4.0 background & rationale
pbmcs

x Any blood remaining in the two 7 mL green-top tubes used to prepare plasma above will be diluted 1:1 with Hank's Balanced Salt Solution ("HBSS", Irvine Scientific, Cat. 9228 or equivalent) and combined with the whole blood from the unused green-top tube in a sterile 50 ml conical centrifuge tube. Peripheral blood mononuclear cells
(PBMC) will then be isolated from the combined whole blood sample by Ficollgradient separation as described below; o Allow Accuspin-Histopaque tubes ("Accuspin", Sigma Cat. A6929 or A0561, for 12 or 100 tubes, respectively) and HBSS to warm to room temperature. Place a Mr. Frosty container in the refrigerator and prepare the freezing media by adding 10% DMSO to fetal calf serum and chill at 4˚C or on ice.  
o Prepare Accuspin tubes by centrifuging at 1000 x g for 1 minute at room temperature (RT) with brakes on. Each tube can process up to 20 mLs of whole blood; prepare the appropriate amount of tubes necessary. After centrifugation, the Histopaque reagent should be below the barrier of the tube. Add 5 mLs of HBSS to the Accuspin tube. Add up to 20 mls of whole blood to each Accuspin tube until all the blood has been distributed.  
o Centrifuge the blood sample at 800 x g for 15 minutes at RT with brakes on LOW. After centrifugation, three layers should be visible above the barrier of the tube: the plasma layer at the top, a cloudy layer in the middle where the PBMC are, and a clear Histopaque reagent layer right below. Using a pipette, remove the upper plasma layer to within 2 cm of the cloudy interphase. Carefully pipette the cloudy PBMC interphase and transfer to a sterile 50 mL centrifuge tube.  
o Add HBSS up to the 45 mL mark in the centrifuge tube with the PBMC and spin at 400 x g for 10 minutes at RT with brakes on. Decant the supernatant and loosen the cell pellet before adding HBSS to the 45-mL mark again for a second wash. Centrifuge at 300 x g for 10 minutes at RT with brakes on. Decant the supernatant, loosen the cell pellet and then add a known volume of HBSS to resuspend the cells for counting. Mix the cell suspension up and down with a pipette several times before removing a small aliquot for cell count.  
Centrifuge the cell suspension one final time at 300 x g for 10 minutes at RT with brakes on.  
PBMC should be frozen down at 0.5 - 1 x 107 cells/vial. Determine the volume of freezing media (fetal calf serum with 10% DMSO) needed to give a 1 x 107 cell/mL suspension. After the last centrifugation is complete, discard supernatant and loosen the cell pellet before adding freezing media slowly, a small volume at a time with mixing in between (vortex at low speed). Aliquot 0.5 - 1 mL of the final cell suspension into individually labeled cryovials. Transfer the cryovials into Mr. Frosty and store at -80˚C. Twenty four hours later, cryovials will be transferred to liquid nitrogen tanks for long-term storage.
4.0 background & rationale
circulating t cell characterization

PD-1 expression on circulating PBMC T cells will be analyzed by flow cytometry. Immune cell populations will be further analyzed via high dimensional flow cytometry to measure levels of Tregs and MDSCs, which will allow for calculating the ratio of effector T cells to Tregs and MDSCs, respectively. Ex vivo isolated T cells will also be analyzed for proliferative capacity (CFSE dilution) and cytokine production (IFN-γ and IL-17) after stimulation with anti-CD3 and/or PMA and Ionomycin.
4.0 background & rationale
serum circulating mirna profiling

20 mL of whole blood will be collected from each patient into 2 Vacutainer red top tubes (no additive) at three timepoints: (1) Screening visit, (2) 21 days after completion of palliative radiotherapy, and (3) at the time of first progression by traditional RECIST1.1 (to capture potential pseudoprogression and only applicable for patients continued on treatment beyond progression), and at end of treatment. Blood will be allowed to clot on ice for 15-20 min, and then centrifuged at 1900g (3000 rpm) for 10 minutes at 4oC using a swinging bucket rotor.  
The upper (yellow) serum phase will then be transferred to four separate labeled cryovials without disturbing the intermediate buffy coat layer. The cryovials will be placed on ice, and then delivered immediately for storage in the Fox North Building, Room N2002, freezer EFRU385. Serum samples will be stored frozen at -80 oC until time of analysis.  
Total RNA will be extracted using Trizol within the Integrative Genomics Core at City of Hope for miRNA-seq. Sequence reads that match human miRNAs in Sanger mirBase will be calculated and analyzed. Circulating whole genome miRNA signatures will be compared to miRNAs sequenced from total RNA-seq from the corresponding tumor biopsy timepoints to aid in biomarker discovery of serum miRNAs that may predict for immunotherapy response or resistance. miR-23a will be of particular interest given its description as a gastric cancer oncomir and function as a repressor of T cell cytotoxicity in preclinical miRNA screens.14,15 miR-34a and miR-200 will be other miRNAs of interest to measure given reporting of their ability to suppress PD-L1 expression in other experimental systems.16,17
4.0 background & rationale
germline mirna biomarker analysis

Weidhass et al recently discovered that a new class of miRNA regulatory network disrupting germline mutations can act as biomarkers of response and toxicity to anti-PD-1 and anti-PDL1 therapy.18 Specifically a panel of germline mutations in 3' untranslated regions (3'-UTR)
and miRNA promoter regions provided 89% specificity for response and 76% specificity for toxicity in a population of 85 patients with melanoma and non-small cell lung cancer treated with anti-PD-1 or anti-PD-L1 agents. Leftover banked PBMC samples from any timepoint will be shipped batched via overnight courier on dry ice to: MiraDx Contact: Joanne Weidhaas, M.D., Ph.D 11600 Wilshire Blvd. Los Angeles, CA 90025 DNA extraction from PBMCs will be performed at MiraDx and targeted sequencing will be performed for the panel of miRNA germline mutation biomarkers. Results will be correlated with RECIST1.1 and irRECIST responses as well as toxicities recorded per NCI CTCAE.
4.0 background & rationale
7.1.2.7.3 correlative studies stool and

