the study Staff to document any protocol deviation identified by the Staff or the site Monitor in the CCR Protocol Deviation Tracking System (PDTS) online application. The entries into the PDTS online application should be timely, complete, and maintained per CCR PDTS user requirements. In addition, any deviation to the protocol should be documented in the participant's source records and reported to the reviewing IRB per their guidelines. OSRO required protocol deviation reporting is consistent with E6(R2) GCP: Integrated Addendum to ICH E6(R1): 4.5 Compliance with Protocol; 5.18.3 (a), and 5.20 Noncompliance; and ICH E3 16.2.2 Protocol deviations.
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9 clinical monitoring

Clinical site monitoring is conducted to ensure:  
that the rights of the participants are protected; that the study is implemented per the approved protocol, Good Clinical Practice and standard operating procedures; and the quality and integrity of study data and data collection methods are maintained.
Monitoring for this study will be performed by NCI CCR Office of Sponsor and Regulatory Oversight (OSRO) Sponsor and Regulatory Oversight Support (SROS) Services contractor. Clinical site monitoring activities will be based on OSRO standards, FDA Guidance E6(R2) Good Clinical Practice: Integrated Addendum to ICH E6(R1) March 2018, and applicable regulatory requirements. Details of clinical site monitoring will be documented in a Clinical Monitoring Plan (CMP) developed by OSRO. CMPs will be protocol-specific, risk-based and tailored to address human subject protections and integrity of the study data. OSRO will determine the intensity and frequency of monitoring based on several factors, including study type, phase, risk, complexity, expected enrollment rate, and any unique attributes of the study and the site. The Sponsor will conduct a periodic review of the CMP to confirm the plan's continued appropriateness. A change to the protocol, significant or pervasive non-compliance with GCP, or the protocol may trigger CMP updates. OSRO SROS Monitoring visits and related activities will be conducted throughout the life cycle of each protocol. The first activity is before the study starts to conduct a Site Assessment Visit
(SAV) (as warranted), followed by a Site Initiation Visit (SIV), Interim Monitoring Visit(s)
(IMVs), and a study Close-Out Visit (COV). Some monitoring activities may be performed remotely, while others will occur at the study site(s). Monitoring visit reports will describe visit activities, observations, and associated action items or follow-up required for resolution of any issues, discrepancies, or deviations. Monitoring reports will be distributed to the study PI, NCI CCR QA, CCR Protocol Support Office, coordinating center (if applicable), and the Sponsor regulatory file. The site Monitor will inform the study team of any deviations observed during monitoring visits. If unresolved, the Monitor will request that the site Staff enter the deviations in the CCR Protocol Deviation Tracking System (PDTS) for deviation reporting to the Sponsor and as applicable per institutional and IRB guidance.
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10 statistical considerations

