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5 correlative studies for research

A description of correlative studies including a brief statement of rationale and processing information is made below.  
| Sample                                                     |                               |                                  |                               |
|------------------------------------------------------------|-------------------------------|----------------------------------|-------------------------------|
| Test/assay                                                 | volume  (approx.)             | Location of  specimen  analysis1 |                               |
| Type of tubea /  tissue sample                             | Collection point              |                                  |                               |
| Immunemonitoring by  FACS                                                            | Blood  Processing Core  (BPC) |                                  |                               |
| Blood, 120                                                 | EDTA (Purple                  | See Study                        |                               |
| mL                                                         | top tubes)                    | Calendar 3.4                     |                               |
| 2 green top  tube (GTT) or  CPT tubes with  sodium citrate | Blood  Processing Core  (BPC) |                                  |                               |
| Serum cytokines  by ELISA                                  | 2mL                           | See Study  Calendar 3.4          |                               |
| Sodium heparin                                             |                               |                                  |                               |
| Tadalafil PK                                               | 4 mL                          | green top tube  (GTT)            | Blood  Processing Core  (BPC) |
| See Study  Calendar 3.4                                    |                               |                                  |                               |  
| Version Date: 01/19/2022 Sample  Test/assay volume  (approx.)   | Location of  specimen  analysis1                                                                                                     |                               |                                          |            |
|-----------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------|-------------------------------|------------------------------------------|------------|
| Type of tubea /  tissue sample                                  | Collection point                                                                                                                     |                               |                                          |            |
| Vancomycin                                                      | Sodium heparin                                                                                                                       |                               |                                          |            |
| Pharmacokinetic                                                 | 4 mL                                                                                                                                 | green top tube                |                                          |            |
| Analysis                                                        | (GTT)                                                                                                                                | Blood  Processing Core  (BPC) |                                          |            |
| See Study  Calendar 3.4                                         |                                                                                                                                      |                               |                                          |            |
| Mixed meal test                                                 | Sodium heparin                                                                                                                       |                               |                                          |            |
| for bile acid levels                                            | 5 mL                                                                                                                                 | green top tube                |                                          |            |
| in blood                                                        | (GTT)                                                                                                                                | Blood  Processing Core  (BPC) |                                          |            |
| See Study  Calendar 3.4                                         |                                                                                                                                      |                               |                                          |            |
| IHC for immune  cell infiltration                               | Tumor sample                                                                                                                         | At time of                    | Laboratory of                            |            |
| biopsies                                                        | Pathology                                                                                                                            |                               |                                          |            |
| Proteomics                                                      | Tumor sample                                                                                                                         | At time of  biopsies          | Greten Lab                               |            |
| Metabolomics                                                    | Tumor sample                                                                                                                         | At time of  biopsies          | Greten Lab Blood  Processing Core  (BPC) |            |
| TCRbeta  sequencing                                             | 10 mL                                                                                                                                | EDTA (Purple                  | See Study                                |            |
| top tube)                                                       | Calendar 3.4                                                                                                                         |                               |                                          |            |
| TCRbeta  sequencing                                             | Tumor sample                                                                                                                         | At time of  biopsies          | Greten Lab                               |            |
| RNA seq                                                         | Tumor sample                                                                                                                         | At time of  biopsies          | Greten Lab                               |            |
| Whole  exome/panel                                              | Tumor sample                                                                                                                         | At time of  biopsies          | Greten Lab                               |            |
| Whole  exome/panel                                              | Blood, 2.5 mL                                                                                                                        | 1 PAX gene                    | See Study  Calendar 3.4                  | Greten Lab |
| DNA tube Feccontainer  tubes or rectal  swabs                   |                                                                                                                                      |                               |                                          |            |
| Fecal bile acid                                                 | Stool sample                                                                                                                         |                               |                                          |            |
| composition                                                     | or rectal swab                                                                                                                       | See Study  Calendar 3.4       | Greten Lab                               |            |
| Vancomycinresistant  enterococci (VRE)  development                                                                 | NIH  Department of  Laboratory  Medicine                                                                                             |                               |                                          |            |
| Feccontainer  tubes or rectal  swabs                            |                                                                                                                                      |                               |                                          |            |
| Stool sample  or rectal swab                                    | See Study  Calendar 3.4                                                                                                              |                               |                                          |            |
| DNA sequencing                                                  | Stool sample                                                                                                                         | Feccontainer                  | See Study  Calendar 3.4                  | Greten Lab |
| of the microbiome                                               | or rectal swab                                                                                                                       | tubes or rectal               |                                          |            |
| a.                                                              | Please note that tubes and media may be substituted based on availability with the  permission of the PI or laboratory investigator. |                               |                                          |            |
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5.2 correlative studies of blood