Patients will be provided a biospecimen bag and sterile swab (BD BBLTM CultureSwabTM
Sterile, Media-free Swabs) and instructed on proper collection from a used toilet tissue paper the required stool sample. Patients will be contacted and reminded by the clinical research coordinator 48-24 hours in advance of the specified protocol visit to perform the stool collection and will be advised to leave the specimen at room temperature until the day of their visit. Patients will also have the option of swabbing and submitting the stool specimen during their protocol visit provided they are reporting daily regular bowel movements. Samples submitted to the clinical research coordinator will be stored promptly and banked at -20 OC
until analysis. Samples will be labeled as "stool" and with the patient's deidentified protocol ID number and date of collection.
4.0 background & rationale
oral sampling

At the prespecified timepoints, patients will be asked during their protocol visit in clinic to perform an oral swab of both inner cheeks and tongue using a provided sterile swab (BD
BBLTM CultureSwabTM Sterile, Media-free Swabs). These samples will also be collected by the clinical research coordinator and will be stored promptly and banked at -20 OC until analysis. Samples will be labeled as "oral swab" and with the patient's deidentified protocol ID number and date of collection.
4.0 background & rationale
microbiomic profiling

Banked stool and oral swab specimens at the time of planned analysis will be shipped via courier overnight on dry ice to the lab of Dr. Greg Caporaso of Northern Arizona University/TGen: J. Gregory Caporaso, Ph.D. Northern Arizona University 1350 S Knoles Drive Flagstaff, AZ 86011, USA Microbial DNA will be extracted from swab samples using the PowerSoil® DNA Isolation Kit (Mobio Carlsbard, CA). A 16S rRNA library for the MiSeq Illumina platform will be prepared according to the protocol from the Earth Microbiome Project (http://www.earthmicrobiome.org/emp-standard-protocols/). Microbiome sequencing data to analyze species diversity will be subsequently analyzed using Quantitative Insights Into Microbial Ecology (QIIME) 1.9.1, biom-format version 2.1.5, VSEARCH version 1.7.0 (https://github.com/torognes/vsearch), SSU-ALIGN 0.1, and FastTree, as well as custom analytic software (source code at https://github.com/caporaso-lab/autism-fmt1) being prepared for release in QIIME 2. 7.1.3 **Laboratory Procedures/Assessments** Laboratory tests for hematology, chemistry, urinalysis, and others are specified in Table 5.  
| Table 5 Laboratory Tests  Hematology Chemistry                                                                                                                                                             | Urinalysis                    | Other                  |                                        |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------|------------------------|----------------------------------------|
| Hematocrit                                                                                                                                                                                                 | Albumin                       | Blood                  | Serum β-human chorionic  gonadotropin† |
| Hemoglobin                                                                                                                                                                                                 | Alkaline phosphatase          | Glucose                | (β-hCG)†                               |
| Platelet count                                                                                                                                                                                             | Alanine aminotransferase      | Protein                | PT (INR)                               |
| (ALT)                                                                                                                                                                                                      |                               |                        |                                        |
| WBC (total and                                                                                                                                                                                             | Aspartate                     | Specific gravity       | aPTT                                   |
| differential)                                                                                                                                                                                              | aminotransferase (AST)        |                        |                                        |
| Red Blood Cell Count                                                                                                                                                                                       | Lactate dehydrogenase         | Microscopic exam (If   | Total thriiodothyronine (T3)           |
| (LDH)                                                                                                                                                                                                      | abnormal)                     |                        |                                        |
| Absolute Neutrophil                                                                                                                                                                                        | Carbon Dioxide ‡              | results are noted      | Free tyroxine (T4)                     |
| Count Absolute Lymphocyte                                                                                                                                                                                  | (CO2 or biocarbonate)         | Urine pregnancy test † | Thyroid stimulating hormone            |
| Count                                                                                                                                                                                                      | (TSH)                         |                        |                                        |
| Uric Acid                                                                                                                                                                                                  | PK                            |                        |                                        |
| Calcium  Chloride                                                                                                                                                                                          | Blood for correlative studies |                        |                                        |
| Glucose  Phosphorus  Potassium  Sodium  Magnesium  Total Bilirubin  Direct Bilirubin (If total  bilirubin is elevated above  the upper limit of normal)  Total protein  Blood Urea Nitrogen                |                               |                        |                                        |
| † Perform on women of childbearing potential only. If urine pregnancy results cannot be confirmed as negative, a  serum pregnancy test will be required.  ‡ If considered standard of care in your region. |                               |                        |                                        |  
Laboratory tests for screening or entry into the Second Course Phase should be performed within 10 days prior to the first dose of treatment. After Cycle 1, pre-dose laboratory procedures can be conducted up to 72 hours prior to dosing. Results must be reviewed by the investigator or qualified designee and found to be acceptable prior to each dose of trial treatment.
4.0 background & rationale
7.1.4 other procedures 7.1.4.1 withdrawal/discontinuation

When a subject discontinues/withdraws prior to trial completion, all applicable activities scheduled for the final trial visit should be performed at the time of discontinuation. Any adverse events which are present at the time of discontinuation/withdrawal should be followed in accordance with the safety requirements outlined in Section 8.0 - Data and Safety Monitoring Plan. Subjects who a) attain a CR or b) complete 24 months of treatment with pembrolizumab may discontinue treatment with the option of restarting treatment if they meet the criteria specified in Section 7.1.5.5. After discontinuing treatment following assessment of CR, these subjects should return to the site for a Safety Follow-up Visit (described in Section 7.1.5.3.1) and then proceed to the Follow-Up Period of the study (described in Section 7.1.5.4).
4.0 background & rationale
7.1.5 visit requirements

Visit requirements are outlined in Section 6.0 - Study Calendar. Specific procedure-related details are provided in Section 7.1 - Trial Procedures.
4.0 background & rationale
7.1.5.1 screening