The primary objectives of this trial are to determine the objective response rates of DAC/THU plus pembrolizumab in patients with NSCLC, EsC, and MPM who have intra-tumoral PD-L1 expression of at least 50% and those who do not. Approximately 30% of NSCLC exhibit PD-L1 expression in 50% or more cancers cells, another 30-50% have PD-L1 expression in 1-49% of cancer cells, and the remaining NSCLC have no PD-L1 expression by IHC (25, 111-113). In contrast, 20-40% of gastroesophageal cancers exhibit PD-L1 expression (25, 111, 112); in the majority of these cancers, PD-L1 is expressed at low levels, and tends not to be expressed on the cancer cells (112). Despite these differences regarding PD-L1 expression, present data indicate that responses to immune checkpoint inhibitors in EsC are comparable to NSCLC with low PDL1 expression (114). Approximately 25%-30% of MPM exhibit some level of PD-L1 expression, with the expression being primarily on tumor-associated immune cells (115). PD-L1 expression tends to be higher on sarcomatoid MPM and lower on the more common epithelioid subtype. Response rates in MPM following PD-1 blockade are comparable to NSCLC with similar levels of PD-L1 expression (99, 100). Patients will be initially enrolled onto a multi-dose level dose escalation portion of the trial following a 3+3 design. Patients will not be stratified for the Phase I component, which will accelerate our ability to define MTD. Thus, for the dose escalation phase, up to 6 patients per dose level may be enrolled, with a total of 12 patients required with a maximum of 2 dose levels. At the MTD, an additional 4 patients will be enrolled to further estimate the safety of the combination. Since published data have demonstrated a response rate of 45% for first-line pembrolizumab alone in NSCLC patients with PD-L1 expression of 50% or greater (95), (98), this trial will try to demonstrate if a response rate in excess of 45% may be realized in NSCLC patients with ≥ 50% PD-L1 expression by the addition of DAC/THU. Briefly, NSCLC patients with high intratumoral
(50% or greater) PD-L1 expression will be evaluated for response as part of a Simon optimal twostage Phase II trial design (116) in order to rule out an unacceptably low PR+CR rate of 35%
(p0=0.35) in favor of an improved response rate of 60% (p1=0.60). With alpha=0.10 (probability of accepting a poor treatment=0.10) and beta=0.20 (probability of rejecting a good treatment=0.20), the first stage will determine the response rate in 10 evaluable patients, including those NSCLC patients among the 10 who are treated at the MTD from the Phase I cohort who have high intra-tumoral PD-L1 expression. If 0 to 4 of the 10 patients have a clinical response, then no further patients will be accrued. If 5 or more of these 10 patients have a response, then accrual would continue until a total of 23 evaluable patients have been treated at the MTD. As it may take up to several months to determine if a patient has experienced a response, a temporary pause in the accrual may be necessary to ensure that enrollment to the second stage is warranted. If there are 5 to 10 patients with a response out of 23 patients, this would be an uninterestingly low response rate. If there were 11 or more of 23 (47.8%) who experienced a response, this would be sufficiently interesting to warrant further study in later trials. Under the null hypothesis (35% response rate), the probability of early termination is 75.2%. NSCLC patients with low (0-49%) intratumoral PD-L1 expression will be evaluated for response using a Simon optimal two-stage Phase II trial design (116) in order to rule out an unacceptably low PR+CR rate of 10% (p0=0.10) in favor of an improved response rate of 30% (p1=0.30). Variable mutational burdens among NSCLC with low PD-L1 expression could affect responses to immune checkpoint inhibitors, potentially making this cohort more heterogeneous than the NSCLC high PD-L1 expresser cohort. For example, NSCLC patients without activating mutations in EGFR, HER2 or ALK with PD-L1 tumor proportion score (TPS) < 1% have an expected response rate of less than 10%, whereas those with activating mutations involving these genes have response rates of 5% or less; patients with PD-L1 TPS of 1-49% have a 17% response rate (25, 103, 104, 113). Therefore, with alpha=0.05 (probability of accepting a poor treatment=0.05) and beta = 0.20 (probability of rejecting a good treatment=0.20), the first stage will determine the response rate in 10 evaluable patients, including those among the 10 who are treated at the MTD