Blood samples will be collected at time points indicated in the Study Calendar 3.4. Blood samples will be initially sent to the Blood Processing Core (BPC) for barcoding and storage. On certain occasions, the blood may also be brought to the Greten lab for processing and analysis
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5.2.1 immune monitoring

We will analyze PBMC for quantitative and functional changes of effector cells by FACS. The effect on (i) CD4 T cell number and activity, (ii) CD8 T cell number and activity, (iii) NK cell number and activity, (iv) Treg number, (v) MDSC: frequency + functional assay, and (vi) the detection of tumor-associated antigens using tetramer assay. These experiments will be done in Blood Processing Core (BPC).
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5.2.2 serum cytokines

We will analyze cytokine profile from sera for cytokines and chemokines by ELISA, that are associated with treatment efficacy and outcome. These experiments will be done in Blood Processing Core (BPC).
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5.2.3 tadalafil pharmacokinetic analysis

Blood samples for the determination of tadalafil plasma trough levels will be obtained from participating patients at time points indicated in Study Calendar 3.4. Bioanalytical measurements will be conducted on an ultra HPLC-MS/MS system by the Clinical Pharmacology Program (CPP)
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5.2.4 vancomycin pharmacokinetic analysis

Blood samples for the determination of vancomycin plasma trough levels will be obtained from participating patients at time points indicated in Study Calendar 3.4. Bioanalytical measurements will be conducted on an ultra HPLC-MS/MS system by the Clinical Pharmacology Program (CPP)
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5.2.5 bile acid analysis

Fasting blood samples will be drawn. At T = 0 min the participants ingest a liquid meal containing 25% of total daily energy expenditure (Boost Plus Vanilla; 1.5 kcal/mL; 49% carbohydrates, 35% fat and 16% protein). Participants are asked to drink the liquid meal within 10 min. Blood samples for determination of plasma bile acid levels will be drawn at time points indicated in Study Calendar **3.4 and Appendix I**. The blood samples will be collected on baseline and ten days after oral vancomycin is taken during the first treatment cycle. Bile acid profile is measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) as described[91]. Moreover, fecal bile acid composition was determined from rectal swab at baseline and 10 days after oral vancomycin treatment, using gas chromatography (GC). The total amount of primary and secondary bile salts is calculated as the sum of the individually quantified bile salts. AUC will be calculated. These experiments will be done in Dr. Figg lab.
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5.2.6 food diary for mixed

It has been well demonstrated the diet has great impact on gut microbiota. Therefore, a food diary for a mixed meal study (**Appendix H**) will be used in this study to assess the dietary composition, in addition to nutrition consult. A specialist will evaluate the diary in detail. The completion of this diary will take 15-30 minutes with the time of consultation included. Patients will have nutrition consult and complete the diary on the same days as bile acid analysis (See Study Calendar 3.4.) Non-English-speaking subjects will complete this instrument with help of an interpreter.
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5.3 tumor studies (non-genetic analysis)

Mandatory tumor biopsies will be performed at baseline and anytime during week 3 of Cycle 2 while patient is still on oral vancomycin. If patient disease progresses before week 3 of Cycle 2, post-treatment biopsy may be performed per PI discretion at the time of progression. It is preferred that at **least two core biopsies ≥18 gauge in diameter and ≥1 cm** in length will be obtained. The use of imaging to facilitate biopsies will be decided by members of the Interventional Radiology team and may include ultrasound, CT scan, PET scan or MRI.  
Tumor biopsies are mandatory on this study, however, biopsies and local anesthesia will be administered only if they are of minimal risk to the participant, as determined by the investigators and Interventional Radiology. If these procedures are not considered to be of minimal risk to the participant, they will not be done. Patient will continue treatment.
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5.3.1 tumor tissue analysis 5.3.1.1