Within 21 days prior to start of treatment, potential subjects will be evaluated to determine that they fulfill the entry requirements as set forth in Section 5.1. Screening procedures may be repeated after consultation with the Sponsor. Results of a test performed prior to the subject signing consent as part of routine clinical management are acceptable in lieu of a screening test if performed within the specified time frame. Screening procedures are to be completed within 21 days prior to the first dose trial treatment except for the following: Laboratory tests and ECOG PS are to be performed within 10 days prior to the first dose of trial treatment. For women of reproductive potential, a urine pregnancy test will be performed within 72 hours prior to the first dose of trial treatment. A serum test may be considered if urine test is not appropriate or inconclusive. Subjects may be rescreened after initially failing to meet the inclusion/exclusion criteria. Results from assessments performed during the initial screening period are acceptable in lieu of a repeat screening test if performed within the specified time frame and the inclusion/exclusion criteria is met. Subjects who are rescreened will retain their original screening number.
4.0 background & rationale
7.1.5.2 treatment period

Visit requirements are outlined in Section 6.0 - Study Calendar. Specific procedure-related details are provided in Section 7.1 - Trial Procedures.
4.0 background & rationale
7.1.5.3 post-treatment visits 7.1.5.3.1 safety

The mandatory Safety Follow-Up Visit should be conducted approximately 30 days after the last dose of trial treatment or before the initiation of a new anti-cancer treatment, whichever comes first. All AEs that occur prior to the Safety Follow-Up Visit should be recorded. Subjects with an AE of Grade > 1 will be followed until the resolution of the AE to Grade 0-1 or until the beginning of a new anti-neoplastic therapy, whichever occurs first. SAEs that occur within 90 days of the end of treatment or before initiation of a new anti-cancer treatment should also be followed and recorded. Subjects who are eligible for retreatment with pembrolizumab (as described in Section 7.1.5.5) may have up to two safety follow-up visits, one after the Treatment Period and one after the Second Course Phase.
4.0 background & rationale
7.1.5.4 follow-up visits

Subjects who discontinue trial treatment for a reason other than disease progression will move into the Follow-Up Phase and should be assessed every 6 weeks (42 ± 7 days) by radiologic imaging to monitor disease status. After 1 year, the imaging time point will occur every 9 weeks (± 7 days). Every effort should be made to collect information regarding disease status until the start of new anti-neoplastic therapy, disease progression, death, end of the study or if the subject begins retreatment with pembrolizumab as detailed in Section 7.1.5.5. Information regarding post-study anti-neoplastic treatment will be collected if new treatment is initiated. Subjects who are eligible to receive retreatment with pembrolizumab according to the criteria in Section 7.1.5.5 will move from the follow-up phase to the Second Course Phase when they experience disease progression. Details are provided in Section 6.2 - Second Course Phase.
4.0 background & rationale
7.1.5.4.1 survival follow-up

Once a subject experiences confirmed disease progression or starts a new anti-cancer therapy, the subject moves into the survival follow-up phase and should be contacted by telephone every 12 weeks to assess for survival status until death, withdrawal of consent, or the end of the study, whichever occurs first.
4.0 background & rationale
7.1.5.5 second course phase (retreatment

Subjects who stop pembrolizumab with SD or better may be eligible for up to one year of additional pembrolizumab therapy if they progress after stopping study treatment. A repeat course of palliative radiotherapy to the primary tumor will not be given during this time. This retreatment is termed the Second Course Phase of this study and is only available if the study remains open and the subject meets the following conditions:
4.0 background & rationale
x either

o Stopped initial treatment with pembrolizumab after attaining an investigatordetermined confirmed CR according to RECIST 1.1, and  
Was treated for at least 24 weeks with pembrolizumab before discontinuing therapy Received at least two treatments with pembrolizumab beyond the date when the initial CR was declared
4.0 background & rationale
or

o Had SD, PR or CR and stopped pembrolizumab treatment after 24 months of study therapy for reasons other than disease progression or intolerability
4.0 background & rationale
and

x Experienced an investigator-determined confirmed radiographic disease progression after stopping their initial treatment with pembrolizumab x Did not receive any anti-cancer treatment since the last dose of pembrolizumab x Has a performance status of 0 or 1 on the ECOG Performance Scale x Demonstrates adequate organ function as detailed in Section 5.1.1 x Female subject of childbearing potential should have a negative urine or serum pregnancy test within 72 hours prior to receiving retreatment with study medication.  
x Female subject of childbearing potential should be willing to use 2 methods of birth control or be surgically sterile, or abstain from heterosexual activity for the course of the study through 120 days after the last dose of study medication (Reference Section 5.5.2). Subjects of child bearing potential are those who have not been surgically sterilized or have been free from menses for > 1 year.  
x Male subject should agree to use an adequate method of contraception starting with the first dose of study therapy through 120 days after the last dose of study therapy.  
x Does not have a history or current evidence of any condition, therapy, or laboratory abnormality that might interfere with the subject's participation for the full duration of the trial or is not in the best interest of the subject to participate, in the opinion of the treating investigator.
Subjects who restart treatment will be retreated at the same dose and dose interval as when they last received pembrolizumab. Patients will not receive another course of palliative radiotherapy to their primary tumor. Treatment will be administered for up to one additional year.  
Visit requirements are outlined in Section 6.2 - Second Course Phase.
4.0 background & rationale
8.0 data and safety monitoring

This is a Risk Level 4 study as defined in the City of Hope Institutional Data and Safety Monitoring Plan [policy dated 07/09/2014]. This determination was made because the study is an investigator-initiated phase II trial and involves a COH IND.
4.0 background & rationale
8.2 monitoring and personnel responsible

The Protocol Management Team (PMT) is responsible for monitoring the data and safety of this study. The PMT consists of the Principal Investigator (PI), Biostatistician, Research Protocol Nurse, and Clinical Research Coordinator. The PMT is required to submit periodic status reports (i.e., the PMT Report) according to the frequency prescribed in the City of Hope Institutional Data and Safety Monitoring Plan [policy dated 07/09/2014]. Important decisions made during PMT meetings (i.e., dose escalation, de-escalation, etc.) only need to be noted in the PMT Report submitted to the Data and Safety Monitoring Committee (DSMC).
4.0 background & rationale
8.3 adverse events and serious