from the Phase I cohort who have low intra-tumoral PD-L1 expression, and if 0 to 1 of the 10 have a clinical response, then no further patients will be accrued. If 2 or more of these 10 patients have a response, then accrual would continue until a total of 29 evaluable patients have been treated in this cohort. As it may take up to several months to determine if a patient has experienced a response, a temporary pause in the accrual may be necessary to ensure that enrollment to the second stage is warranted. If there are 2 to 5 patients with a response out of 29 patients, this would be an uninterestingly low response rate. If there were 6 or more of 29 (20.7%) who experienced a response, this would be sufficiently interesting to warrant further study in later trials. Under the null hypothesis (10% response rate), the probability of early termination is 73.6%. While accrual of NSCLC patients is underway, up to 10 EsC patients including any from the Phase I component after the MTD has been identified, will be accrued to examine responses in EsC patients treated with DAC-THU at the MTD and pembrolizumab. Since it is anticipated that most if not all of these 10 patients will have low or no PD-L1 expression in their tumor tissues, and because response rates to immune checkpoint inhibitors in EsC appear to be comparable to NSCLC with low PD-L1 expression, criteria similar to those reported for the NSCLC low PD-L1 cohort will be used to identify a potential signal of activity. Specifically, if there are 2 or more responses in these 10 patients, this might warrant amendment of the current protocol to enable expansion of this cohort with appropriate statistical criteria, or to justify a larger, focused Phase II trial. Additionally, up to 10 MPM patients including any from the Phase I component after the MTD has been identified, will be accrued to examine responses in MPM patients treated with DAC-THU at the MTD and pembrolizumab. Since it is anticipated that most if not all of these 10 patients will have low PD-L1 expression in their tumor tissues, and because response rates to immune checkpoint inhibitors in MPM patients appear to be comparable to NSCLC with low PD-L1 expression, criteria similar to those reported for the NSCLC low PD-L1 cohort will be used to identify a potential signal of activity. Specifically, if there are 2 or more responses in these 10 patients, this might warrant amendment of the current protocol to enable expansion of this cohort with appropriate statistical criteria, or to justify a larger, focused Phase II trial.  
It is expected that approximately 2-4 patients per month may enroll onto this trial. Thus, it is expected that 24-30 months may be required in order to enroll up to 78 evaluable patients. Assuming two dose levels will be used to define MTD, the Phase I component of the trial will require 16 patients. The Phase II portion of the trial may require 29 NSCLC patients for the PDL1 high, and 23 NSCLC patients for the PD-L1 low cohorts, plus 10 EsC patients, plus 10 MPM
patients, less 10 patients treated at the MTD in the Phase I portion of the protocol; hence 16+29+23+10+10-10=78 patients In order to allow for a small number of inevaluable patients, the accrual ceiling will be set at 85 patients, acknowledging that the trial can be completed with 60 or fewer patients if response goals for each cohort are not met.  
Twelve *months after Amendment E has been implemented, accrual will be assessed. If the trial* is not on track for accrual targets, e.g., if fewer than 12 patients have been accrued within 12 months, the protocol will either be terminated, or the trial will be opened at an additional site to accelerate patient accrual, and ensure timely completion of this trial. Pharmacokinetic analysis will be conducted using non-compartmental methods. Pharmacodynamic analyses, as well as analyses of molecular endpoints in tumor tissues and PBMC before and after treatment will be performed, with the primary focus being with respect to results at the MTD. For pharmacodynamic endpoints, descriptive statistics will be used for each endpoint. Briefly, pharmacodynamic and molecular endpoints in target tissues will be assessed before and after DAC-THU/pembrolizumab treatment, with the primary focus being comparison of microarray, qRT-PCR and IHC results relative to profiles identified in our preclinical studies. It is anticipated that non-parametric statistical methods will be used to compare paired results before and after treatment (but not between groups), as well as to perform comparisons or correlations among parameters. As the exact set of comparisons and analyses to be performed will be determined following completion of the trial and will be based on limited numbers of patients, the analyses will be considered exploratory and hypothesis generating rather than definitive.
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10.1 methods of analysis