IHC will be performed on tumor tissue for assessment of immune cell infiltration (e.g. CD3+ CD4/8 cells, PDL1 etc.). Immunohistochemistry will be performed in the Laboratory of Pathology under the direction of Dr. David Kleiner.
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5.3.1.2 omics study

Tumor samples will be collected for proteomics and metabolomics studies to investigate the differential expression of protein and metabolite that might be associated with the treatment outcome or efficacy. These experiments will be done in Dr. Greten lab.
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5.3.2 stool collection and processing

Stool samples or rectal swabs will be collected by the patients at time points indicated in the Study Calendar 3.4. Specimens will be frozen and kept at -20°C until analysis, which will be done in batches. Samples will be collected for bile acid composition (Section **5.2.5**), Vancomycin-resistant enterococci (VRE) development DNA and sequencing of the microbiome (Section **5.5.3**).
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5.3.2.1 vancomycin-resistant enterococci (vre) development

Stool specimen will be collected to evaluate VRE development given prolonged vancomycin exposure increase the odds of VRE. The protocol will evaluate VRE incidence as one of safety monitor.
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5.4 sample storage tracking and

Samples will be ordered in CRIS and tracked through a Clinical Trial Data Management system. Should a CRIS screen not be available, the CRIS downtime procedures will be followed. All samples will be sent to Blood Processing Core (BPC) for processing and storage until they are distributed to the designated place of analysis as described in the protocol. Samples will not be sent outside NIH without appropriate approvals and/or agreements, if required. All samples will be barcoded, with data entered and stored in the secure databases. These databases create a unique barcode ID for every sample and sample box, which cannot be traced back to patients without database 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.).  
Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -800C according to stability requirements. 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 database. 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. 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.
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5.4.1 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, contact NCIBloodcore@mail.nih.gov. 5.4.2 Samples Managed by the Laboratory of Dr. Tim Greten Contact information:
Sophie Wang Building 10 Rm 3B44 Phone: 240-858-3218 E-mail: sophie.wang@nih.gov
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5.4.3 protocol completion/sample destruction

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 in sections above. The study will remain open so long as sample or data analysis continues. Samples from consenting subjects will be stored until they are no longer of scientific value or if a subject withdraws consent for their continued use, at which time they will be destroyed. If the 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. Reporting will be per the requirements of section 7.2.
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5.5 samples for genetic/genomic analysis

PBMC and tumor samples will be collected for TCR beta sequencing performed in Blood Processing Core (BPC) and Dr. Greten' lab to analyze the T cell response.
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5.5.2 whole exome/panel (tumor/normal) and

Whole exome will be done on tumor and blood sample. RNAseq will be done on tumor for expressed somatic mutations and neoantigen discovery. Tumor material for these analyses will be sent to the laboratory of Dr Greten. Specifically, the expression of different signaling pathway, including interferon gamma signaling, immune cell functional molecular, e.g. perforin and granzyme, will be investigated
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5.5.3 microbial dna sequence

Samples sequenced to study microbial communities will contain human DNA sequences. However, bioinformatic analyses of the sequences will remove sequences containing significant alignment to publicly available human DNA sequences. These data will be coded and stored at the NIH Intramural Sequencing Center (NISC) per standard protocols for intramural sequencing. The resulting sequences will be analyzed. Publicly available human DNA sequences likely do not include all possible DNA sequences and could result in fragments of human DNA sequences being incorporated into the microbial DNA analysis. Microbial DNA sequence from this study will be published and posted on the web. Coded information obtained from sequencing samples will be placed into an open access (public) scientific database such as Genbank: http://www.ncbi.nlm.nih.gov/Genbank/. For example, we will deposit 16S rRNA gene sequences in Genbank. Microbial sequencing is also deposited in the *Short Read Trace Archive* that is a component of Genbank. All of the data will be coded to minimize any risk of identifying an individual. Samples will be reported without any unique identifiable features of the subject.
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5.5.4 management of results