The PI will be responsible for determining the event name, assessing the severity (i.e., grade), expectedness, and attribution of all adverse events. Adverse Event (AE) - An adverse event is any untoward medical experience or change of an existing condition that occurs during or after treatment, whether or not it is considered to be related to the protocol intervention. Reporting Non-serious Adverse Events - Adverse events will be collected after the patient is given the study treatment or any study related procedures. Adverse events will be monitored by the PMT. Adverse events that do not meet the criteria of serious OR are not unanticipated problems will be reported only in the PMT Report. Serious Adverse Event (SAE) [Modified from the definition of unexpected adverse drug experience in 21 CFR 312.32] - defined as any expected or *unexpected adverse event*s that result in any of the following outcomes:
x Death x Is life-threatening experience (places the subject at immediate risk of death from the event as it occurred)
x Unplanned hospitalization (equal to or greater than 24 hours) or prolongation of existing hospitalization x A persistent or significant disability/incapacity x A congenital anomaly/birth defect x Secondary malignancy x Any other adverse event that, based upon appropriate medical judgment, may jeopardize the subject's health and may require medical or surgical intervention to prevent one of the outcomes listed above (examples of such events include allergic bronchospasm requiring intensive treatment in the emergency room or at home, blood dyscrasias of convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse).  
Reporting Serious Adverse Events - begins after study treatment or any study related procedures. All SAEs occurring during this study, whether observed by the physician, nurse, or reported by the patient, will be reported according to the approved City of Hope's Institutional policy [policy effective date: 05/14/14]. Serious Adverse Events that require expedited reporting will be submitted electronically using iRIS.
4.0 background & rationale
adverse event name and severity

The PI will determine the adverse event name and severity (grade) by using the CTCAE version 4.0. Expected **Adverse Event** - Any event that does not meet the criteria for an unexpected event, OR is an expected natural progression of any underlying disease, disorder, condition, or predisposed risk factor of the research participant experiencing the adverse event. Unexpected **Adverse Event** [21 CFR 312.32 (a)] - An adverse event is unexpected if it is not listed in the investigator's brochure and/or package insert; is not listed at the specificity or severity that has been observed; is not consistent with the risk information described in the protocol and/or consent; is not an expected natural progression of any underlying disease, disorder, condition, or predisposed risk factor of the research participant experiencing the adverse event.
4.0 background & rationale
adverse event attribution

The following definitions will be used to determine the causality (attribution) of the event to the study agent or study procedure.  
Definite - The AE is clearly related to the investigational agent or study procedure and unrelated to any other cause. Probable - The AE is likely related to the investigational agent or study procedure and unlikely related to other cause(s). Possible -The AE may be related to the investigational agent or study procedure and may be related to another cause(s). Unlikely -The AE is doubtfully related to the investigational agent or study procedure and likely related to another cause(s). Unrelated -The AE is clearly not related to the investigational agent or study procedure and is attributable to another cause(s).
4.0 background & rationale
coh held ind

Serious Adverse Events meeting the requirements for expedited reporting to the Food and Drug Administration (FDA), as defined in 21 CFR 312.32, will be reported as an IND safety report using the MedWatch Form FDA 3500A for Mandatory Reporting. The criteria that require reporting using the Medwatch 3500A are:  
x Any unexpected fatal or life threatening adverse experience associated with use of the drug must be reported to the FDA no later than 7 calendar days after initial receipt of the information [21 CFR 312.32(c)(2)]
x Any adverse experience associated with use of the drug that is both serious and unexpected must be submitted no later than 15 calendar days after initial receipt of the information [21 CFR 312.32(c)(1)]
x Any follow-up information to a study report shall be reported as soon as the relevant information becomes available. [21 CFR 312.32(d)(3)]
The PI or designee will be responsible for contacting the Office of IND Development and Regulatory Affairs (OIDRA) at COH to ensure prompt reporting of safety reports to the FDA. OIDRA will assist the PI with the preparation of the report and submit the report to the FDA in accordance with the approved City of Hope's Institutional policy [policy effective date: 05/14/14].
4.0 background & rationale
8.4 deviations and unanticipated problems

Deviation - A deviation is a divergence from a specific element of a protocol that occurred without prior IRB approval. Investigators may deviate from the protocol to eliminate immediate hazard(s) for the protection, safety, and well-being of the study subjects without prior IRB approval. For any such deviation, the PI will notify the COH DSMC and IRB within 5 calendar days of its occurrence via iRIS in accordance with the Clinical Research Protocol Deviation policy [policy effective date: 11/07/11].
4.0 background & rationale
single subject exception (sse)

An SSE is a planned deviation, meaning that it involves circumstances in which the specific procedures called for in a protocol are not in the best interests of a specific patient. It is a deviation that is anticipated and receives prior approval by the PI and the IRB. The SSE must be submitted as a "Single Subject Exception Amendment Request" via iRIS in accordance with IRB guidelines and the Clinical Research Protocol Deviation policy [policy effective date: 11/07/11]. An IRB approved SSE does not need to be submitted as a deviation to the DSMC. Unanticipated Problem (UP) - Any incident, experience, or outcome that **meets all three** of the following criteria:
1. Unexpected (in terms of nature, severity, or frequency) given the following: a) the research procedures described in the protocol-related documents such as the IRB approved research protocol, informed consent document or Investigator Brochure (IB); and b) the characteristics of the subject population being studied; AND
2. Related or possibly related to participation in the research (possibly related means there is a reasonable possibility that the incident, experience, or outcomes may have been caused by the drugs, devices or procedures involved in the research); AND
3. Suggests that the research places subjects or others at greater risk of harm (including physical, psychological, economic, or social harm) than previously known or recognized.  
Any UP that occurs during study conduct will be reported to the DSMC and IRB in accordance with the City of Hope's Institutional policy [policy effective date: 05/14/14] using iRIS.
4.0 background & rationale
8.5 definition of an overdose