Response rates will be calculated as the percent of patients whose best response is a CR or PR. Toxicity information recorded will include the type, severity, time of onset, time of resolution, and the probable association with the study regimen. Tables will be constructed to summarize the observed incidence by severity and type of toxicity.
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11 human subjects protections 11.1

The patients to be entered on this protocol have advanced NSCLC, EsC, or MPM and have limited life expectancies. This population was selected because of the unknown outcome of this treatment in terms of its effectiveness. The experimental treatment has a chance to provide clinical benefit although this is unknown. Subjects of both genders and all racial/ethnic groups are eligible. Efforts will be made to extend accrual to a representative population, but in this preliminary study, a balance must be struck between patient safety considerations and limitations on the number of individuals exposed to potentially toxic and/or ineffective treatments on the one hand and the need to explore gender and ethnic aspects of clinical research on the other hand. If differences in outcome that correlate with gender or ethnic identity are noted, accrual may be expanded, or a follow-up study may be written to investigate these differences more fully. One strategy for recruitment may be to distribute an IRB approved protocol recruitment letter to General and Oncology physicians and nurses.
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11.2 participation of children

It is anticipated that most children with cancer will not have disease that is appropriate for study. Because the effects of DAC-THU have not been evaluated in children, individuals <18 will be excluded from this study until more data are available.
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11.3 participation of subjects unable

Adults unable to give consent are excluded from enrolling in the protocol. However, re-consent may be necessary and there is a possibility, though unlikely, that subjects could become decisionally impaired. For this reason and because there is a prospect of direct benefit from research participation (Section **11.4**), all subjects will be offered the opportunity to fill in their wishes for research and care, and assign a substitute decision maker on the "NIH Advance Directive for Health Care and Medical Research Participation" form so that another person can make decisions about their medical care in the event that they become incapacitated or cognitively impaired during the course of the study. Note: The PI or AI will contact the NIH Ability to Consent Assessment Team (ACAT) for evaluation to assess ongoing capacity of the subjects and to identify an LAR, as needed. Please see Section **11.5.1** for consent procedure.
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11.4 evaluation of benefits and

The risks to the patient participating in this trial are anticipated to be small and are primarily the risks associated with administering the DAC-THU with pembrolizumab. These potential risks are addressed in Sections 13.1.9, **13.2.8** and **13.3.6**. There is the potential risk of not responding to the investigational treatment, and being unfit to receive and potentially benefit from standard of care cytotoxic chemotherapy. Risks and benefits will be carefully discussed with the patient at the time consent is obtained. Patients will be monitored throughout the study in order to limit the consequences of any adverse events. 11.4.1.2 Blood Sampling Side effects of blood draws include pain and bruising, lightheadedness, and rarely, fainting. 11.4.1.3 Urine Collection There is no physical risk involved with urine collection. 11.4.1.4 Biopsies The risks associated with biopsies are pain and small amounts of bleeding at the biopsy site, and rarely, a small infection at the site. Biopsy sites usually heal very well and with very little scarring. In order to minimize pain, local anesthesia will be used; side effects of the local anesthesia may include a mild burning sensation when the numbing medicine is injected into the skin. 11.4.1.5 Conscious Sedation The common side effects of conscious sedation include drowsiness, delayed reflexes, hypotension, headache, and nausea. These are generally mild and last no more than a few hours. 11.4.1.6 Pulmonary Function Tests These tests are safe and side effects are unlikely, but may include brief light headedness or slight soreness of the chest. 11.4.1.7 Scans and Contrast The most common discomfort is the length of time a patient must lay still during a scan. Patients may also become uncomfortable with the closed space of the machines. There is a small risk of reaction in scans involving contrast. Common reactions include pain in the vein where the contrast was given, a metallic or bitter taste in the mouth, headache, nausea and a warm or flushing feeling that lasts from 1-3 minutes. In very rare cases, severe reactions that affect breathing, heart rhythm or blood pressure have occurred. An IV catheter may need to be inserted for administration of the contrast agent or anesthetic, which may cause pain at the site where the IV is placed and there is a small risk of bruising or infection, or inflammation of the skin and vein with pain and swelling.
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11.4.1.8 risks of exposure to

This research study involves the potential for 5 CT CAP scans, 5 PET/CT scans, 5 brain CT scans as well as 2 CT-guided biopsies over the course of the first year on study. This radiation exposure is not required for medical care and is for research purposes only. Subjects will be exposed to approximately 13.6 rem. This amount of radiation is above the guideline of 5 rem per year and will expose the subject to the roughly the same amount of radiation as 45.3 years of background radiation.
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11.4.2 benefits

There may be some direct benefit to patients who participate in this trial since it is anticipated that DAC-THU with pembrolizumab may cause tumor stabilization or some tumor regression. The greatest benefit will be the information regarding the feasibility, toxicity and dosing of DAC-THU with pembrolizumab administration, as well as the information on changes in the epigenome of lung and gastroesophageal cancers and systemic immunity to up-regulated CTAs.
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11.5 consent process and documentation