Subjects will be contacted if a clinically actionable gene variant is discovered. Clinically actionable findings for the purpose of this study are defined as disorders appearing in the American College of Medical Genetics and Genomics recommendations for the return of incidental findings that is current at the time of primary analysis. (A list of current guidelines is maintained on the CCR intranet: https://ccrod.cancer.gov/confluence/display/CCRCRO/Incidental+Findings+Lists. Subjects will be contacted at that time with a request to provide a blood sample to be sent to a CLIA certified laboratory.
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5.5.5 genetic counseling

If the research findings are verified in the CLIA certified lab, the subject will be offered the opportunity to come to NIH (at our expense) to have genetic education and counseling with the NCI Genetics Branch to explain this result. If the subject does not want to come to NIH, a referral to a local genetic healthcare provider will be provided (at their expense). This is the only time during the course of the study that incidental findings will be returned. No interrogations regarding clinically actionable findings will be made after the primary analysis.
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6 data collection and evaluation

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. 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 of cycle 1 through 90 days after the study agent (s) was/were administered. Beyond 90 days after the last intervention, only adverse events which are serious and related to the study intervention need to be recorded.  
An abnormal laboratory value will be recorded in the database as an AE only if the laboratory abnormality is characterized by any of the following: - Results in discontinuation from the study  
- Is associated with clinical signs or symptoms - Requires treatment or any other therapeutic intervention - Is associated with death or another serious adverse event, including hospitalization. - Is judged by the Investigator to be of significant clinical impact
If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the patient's outcome. Adverse Events of grade 1 will not be collected. The results of the full diagnostic workup, blood and sputum culture, hematological parameters etc. will be captured in the eCRF. Information on all concomitant medications, administered blood products, as well as interventions occurring during the study must be recorded on the patient's eCRF in C3D. 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 breech 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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6.2 data sharing plans 6.2.1

The PI will share coded linked human data generated in this research for future research in a NIH-funded or approved public repository clinicaltrials.gov, dbGaP and GenBank  
in BTRIS
in publication and/or public presentations 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 should be re-evaluated for response every 8 weeks (+/- 1 week). Whilst immune-related RECIST criteria (**Appendix E**) will be taken into consideration regarding continuation of therapy in the event of growth (with a requirement for confirmation of PD), standard RECIST criteria will be the primary method used for evaluation of the primary endpoint. 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) guideline (version 1.1) [92, 93] and Modified Immune-related response criteria (Section **6.3.3.5**). 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.
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6.3.1 disease parameters

Measurable disease: Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as:
By chest x-ray: >20 mm;  
By CT scan:
o Scan slice thickness 5 mm or under as >10 mm with CT scan 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). 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. 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/pneumonitis, 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. 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. 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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6.3.2 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. 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. 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. Conventional CT and MRI: This guideline has defined measurability of lesions on CT scan based on the assumption that CT slice thickness is 5 mm or less. If CT scans have slice thickness greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness. MRI is also acceptable in certain situations (e.g. for body scans). Use of MRI remains a complex issue. MRI has excellent contrast, spatial, and temporal resolution; however, there are many image acquisition variables involved in MRI, which greatly impact image quality, lesion conspicuity, and measurement. Furthermore, the availability of MRI is variable globally. As with CT, if an MRI is performed, the technical specifications of the scanning sequences used should be optimized for the evaluation of the type and site of disease. Furthermore, as with CT, the modality used at follow-up should be the same as was used at baseline and the lesions should be measured/assessed on the same pulse sequence. It is beyond the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body parts, and diseases. Ideally, the same type of scanner should be used, and the image acquisition protocol should be followed as closely as possible to prior scans. Body scans should be performed with breath-hold scanning techniques, if possible. Ultrasound: Ultrasound is not useful in assessment of lesion size and should not be used as a method of measurement. Ultrasound examinations cannot be reproduced in their entirety for independent review at a later date and, because they are operator dependent, it cannot be guaranteed that the same technique and measurements will be taken from one assessment to the next. If new lesions are identified by ultrasound during the study, confirmation by CT or MRI is advised. If there is concern about radiation exposure at CT, MRI may be used instead of CT in selected instances. Endoscopy, Laparoscopy: The utilization of these techniques for objective tumor evaluation is not advised. However, such techniques may be useful to confirm complete pathological response when biopsies are obtained or to determine relapse in trials where recurrence following complete response (CR) or surgical resection is an endpoint. Cytology, Histology: These techniques can be used to differentiate between partial responses (PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as germ cell tumors, where known residual benign tumors can remain). The cytological confirmation of the neoplastic origin of any effusion that appears or worsens during treatment when the measurable tumor has met criteria for response or stable disease is mandatory to differentiate between response or stable disease (an effusion may be a side effect of the treatment) and progressive disease.
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6.3.3 response criteria 6.3.3.1 evaluation

Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum of diameters. Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions). Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum of diameters while on study.
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6.3.3.2 evaluation of non-target lesions

Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (<10 mm short axis). Note: If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response. Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits. Progressive Disease (PD): Appearance of one or more new lesions and/or *unequivocal progression* of existing non-target lesions. *Unequivocal progression* should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase. Although a clear progression of "non-target" lesions only is exceptional, the opinion of the treating physician should prevail in such circumstances, and the progression status should be confirmed at a later time by the review panel (or Principal Investigator).
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6.3.3.3 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  Non-Target  New  Overall  Lesions Lesions Lesions Respons e   | Best Overall Response when  Confirmation is Required*                                                                                                                                                                                                                                                                        |           |                                                   |                        |
|-----------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------|---------------------------------------------------|------------------------|
| CR                                                                                                                                | CR                                                                                                                                                                                                                                                                                                                           | No        | CR                                                | >4 wks. Confirmation** |
| CR                                                                                                                                | Non-CR/Non-PD                                                                                                                                                                                                                                                                                                                | No        | PR                                                |                        |
| CR                                                                                                                                | Not evaluated                                                                                                                                                                                                                                                                                                                | No        | PR                                                |                        |
| PR                                                                                                                                | Non-CR/Non                                                                                                                                                                                                                                                                                                                              |           |                                                   |                        |
| PD/not evaluated                                                                                                                  | No                                                                                                                                                                                                                                                                                                                           | PR        | >4 wks. Confirmation**                            |                        |
| SD                                                                                                                                | Non-CR/Non                                                                                                                                                                                                                                                                                                                              |           |                                                   |                        |
| PD/not evaluated                                                                                                                  | No                                                                                                                                                                                                                                                                                                                           | SD        | Documented at least once >4  wks. from baseline** |                        |
| PD                                                                                                                                | Any                                                                                                                                                                                                                                                                                                                          | Yes or No | PD                                                |                        |
| Any                                                                                                                               | PD***                                                                                                                                                                                                                                                                                                                        | Yes or No | PD                                                | no prior SD, PR or CR  |
| Any                                                                                                                               | Any                                                                                                                                                                                                                                                                                                                          | Yes       | PD                                                |                        |
| *                                                                                                                                 | See RECIST 1.1 manuscript for further details.                                                                                                                                                                                                                                                                               |           |                                                   |                        |
| **                                                                                                                                | 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.                                                                                                                                                                                                         |           |                                                   |                        |
| Note:                                                                                                                             | 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. |           |                                                   |                        |  
| For Patients with Non-Measurable Disease (i.e., Non-Target Disease) Non-Target Lesions New Lesions Overall Response CR No CR Non-CR/non-PD No Non-CR/non-PD* Not all evaluated No not evaluated Unequivocal PD Yes or No PD Any Yes PD   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Non-Target Lesions New Lesions **Overall Response**
* 'Non-CR/non-PD' is preferred over 'stable disease' for non-target disease since SD is increasingly used as an endpoint for assessment of efficacy in some trials so to assign this category when no lesions can be measured is not advised
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6.3.3.4 duration of response

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.  
Duration of stable disease: Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements.
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6.3.3.5 modified immune-mediated response criteria