For purposes of this trial, an overdose of pembrolizumab will be defined as any dose of 1,000 mg or greater (≥5 times the indicated dose). No specific information is available on the treatment of overdose of pembrolizumab. Appropriate supportive treatment should be provided if clinically indicated. In the event of overdose, the subject should be observed closely for signs of toxicity. Appropriate supportive treatment should be provided if clinically indicated. If an adverse event(s) is associated with ("results from") the overdose of a Merck product, the adverse event(s) is reported as a serious adverse event, even if no other seriousness criteria are met. If a dose of Merck's product meeting the protocol definition of overdose is taken without any associated clinical symptoms or abnormal laboratory results, the overdose is reported as a non-serious Event of Clinical Interest (ECI), using the terminology "accidental or intentional overdose without adverse effect." All reports of overdose with and without an adverse event must be reported within 2 working days hours to Merck Global Safety. (Attn: Worldwide Product Safety; FAX 215 993-1220)
4.0 background & rationale
8.6 reporting of pregnancy and

Although pregnancy and lactation are not considered adverse events, it is the responsibility of investigators or their designees to report any pregnancy or lactation in a subject (spontaneously reported to them), including the pregnancy of a male subject's female partner that occurs during the trial or within 120 days of completing the trial completing the trial, or 30 days following cessation of treatment if the subject initiates new anticancer therapy, whichever is earlier. All subjects and female partners of male subjects who become pregnant must be followed to the completion/termination of the pregnancy. Pregnancy outcomes of spontaneous abortion, missed abortion, benign hydatidiform mole, blighted ovum, fetal death, intrauterine death, miscarriage and stillbirth must be reported as serious events (Important Medical Events). If the pregnancy continues to term, the outcome (health of infant) must also be reported. Such events must be reported within 2 working days to Merck Global Safety. (Attn: Worldwide Product Safety; FAX 215 993-1220)
4.0 background & rationale
8.7 reporting of adverse events

A serious adverse event is any adverse event occurring at any dose or during any use of Merck's product that:
x Results in death; x Is life threatening; x Results in persistent or significant disability/incapacity; x Results in or prolongs an existing inpatient hospitalization; x Is a congenital anomaly/birth defect; x Is a new cancer (that is not a condition of the study);
x Is associated with an overdose; x Is another important medical event Any serious adverse event, or follow up to a serious adverse event, including death due to any cause other than progression of the cancer under study that occurs to any subject from the time the consent is signed through 90 days following cessation of treatment, or the initiation of new anti-cancer therapy, whichever is earlier, whether or not related to Merck product, must be reported within 24 hours to Merck Global Safety. Non-serious Events of Clinical Interest will be forwarded to Merck Global Safety and will be handled in the same manner as SAEs. Additionally, any serious adverse event, considered by an investigator who is a qualified physician to be related to Merck product that is brought to the attention of the investigator at any time outside of the time period specified in the previous paragraph also must be reported immediately to the Sponsor and to Merck.
4.0 background & rationale
sae reports and any other

A copy of all 15 Day Reports and Annual Progress Reports is submitted as required by FDA, European Union (EU), Pharmaceutical and Medical Devices agency (PMDA) or other local regulators. Investigators will cross reference this submission according to local regulations to the Merck Investigational Compound Number (IND, CSA, etc.) at the time of submission. Additionally investigators will submit a copy of these reports to Merck & Co., Inc. (Attn: Worldwide Product Safety; FAX 215 993-1220) at the time of submission to FDA. All subjects with serious adverse events must be followed up for outcome.
4.0 background & rationale
9.0 statistical analysis plan 9.1

The primary objective of this study is to establish that the combination of pembrolizumab and a palliative course of traditional external beam multifractionated RT in patients with metastatic gastric, esophageal, and/or GEJ cancers will lead to an increase in tumor infiltrating cytotoxic T-cells and circulating cytotoxic T cells and a reduction in immunosuppressive Tregs and MDSCs in non-irradiated sites. Each patient will serve as their own control, and tumor cytotoxic T cell infiltration and immunosuppressive Tregs will be compared pre- and post-treatment. As variability of the difference in the T cell populations pre- and post-treatment is unknown and still being established, the power calculation is based on the concept of "effect size" in this biomarker analysis study. Specifically, a sample-size of 14 patients will have 80% power to detect an effect size of 0.71 using a paired t-test with a 0.05 one-sided significance level. The effect size is the mean difference divided by the standard deviation of the difference. A positive biological signal combined with acceptable secondary endpoints (response and toxicity) are sought for a clear indication of promise of this combination.
4.0 background & rationale
9.2 secondary objectives

Secondary objectives such as ORR per RECIST 1.1 and irRECIST, PFS, and OS are exploratory, though will also be documented, along with toxicity. All patients who receive at least one dose of pembrolizumab will be included in the safety analysis. Stool/oral microbiomic profiling and germline miRNA biomarker panel analyses will also be correlated with secondary outcome measures of ORR and toxicities. While overlapping toxicities are not anticipated with the combination of pembrolizumab and RT, the initial treatment of patients within this trial warrants a careful review of toxicity:
x Toxicity-based Interim Analysis: A study hold will be placed for re-evaluation at the proposed starting dose as accrual proceeds on this phase II study according to the following rule: The study will hold pending an amendment if 3 patients discontinue therapy after cycle 1 due to treatment-related toxicity. This rule has a 97% chance of holding the study if the true discontinuation rate due to toxicity on cycle 1 is 50%. It has a 15% chance of holding if the true discontinuation rate on cycle 1 is 15%.  
x Treatment-related mortality stopping rule: Any treatment-related death will hold the study accrual pending review by the PI, Sponsor, and DSMB/IRB.  
With 14 patients, there is 96% power to detect at least one responder if the true response rate is 21% or higher, which is in the range of 22% in the phase I trial of pembrolizumab among gastric cancer patients with PD-L1 overexpression. As a result, failure to observe any response in the 14 patients would result in lack of enthusiasm for the combination of pembrolizumab and RT in this patient population. PFS and OS will be determined using the Kaplan-Meier method.
4.0 background & rationale
10.0 labeling packaging storage and