The informed consent document will be provided as a physical or electronic document to the participant or consent designee(s) as applicable for review prior to consenting. A designated study investigator will carefully explain the procedures and tests involved in this study, and the associated risks, discomforts and benefits. In order to minimize potential coercion, as much time as is needed to review the document will be given, including an opportunity to discuss it with friends, family members and/or other advisors, and to ask questions of any designated study investigator. A signed informed consent document will be obtained prior to entry onto the study. The initial consent process as well as re-consent, when required, may take place in person or remotely (e.g., via telephone or other NIH approved remote platforms used in compliance with policy, including HRPP Policy 303) per discretion of the designated study investigator and with the agreement of the participant/consent designee(s). Whether in person or remote, the privacy of the subject will be maintained. Consenting investigators (and participant/consent designee, when in person) will be located in a private area (e.g., clinic consult room). When consent is conducted remotely, the participant/consent designee will be informed of the private nature of the discussion and will be encouraged to relocate to a more private setting if needed. Consent will be documented with required signatures on the physical document (which includes the printout of an electronic document sent to participant) or as described below, with a manual (non-electronic) signature on the electronic document. When required, witness signature will be obtained similarly as described for the investigator and participant.
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manual (non-electronic) signature on electronic

When a manual signature on an electronic document is used for the documentation of consent at the NIH Clinical Center, this study will use the following to obtain the required signatures:  
Adobe platform (which is not 21 CFR Part 11 compliant); or, iMedConsent platform (which is 21 CFR Part 11 compliant)
During the consent process, participants and investigators will view individual copies of the approved consent document on screens at their respective locations (if remote consent); the same screen may be used when in the same location, but is not required. Both the investigator and the participant will sign the document using a finger, stylus or mouse. Note: Refer to the CCR SOP PM-2, Obtaining and Documenting the Informed Consent Process for additional information (e.g., verification of participant identity when obtaining consent remotely) found here.
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11.5.1 consent process for adults

For participants addressed in Section **11.3**, an LAR will be identified consistent with Policy 403 and informed consent obtained from the LAR, as described in Section **11.5**.
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12 regulatory and operational considerations

This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable cause. Written notification, documenting the reason for study suspension or termination, will be provided by the suspending or terminating party to investigators, funding agencies, the Investigational New Drug (IND) sponsor and regulatory authorities, as applicable. If the study is prematurely terminated or suspended, the Principal Investigator (PI) will promptly inform study participants, the Institutional Review Board (IRB), and sponsor and will provide the reason(s) for the termination or suspension. Study participants will be contacted, as applicable, and be informed of changes to study visit schedule. Circumstances that may warrant termination or suspension include, but are not limited to:
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12.2 quality assurance and quality

The clinical site will perform internal quality management of study conduct, data and biological specimen collection, documentation and completion. An individualized quality management plan will be developed to describe a site's quality management. Quality control (QC) procedures will be implemented beginning with the data entry system and data QC checks that will be run on the database will be generated. Any missing data or data anomalies will be communicated to the site(s) for clarification/resolution. Following written Standard Operating Procedures (SOPs), the monitors will verify that the clinical trial is conducted and data are generated and biological specimens are collected, documented (recorded), and reported in compliance with the protocol, International Conference on Harmonization Good Clinical Practice (ICH GCP), and applicable regulatory requirements (e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)). The investigational site will provide direct access to all trial related sites, source data/documents, and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local and regulatory authorities.
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12.3 conflict of interest policy

The independence of this study from any actual or perceived influence, such as by the pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who have a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, persons who have a perceived conflict of interest will be required to have such conflicts managed in a way that is appropriate to their participation in the design and conduct of this trial. The study leadership in conjunction with the National Cancer Institute has established policies and procedures for all study group members to disclose all conflicts of interest and will establish a mechanism for the management of all reported dualities of interest.  
Determination of unexpected, significant, or unacceptable risk to participants Demonstration of efficacy that would warrant stopping Insufficient compliance to protocol requirements Data that are not sufficiently complete and/or evaluable Determination that the primary endpoint has been met Determination of futility Study may resume once concerns about safety, protocol compliance, and data quality are addressed, and satisfy the sponsor, IRB and as applicable, Food and Drug Administration (FDA).
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12.4 confidentiality and privacy