Modified immune-mediated response criteria (imRC) will also be employed in this study. This new classification is based on the recent learning from clinical studies with cancer immunotherapies that even if some new lesions appear at the beginning of a treatment or if the total tumor burden does not increase substantially, tumor regressions or stabilizations might still occur later. The irRC were created using bi-dimensional measurements (as previously widely used in the World Health Organization criteria). For this trial, the concepts of the irRC are combined with RECIST 1.1 to come up with the modified imRC. Please refer to **Appendix E** for further details.
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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 should have access to a copy of the CTCAE version 5.0. A copy of the CTCAE version 5.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm\#ctc_50).
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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 Dr. Dahut 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. All data will be collected in a timely manner and reviewed by the principal investigator or a lead associate investigator. 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 protocol/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

As death due to disease progression is part of the study objectives (OS), and captured as an endpoint in this study, death due to disease progression will not be reported in expedited manner to the sponsor. However, if there is evidence suggesting a causal relationship between the study drug and the event, report the event in an expedited manner according to section 8.3. 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 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 safety reporting to the

All events listed below must be reported in the defined timeline to CCRsafety@mail.nih.gov. The CCR Office of Regulatory Affairs will send all reports to the manufacturer as described below.
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8.5.1 death reporting

All deaths that occur during the study, or within 90-day post-last dose of nivolumab/oral vancomycin/tadalafil safety follow-up period must be reported to BMS as follows:
Death that is clearly the result of disease progression should be documented but should not be reported as an SAE.  
Where death is not due (or not clearly due) to progression of the disease under study, the AE causing the death must be reported to BMS as a SAE within 24 hours of Sponsor awareness. The report should contain a comment regarding the co-involvement of progression of disease, if appropriate, and should assign main and contributory causes of death.  
Deaths with an unknown cause should always be reported as a SAE.
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8.5.2 reporting of serious adverse

All SAEs will be reported, whether considered causally related to the investigational product, or to the study procedure(s). The reporting period for SAEs is the period immediately following the time that written informed consent is obtained through 90 days after the last dose of nivolumab/oral vancomycin/tadalafil or until the initiation of alternative anticancer therapy. Sponsor must inform the FDA, via a MedWatch, of any serious or unexpected adverse events. SAEs and pregnancies must be reported to BMS within one business day of Sponsor becoming aware of the event. SAEs must be recorded on MedWatch or equivalent form. Pregnancies must be reported and submitted to BMS. BMS will perform due diligence follow-up using the BMS pregnancy Form (**Appendix G**) which the investigator must complete. It is the responsibility of the sponsor to compile all necessary information and ensure that the FDA receives a report according to the FDA reporting requirement timelines and to ensure that these reports are also submitted to BMS at the above time points. * A cover page should accompany the MedWatch form indicating the following:  
- "Notification from an Investigator Sponsored Study"
- The investigator IND number assigned by the FDA  
- The investigator's name and address - The trial name/title and BMS ISS reference number (CA209-8R3) (BMS only)
* Sponsor must also indicate, either in the SAE report or the cover page, the causality of events in relation to all study medications and if the SAE is related to disease progression, as determined by the principal investigator. * Send SAE report and accompanying cover page by way of email to BMS's designated mailbox: Worldwide.Safety@BMS.com. SAE FAX Number: +1 609-818-3804 If a non-serious AE becomes serious, this and another relevant follow-up information must also be provided to BMS, and the FDA. For all infusion reactions, SAEs should be reported to BMS Patient safety.
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8.5.3 drug induced liver injury

Wherever possible, timely confirmation of initial liver-related laboratory abnormalities should occur prior to the reporting of a potential DILI event. All occurrence of potential DILIs, meeting the defined criteria, must be reported as SAEs (See Section 8.3 for reporting details). The following definition takes into account anticipated baseline compromise of liver function in patients. Potential drug induced liver injury is defined as: 1) Concurrent ALT > 10 times upper limit of normal (ULN) AND 2) Total bilirubin > 2 times ULN or baseline value (if elevated bilirubin at study entry),
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and