The investigator shall take responsibility for and shall take all steps to maintain appropriate records and ensure appropriate supply, storage, handling, distribution and usage of investigational product in accordance with the protocol and any applicable laws and regulations. Clinical Supplies will be provided by Merck as summarized in Table 7.  
| Product Name & Potency    | Dosage Form                      |
|---------------------------|----------------------------------|
| Pembrolizumab 50 mg       | Lyophilized Powder for Injection |
| Pembrolizumab 100 mg/ 4mL | Solution for Injection           |
4.0 background & rationale
10.2 packaging and labeling information

Clinical supplies will be affixed with a clinical label in accordance with regulatory requirements.
4.0 background & rationale
10.3 clinical supplies disclosure

This trial is open-label; therefore, the subject, the trial site personnel, the Sponsor and/or designee are not blinded to treatment. Drug identity (name, strength) is included in the label text; random code/disclosure envelopes or lists are not provided.
4.0 background & rationale
10.4 storage and handling requirements

Clinical supplies must be stored in a secure, limited-access location under the storage conditions specified on the label. Receipt and dispensing of trial medication must be recorded by an authorized person at the trial site. Clinical supplies may not be used for any purpose other than that stated in the protocol.
4.0 background & rationale
10.5 returns and reconciliation

The investigator is responsible for keeping accurate records of the clinical supplies received from Merck or designee, the amount dispensed to subjects, and the amount remaining at the conclusion of the trial. Upon completion or termination of the study, all unused and/or partially used investigational product will be destroyed at the site per institutional policy. It is the Investigator's responsibility to arrange for disposal of all empty containers, provided that procedures for proper disposal have been established according to applicable federal, state, local and institutional guidelines and procedures, and provided that appropriate records of disposal are kept.
4.0 background & rationale
11.0 data reporting 11.1 data

The original data collection forms will be sent to the CTO and stored in a locked area. This protocol uses Electronic Data Collection per institutional policy for investigator-initiated trials, and the data will be stored in encrypted, password protected, secure computers that meet all HIPAA requirements. When results of this study are reported in medical journals or at meetings, identification of those taking part will not be disclosed. Medical records of subjects will be securely maintained in the strictest confidence, according to current legal requirements. They will be made available for review, as required by the FDA, HHS, or other authorized users such as the NCI, under the guidelines established by the Federal Privacy Act and rules for the protection of human subjects.
4.0 background & rationale
11.1.2 subject consent form

At the time of registration, the original signed and dated Informed Consent form, HIPAA research authorization form, and the California Experimental Subject's Bill of Rights (for the medical record) and three copies (for the subject, the research record, and the Coordinating Center) must be available. All Institutional, NCI, Federal, and State of California requirements will be fulfilled.
4.0 background & rationale
11.1.3 data collection forms and

All data will be collected 2 weeks prior using MediData Rave. Data will be sent to the location identified in Section 11.1.1 and stored in a secure location.
4.0 background & rationale
11.1.3.1 eligibility checklist

The Eligibility Checklist must be completed by a protocol nurse or clinical research associate and signed by an authorized investigator prior to registering the subject.
4.0 background & rationale
11.1.3.2 prior therapy forms and

Within 2 weeks of registration, the clinical research associate will submit data in regards to prior therapies the patient has received, if applicable.
4.0 background & rationale
12.0 human subject issues 12.1

In accordance with City of Hope policies, an Institutional Review Board (IRB) that complies with the federal regulations at 45 CFR 46 and 21 CFR 50, 56 and State of California Health and Safety code, Title 17, must review and approve this protocol and the informed consent form prior to initiation of the study. All institutional, NCI, Federal, and State of California regulations must be fulfilled. Any documents that the IRB may need to fulfill its responsibilities (such as protocol, protocol amendments, Investigator's Brochure, consent forms, information concerning patient recruitment, payment or compensation procedures, or other pertinent information) will be submitted to the IRB. The IRB's written unconditional approval of the study protocol and the informed consent document will be in the possession of the investigator before the study is initiated. The IRB will be informed of revisions to other documents originally submitted for review; serious unexpected or unanticipated adverse experiences occurring during the study, and any additional adverse experiences in accordance with the standard operating procedures and policies of the IRB; new information that may affect adversely the safety of the patients of the conduct of the study; an annual update and/or request for re-approval; and when the study has been completed. Any amendment to the protocol document and accompanying informed consent document/template, as developed and provided by the PI, will require review and approval by the COH IRB before the changes are implemented in the study.
4.0 background & rationale
12.2 recruitment of subjects

The Gastrointestinal Medical Oncology Division will identify patients undergoing treatment for metastatic gastroesophageal cancer at City of Hope.
4.0 background & rationale
12.4 confidentiality

Participant confidentiality is strictly held in trust by the investigators, study staff, and the sponsor(s) and their agents. This confidentiality is extended to cover testing of biological samples in addition to any study information relating to participants. This research will be conducted in compliance with federal and state requirements relating to protected health information (PHI), including the requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA). HIPAA regulations require a signed participant authorization informing the participant of the nature of the PHI to be collected, who will have access to that information and why, who will use or disclose that information, ant the rights of a research participant to revoke their authorization for use of their PHI. In the event that a participant revokes authorization to collect or use PHI, the investigator, by regulation, retains the ability to use all information collected prior to the revocation of participant authorization. For participants that have revoked authorization to collect or use PHI, attempts should be made to obtain permission to collect at least vital status (i.e. that the participant is alive) at the end of their scheduled study period. Release of research results should preserve the privacy of medical information and must be carried out in accordance with Department of Health and Human Services Standards for Privacy of Individually Identifiable Health Information, 45 CFR 164.508. When results of this study are reported in medical journals or at meetings, identification of those taking part will not be disclosed and no identifiers will be used. Medical records of participants will be securely maintained in the strictest confidence, according to current legal requirements. Data will be entered, analyzed and stored in encrypted, password protected, secure computers that meet all HIPAA requirements. All data capture records, drug accountability records, study reports and communications will identify the patient by initials and the assigned patient number. Source documents provided to coordinating center for the purpose of auditing or monitoring will be de-identified and labeled with the study number, participant ID, and participant initials. The investigator/institution will permit direct access to source data and documents by sponsor representatives, the FDA, and other applicable regulatory authorities. The access may consist of trial-related monitoring/ auditing, IRB reviews, and FDA/regulatory authority inspections. The participant's confidentiality will be maintained and will not be made publicly available to the extent permitted by the applicable laws and regulations.
4.0 background & rationale
12.5 financial obligations and compensation