Participant confidentiality and privacy is strictly held in trust by the participating investigators, their staff, and the sponsor(s). This confidentiality is extended to cover testing of biological samples and genetic tests in addition to the clinical information relating to participants. Therefore, the study protocol, documentation, data, and all other information generated will be held in strict confidence. No information concerning the study or the data will be released to any unauthorized third party without prior written approval of the sponsor. All research activities will be conducted in as private a setting as possible. The study monitor, other authorized representatives of the sponsor, representatives of the Institutional Review Board (IRB), and/or regulatory agencies may inspect all documents and records required to be maintained by the investigator, including but not limited to, medical records (office, clinic, or hospital) and pharmacy records for the participants in this study. The clinical study site will permit access to such records. The study participant's contact information will be securely stored at the clinical site for internal use during the study. At the end of the study, all records will continue to be kept in a secure location for as long a period as dictated by the reviewing IRB, Institutional policies, or sponsor requirements. Study participant research data, which is for purposes of statistical analysis and scientific reporting, will be stored at the NCI CCR. This will not include the participant's contact or identifying information. Rather, individual participants and their research data will be identified by a unique study identification number. The study data entry and study management systems used by the clinical site and by NCI CCR research staff will be secured and password protected. At the end of the study, all study databases will be archived at the NIH. To further protect the privacy of study participants, a Certificate of Confidentiality has been issued by the National Institutes of Health (NIH). This certificate protects identifiable research information from forced disclosure. It allows the investigator and others who have access to research records to refuse to disclose identifying information on research participation in any civil, criminal, administrative, legislative, or other proceeding, whether at the federal, state, or local level. By protecting researchers and institutions from being compelled to disclose information that would identify research participants, Certificates of Confidentiality help achieve the research objectives and promote participation in studies by helping assure confidentiality and privacy to participants.
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13.1.2 description

Decitabine is an analog of 2'-deoxycytidine in which the carbon at position 5 in the pyridine ring has been replaced by nitrogen. In acid media, decitabine undergoes cleavage at the glycosidic bond to yield 5-azacytosine and deoxyribose. In acid media, a rapid, reversible opening of the 5-azacytosine ring occurs between positions 1 and 6, producing N-(formylamidino)-N'-D-2'-deoxyribofuranosylurea which decomposes with the loss of a formyl group. Decitabine is enzymatically phosphorylated in sequence by deoxycytidine kinase, deoxycytidylic kinase, and nucleoside diphosphonucleoside kinase to the active forms, 5-Aza-dCMP, 5-Aza-dCDP, 5-Aza-dCTP, respectively. The latter form is incorporated into DNA. Decitabine is enzymatically deaminated by cytidine deaminase; phosphorylated decitabine nucleoside (5-Aza-dCMP) is catabolized by deoxycytidylic deaminase. 13.1.2.1 Mode of Action Inhibition of DNA methylation, and induction of cellular differentiation. 13.1.3 Chemical Names  
(1) 1-(2'-deoxy-D-ribofuranosyl)-5-azacytosine (2) 3,5-Triazin-2(1H)-one,4-amino-l-(2-deoxy-ß-D-erythro-pentofuranosyl)- (3) 4-Amino-l-(2-deoxy-ß-D-erythro-entofuranosyl)-1,3,5-triazin-2(1H)-one (4) 5-aza-2'-deoxycytidine
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13.1.4 chemical identification

Empiric formula C8Hl2N404 Molecular weight 228.21 daltons CAS registry No. 2353-33-5 pertains to free base.
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13.1.5 formulation

Decitabine is manufactured by IriSys LLC (San Diego, CA) and supplied by DCTD as a freeze-dried powder in 20 mL-capacity vials containing DAC 50 mg with 68 mg potassium phosphate, and NaOH. Decitabine will be encapsulated by IriSys LLC under contract with the NIH CC Pharmacy per federal Good Manufacturing Practice guidelines. In this formulation decitabine is supplied as capsules containing 5 mg of decitabine per capsule, in bottles containing 30 capsules, together with a drying agent. The label on the bottles includes the following information: the (i) name of the drug; (ii) quantity of drug per capsule; (iii) date of packaging; (iv) recommended storage temperature; (v) lot number of bulk drug used to generate the capsules
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13.1.6 stability