The investigational drug, pembrolizumab, will be provided free of charge by City of Hope and Merck. Should this drug or device become commercially available during the course of treatment, the research participant and/or the insurance carrier may be asked to pay for the costs of the drug. The standard of care drug(s) and procedures provided will be the responsibility of the research participant and/or the insurance carrier. The research participant will be responsible for all copayments, deductibles, and other costs of treatment and diagnostic procedures as set forth by the insurance carrier. The research participant and/or the insurance carrier will be billed for the costs of treatment and diagnostic procedures in the same way as if the research participant were not in a research study. However, neither the research participant nor the insurance carrier will be responsible for the research procedures related to this study. In the event of physical injury to a research participant, resulting from research procedures, appropriate medical treatment will be available at the City of Hope to the injured research participant, however, financial compensation will not be available. The research participant will not be paid for taking part in this study.
4.0 background & rationale
12.6 informed consent processes

The Principal Investigator or IRB approved named designate will explain the nature, duration, purpose of the study, potential risks, alternatives and potential benefits, and all other information contained in the informed consent document. In addition, they will review the experimental subject's bill of rights and the HIPAA research authorization form. Prospective participants will be informed that they may withdraw from the study at any time and for any reason without prejudice, including as applicable, their current or future care or employment at City of Hope or any relationship they have with City of Hope. Prospective participants will be afforded sufficient time to consider whether or not to participate in the research. After the study has been fully explained, written informed consent will be obtained from either the prospective participant or his/her guardian or legal representative before study participation. The method of obtaining and documenting the informed consent and the contents of the consent must comply with the ICH-GCP and all applicable regulatory requirements. Before implementing any study procedure, informed consent shall be documented by the use of a written consent form approved by the IRB and signed and dated by the prospective participant or his/her legally authorized representative at the time of consent. A copy of the signed informed consent will be given to the participant or his/her legally authorized representative. The original signed consent must be maintained by the investigator and available for inspection sponsor designated representatives, or regulatory authority at any time. Informed consent is a process that is initiated prior to the individual agreeing to participate in the study and continues throughout study participation. REFERENCES 1. Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet 2010;376:687-97. 2. Wilke H, Muro K, Van Cutsem E, et al. Ramucirumab plus paclitaxel versus placebo plus paclitaxel in patients with previously treated advanced gastric or gastro-oesophageal junction adenocarcinoma (RAINBOW): a double-blind, randomised phase 3 trial. The Lancet Oncology 2014;15:1224-35. 3. Cunningham D, Starling N, Rao S, et al. Capecitabine and oxaliplatin for advanced esophagogastric cancer. N Engl J Med 2008;358:36-46. 4. Muro K, Bang Y-J, Shankaran V, et al. Relationship between PD-L1 expression and clinical outcomes in patients (Pts) with advanced gastric cancer treated with the anti-PD-1 monoclonal antibody pembrolizumab (Pembro; MK-3475) in KEYNOTE-012. ASCO Meeting Abstracts 2015;33:3. 5. Doi T, Piha-Paul SA, Jalal SI, et al. Pembrolizumab (MK-3475) for patients (pts) with advanced esophageal carcinoma: Preliminary results from KEYNOTE-028. ASCO Meeting Abstracts 2015;33:4010. 6. Carbognin L, Pilotto S, Milella M, et al. Differential Activity of Nivolumab, Pembrolizumab and MPDL3280A according to the Tumor Expression of Programmed DeathLigand-1 (PD-L1): Sensitivity Analysis of Trials in Melanoma, Lung and Genitourinary Cancers. PLoS ONE 2015;10:e0130142. 7. Demaria S, Ng B, Devitt ML, et al. Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated. International journal of radiation oncology, biology, physics 2004;58:862-70. 8. Park SS, Dong H, Liu X, et al. PD-1 Restrains Radiotherapy-Induced Abscopal Effect. Cancer Immunol Res 2015. 9. Dovedi SJ, Adlard AL, Lipowska-Bhalla G, et al. Acquired Resistance to Fractionated Radiotherapy Can Be Overcome by Concurrent PD-L1 Blockade. Cancer Research 2014;74:5458-68. 10. Twyman-Saint Victor C, Rech AJ, Maity A, et al. Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer. Nature 2015;520:373-7. 11. Snyder A, Makarov V, Merghoub T, et al. Genetic Basis for Clinical Response to CTLA-4 Blockade in Melanoma. New England Journal of Medicine 2014;371:2189-99. 12. Lin D-C, Hao J-J, Nagata Y, et al. Genomic and molecular characterization of esophageal squamous cell carcinoma. Nat Genet 2014;46:467-73. 13. The Cancer Genome Atlas Research N. Comprehensive molecular characterization of gastric adenocarcinoma. Nature 2014;513:202-9. 14. Ma G, Dai W, Sang A, Yang X, Gao C. Upregulation of microRNA-23a/b promotes tumor progression and confers poor prognosis in patients with gastric cancer. International journal of clinical and experimental pathology 2014;7:8833-40. 15. Lin R, Chen L, Chen G, et al. Targeting miR-23a in CD8+ cytotoxic T lymphocytes prevents tumor-dependent immunosuppression. The Journal of clinical investigation 2014;124:5352-67. 16. Wang X, Li J, Dong K, et al. Tumor suppressor miR-34a targets PD-L1 and functions as a potential immunotherapeutic target in acute myeloid leukemia. Cellular Signalling 2015;27:443-52.  
17. Chen L, Gibbons DL, Goswami S, et al. Metastasis is regulated via microRNA200/ZEB1 axis control of tumour cell PD-L1 expression and intratumoral immunosuppression.  
Nat Commun 2014;5. 18. Weidhaas JB, Telesca D, Kalbasi A, Salzman D, Ribas A. Germ-line biomarkers disrupting microRNA regulatory pathways to predict toxicity and response to anti-PD-1 and anti-PD-L1 therapies. Journal of Clinical Oncology 2017;35:3040-.
4.0 background & rationale
appendix 1 ecog perfomance status