Powder: The glass bottles containing decitabine should be stored at -20°C. The drug substance has been tested after 18 months of storage at -20°C and no change in appearance, assay or impurities was observed. Decitabine is stable as a dry powder, however, degrades in a pH dependent manner in aqueous solution (optimal pH for stability is 7.0). Decitabine is hydrolyzed in aqueous solution at pH 7.4 at a rate of approximately 2.5% every 7 hours at 4°C. Capsules: Shelf life stability testing of the intact bottles is on-going.
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13.1.7 storage

Powder: Containers should be tightly closed and stored in the freezer at -20°C. Capsules: Bottles should be tightly closed and stored at refrigerated temperatures (2-8°C).
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13.1.9 toxicities

There is substantial information regarding the toxicity of decitabine in humans from clinical trials in patients with MDS and AML, including relapsed or poor prognosis cases. Some of this information is summarized in the package insert for this FDA approved drug. In almost all studies, the patients received doses much higher than the decitabine doses planned in this study. Leukopenia was a major toxicity and nausea, or vomiting were common non-hematologic toxicities. A better guide to anticipated side-effects in this clinical trial are observations from clinical studies of decitabine repositioned for non-cytotoxic DNMT1-depletion for sickle cell disease (SCD), -thalassemia intermedia and MDS. In these clinical trials there were no episodes of non-hematologic CTCAE Grade 2 or higher toxicity from decitabine. In the SCD and -
thalassemia trials, consistent with a non-cytotoxic mechanism of action, the main side-effect was an increase in the platelet count (as opposed to the usual side-effect of thrombocytopenia seen with cytotoxic treatments). Cytotoxicity/DNA damage assays based on bone marrow morphological examination, bone marrow DNA content analysis, VDJ recombination assay, erythrocyte micronucleus assay and gamma-H2AX assay were also negative. These assays did not reveal evidence of DNA damage or cytotoxicity. The increase in the platelet count was not associated with any clinical adverse events. Other common side effects are fatigue, fever, nausea, cough, petechiae, constipation, diarrhea, and hyperglycemia.
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expected side effects in current

1. Thrombocytosis: Decitabine-induced shifts in differentiation include increased erythropoiesis and megakaryopoiesis (60, 61). Therefore, increases in platelet counts are expected, especially in patients who are hyposplenic (e.g., patients with SCD) or asplenic (many patients with -
thalassemia). Although it is intuitive to expect an association between platelet count and thrombosis risk, this has not been evident in clinical observations and studies, whereas qualitative RBC defects from underlying disease can be pro-thrombotic (60, 117-124). Hence, in a SCD study, SC decitabine-induced improvement in multiple indices of RBC pathology was accompanied by improvement in multiple markers of coagulation pathway activity, despite concurrent platelet count increases to > 800x109/L (60). Thus, it is possible that decitabine-induced improvements in RBC phenotype could reduce thrombophilia despite concurrent platelet count increases. Coagulation pathway evaluation (for example by measurement of D-dimer levels) should probably be a component of studies with decitabine to treat hemoglobinopathies.  
2. Neutropenia: Decitabine induced shifts in differentiation include favoring erythroidmegakaryocyte progenitor production over granulocyte-monocyte progenitor production by hematopoietic stem cells (60, 61). Hence, even at low, non-cytotoxic concentrations, a downward shift in neutrophil counts is expected. Of note, the current standard of care for symptomatic SCD is DNA damaging, cytotoxic doses of HU. Consequently, in SCD subjects treated with HU,
hematologic toxicities are pancellular, with neutrophil recovery taking up to 2 weeks (AHFS Drug Information, 2000).  
3. Lymphopenia: Decitabine induced shifts in hematopoietic differentiation may also reduce lymphocyte production.  
3. Testicular toxicity: Pre-clinical studies suggest an effect on sperm-counts can occur even with the intended low, non-cytotoxic concentrations of decitabine. This toxicity appears reversible. Of note, the current standard of care for symptomatic SCD with HU also causes testicular toxicity (AHFS Drug Information, 2000).  
4. Teratogenicity: Pre-clinical data indicates that there is a teratogenic risk even at low doses of decitabine. Therefore, all clinical studies with decitabine must take into account, and incorporate precautions, to address a risk for teratogenicity. Current standard therapy for SCD with either HU
or stem cell transplantation also has teratogenic risks (pregnancy category D).
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13.2 tetrahydrouridine