| Grade                                                                                                                                                                                                                                                                                                                        | Description                                                                                                                                                                                      |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 0                                                                                                                                                                                                                                                                                                                            | Normal activity. Fully active, able to carry on all pre-disease  performance without restriction.  Symptoms, but ambulatory. Restricted in physically strenuous                                  |
| 1                                                                                                                                                                                                                                                                                                                            | activity, but ambulatory and able to carry out work of a light or  sedentary nature (e.g., light housework, office work).  In bed <50% of the time. Ambulatory and capable of all self-care, but |
| 2                                                                                                                                                                                                                                                                                                                            | unable to carry out any work activities. Up and about more than 50%  of waking hours.                                                                                                            |
| 3                                                                                                                                                                                                                                                                                                                            | In bed >50% of the time. Capable of only limited self-care, confined  to bed or chair more than 50% of waking hours.                                                                             |
| 4                                                                                                                                                                                                                                                                                                                            | 100% bedridden. Completely disabled. Cannot carry on any self-care.  Totally confined to bed or chair.                                                                                           |
| 5                                                                                                                                                                                                                                                                                                                            | Dead.                                                                                                                                                                                            |
| * As published in Am. J. Clin. Oncol.: Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E.,  McFadden, E.T., Carbone, P.P.: Toxicity And Response Criteria Of The Eastern Cooperative Oncology  Group. Am J Clin Oncol 5:649-655, 1982. The Eastern Cooperative Oncology Group, Robert Comis  M.D., Group Chair. |                                                                                                                                                                                                  |
4.0 background & rationale
appendix 2 response evaluation criteria

RECIST version 1.1* will be used in this study for assessment of tumor response. While either CT or MRI may be utilized, as per RECIST 1.1, CT is the preferred imaging technique in this study. * As published in the European Journal of Cancer: E.A. Eisenhauer, P. Therasse, J. Bogaerts, L.H. Schwartz, D. Sargent, R. Ford, J. Dancey, S. Arbuck, S. Gwyther, M. Mooney, L. Rubinstein, L. Shankar, L. Dodd, R. Kaplan, D. Lacombe, J. Verweij. New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1). Eur J Cancer. 2009 Jan;45(2):228-47. irRECIST** will be utilized to account for the unique tumor responses associated with immunotherapeutics. **As published in Clinical Cancer Research:
Nishino M, Giobbie-Hurder A, Gargano M, Suda M, Ramaiya NH, Hodi FS.  
Developing a common language for tumor response to immunotherapy: immune-related response criteria using unidimensional measurements. Clin Cancer Res. 2013 Jul 15;19(14):3936-43. Epub 2013 Jun 6.
4.0 background & rationale
appendix 3 events of clinical

The most recent version (5.0, 12/18/2014) of the Events of Clinical Interest (ECI) Guidance Document provides study sites with further guidance on the identification and management of ECI for the pembrolizumab program and reporting guidelines to Merck.
4.0 background & rationale
appendix 1 ecog perfomance status

| Grade                                                                                                                                                                                                                                                                                                                        | Description                                                                                                                                                                                      |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 0                                                                                                                                                                                                                                                                                                                            | Normal activity. Fully active, able to carry on all pre-disease  performance without restriction.  Symptoms, but ambulatory. Restricted in physically strenuous                                  |
| 1                                                                                                                                                                                                                                                                                                                            | activity, but ambulatory and able to carry out work of a light or  sedentary nature (e.g., light housework, office work).  In bed <50% of the time. Ambulatory and capable of all self-care, but |
| 2                                                                                                                                                                                                                                                                                                                            | unable to carry out any work activities. Up and about more than 50%  of waking hours.                                                                                                            |
| 3                                                                                                                                                                                                                                                                                                                            | In bed >50% of the time. Capable of only limited self-care, confined  to bed or chair more than 50% of waking hours.                                                                             |
| 4                                                                                                                                                                                                                                                                                                                            | 100% bedridden. Completely disabled. Cannot carry on any self-care.  Totally confined to bed or chair.                                                                                           |
| 5                                                                                                                                                                                                                                                                                                                            | Dead.                                                                                                                                                                                            |
| * As published in Am. J. Clin. Oncol.: Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E.,  McFadden, E.T., Carbone, P.P.: Toxicity And Response Criteria Of The Eastern Cooperative Oncology  Group. Am J Clin Oncol 5:649-655, 1982. The Eastern Cooperative Oncology Group, Robert Comis  M.D., Group Chair. |                                                                                                                                                                                                  |
4.0 background & rationale
appendix 2 response evaluation criteria

RECIST version 1.1* will be used in this study for assessment of tumor response. While either CT or MRI may be utilized, as per RECIST 1.1, CT is the preferred imaging technique in this study. * As published in the European Journal of Cancer: E.A. Eisenhauer, P. Therasse, J. Bogaerts, L.H. Schwartz, D. Sargent, R. Ford, J. Dancey, S. Arbuck, S. Gwyther, M. Mooney, L. Rubinstein, L. Shankar, L. Dodd, R. Kaplan, D. Lacombe, J. Verweij. New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1). Eur J Cancer. 2009 Jan;45(2):228-47. irRECIST** will be utilized to account for the unique tumor responses associated with immunotherapeutics. **As published in Clinical Cancer Research:
Nishino M, Giobbie-Hurder A, Gargano M, Suda M, Ramaiya NH, Hodi FS.  
Developing a common language for tumor response to immunotherapy: immune-related response criteria using unidimensional measurements. Clin Cancer Res. 2013 Jul 15;19(14):3936-43. Epub 2013 Jun 6.
4.0 background & rationale
appendix 3 events of clinical

The most recent version (5.0, 12/18/2014) of the Events of Clinical Interest (ECI) Guidance Document provides study sites with further guidance on the identification and management of ECI for the pembrolizumab program and reporting guidelines to Merck.