13.2.1 Names Chemical Name: 2(1H)-pyrimidinone, tetrahydro-4-hydroxy-1.beta.-D-ribofuranosyl-
Other Names: THU, H4U
CAS Registry Number: 18771-50-1 Molecular Formula: C9H16N2O6 M.W.: 248 Approximate Solubility: The drug is highly soluble in water, up to 200 mg/mL.  
13.2.2 Mode of Action: Cytidine deaminase inhibitor
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13.2.3 how supplied

Tetrahydrouridine is manufactured by KP Pharmaceutical Technology Inc (Bloomington, IN, USA) and will be purchased from the Cleveland Clinic. THU is supplied as capsules containing 250 mg of THU per capsule, in bottles containing 8 capsules per bottle together with a drying agent.  
The label on the bottles includes the following information: the (i) name of the drug; (ii) quantity of drug per bottle; (iii) date of manufacture; (iv) recommended storage temperature; (v) manufacturer's lot number.
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13.2.4 storage

Capsules: Bottles should be tightly closed and stored at refrigerated temperatures 2°-8°C (35-46°F) 13.2.5 Stability: Capsules: Shelf life stability testing of the intact bottles is on-going. 13.2.6 Route of Administration: Oral 13.2.7 Method of Administration: Please see Section 3.2.1.
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13.2.8 toxicities

Most reports discuss the use of THU co-administered with a cytidine analogue in order to inhibit the metabolism of that cytidine-based therapeutic, and the side-effects are that of the active agent cytidine analogue therapeutic. The single dose toxicology of THU was evaluated in dogs and Rhesus monkeys at intravenous doses of up to 1000 mg/kg: with the exception of local inflammation at the injection site, no notable toxic effects were attributed to the administration of THU (125). Although there is only limited non-clinical toxicology data with THU alone, the data that exists suggests that THU by itself does not pose a risk for toxicity, even at doses considerably higher than the dose in the planned study.
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13.3.1 description

Pembrolizumab is a humanized monoclonal IgG4 antibody directed against the human cell surface receptor, programmed death-1, (PD-1). Following administration, pembrolizumab binds to PD-1 and blocks the interaction between PD-1 and its ligands.
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13.3.2 how supplied

Keytruda (pembrolizumab) will be purchased by the NIH Clinical Center Pharmacy Department from commercial sources. Pembrolizumab is supplied in a single-use 50 mg vial as pembrolizumab lyophilized powder and as a single use 25 mg/mL ready-to-use solution containing 4 mL (100 mg/vial).
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13.3.3 reconstitution/dose preparation

The lyophilized powder is reconstituted with 2.3 mL Sterile Water for injection, USP, and the resultant concentration is 25 mg/mL. Diluent should be directed against the side of the vial to avoid excess foaming. Swirl contents gently until completely dissolved. Let contents sit for up to 5 minutes to allow all bubbles to clear. Do not shake. The ready-to-use solution or reconstituted lyophilized powder must be further diluted prior to administration. It may be diluted in 0.9% Sodium Chloride injection, USP or 5% Dextrose Injection, USP. The final concentration of the dilution solution should be between 1 mg/ml to 10 mg/ml.
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13.3.4 stability/storage

The reconstituted lyophilized power is stable for up to 6 hours at room temperature or up to 24 hours at when stored under refrigeration (2-8°C or 36-46°F). The diluted solution is stable for up to 6 hours at room temperature or up to 24 hours at when stored under refrigeration (2-8°C or 36-46°F).
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13.3.5 administration

Pembrolizumab will be given at a fixed dose of 200 mg as an intravenous infusion over 30 minutes through an intravenous line containing a sterile, non-pyrogenic, low-protein binding 0.2 micron to 5 micron in-line or add-on filter.
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13.3.6 toxicities

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