3) No other immediately apparent possible causes of AT elevation and hyperbilirubinemia, including, but not limited to, viral hepatitis, pre-existing chronic or acute liver disease, or the administration of other drug(s) known to be hepatotoxic.
Drug induced liver injury (DILI) in a study subject, with or without associated clinical manifestations, is required to be reported as "hepatic function abnormal" within 24 hours of Sponsor knowledge of the event to the BMS Patient Safety using the designated Safety e-mailbox Worldwide.Safety@BMS.com unless a definitive underlying diagnosis for the abnormality (e.g., cholelithiasis or bile duct obstruction) that is unrelated to investigational product has been confirmed. If the definitive underlying diagnosis for the abnormality has been established and is unrelated to investigational product, the decision to continue dosing of the study subject will be based on the clinical judgment of the investigator. If no definitive underlying diagnosis for the abnormality is established, dosing of the study subject must be interrupted immediately. Follow-up investigations and inquiries must be initiated by the investigational site without delay. Each reported event of hepatic function abnormality will be followed by the investigator and evaluated by the sponsor and BMS.
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8.6 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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8.6.1 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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8.6.2 paternal exposure

Male patients should refrain from fathering a child or donating sperm during the study and for 7 months after the last dose of study drug (s). 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 7 months after the last dose should, if possible, be followed up and documented.
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8.7 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. 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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8.8 sponsor protocol non-adherence reporting

Protocol non-adherence is defined as any noncompliance with the clinical trial protocol, 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 non-adherence identified by the Staff or the site Monitor on the OSRO Site Protocol Non-Adherence Log. The protocol-specific, cumulative non-adherence log should be maintained in the site essential documents file and provided to OSRO as required over the duration of the study. In addition, any non-adherence to the protocol should be documented in the participant's source records and reported to the local IRB per their guidelines. OSRO required protocol non-adherence 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

As a sponsor for clinical trials, FDA regulations require the CCR to maintain a monitoring program. The CCR's program allows for confirmation of: study data, specifically data that could affect the interpretation of primary and secondary study endpoints; adherence to the protocol, regulations, ICH E6, and SOPs; and human subjects protection. This is done through independent verification of study data with source documentation focusing on:
Informed consent process  
Eligibility confirmation Drug administration and accountability Adverse events monitoring
Response assessment.  
The monitoring program also extends to multi-site research when the CCR is the coordinating center. This trial will be monitored by personnel employed by a CCR contractor. Monitors are qualified by training and experience to monitor the progress of clinical trials. Personnel monitoring this study will not be affiliated in any way with the trial conduct. 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 that 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) Monitoring 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. The intensity and frequency of monitoring will be based on several factors, including study type, phase, risk, complexity, expected enrollment rate, and any unique attributes of the study and the site. OSRO Monitoring visits and related activities will be conducted throughout the life cycle of each protocol, with the first activity being before study start 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 take place at the study site(s). Monitoring visit reports will describe visit activities, observations, findings of protocol non-adherence and associated action items or follow-up required for resolution of findings. Monitoring reports will be distributed to the study PI, NCI CCR QA, coordinating center (if applicable) and the OSRO regulatory file. If protocol non-adherence is identified by the Monitor (i.e., any noncompliance with the clinical trial protocol, GCP, or protocol-specific procedural requirements on the part of the participant, the Investigator, or the site Staff) the Monitor will note the observation, review with site Staff and if unresolved, request that the Staff document the non-adherence on the protocol-specific OSRO Site Protocol Non-Adherence Log (see Section **8.8**).
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10 statistical considerations 10.1 study

The primary objective of this pilot trial is to preliminarily determine the best overall response according to RECIST 1.1 to combined treatment of nivolumab, oral vancomycin and tadalafil in the treatment of patients with HCC or non-HCC liver dominant metastases from CRC and PDAC.
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10.1.2 secondary objectives

To assess the safety and tolerability of nivolumab in combination with oral vancomycin and tadalafil in patients with refractory primary HCC or liver dominant metastatic cancer from CRC or PDAC.  
To assess overall survival (OS) of nivolumab combined with oral vancomycin and tadalafil in patients with refractory HCC or liver dominant metastatic cancer from CRC or PDAC.
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10.2 sample size determination

Separately in HCC and Non-HCC patients, a small one-stage phase II design will be utilized: In HCC cohort 12 and in Non-HCC cohort 13 evaluable patients will be enrolled. With 12/13 patients, an exact binomial test with a one-sided 0.10 significance level will have 84% power to detect the difference between an unacceptably low 4% response rate (PR + CR) and a desirable, targeted response rate (PR + CR) of 25%. In practice, if there are at least 2 responses among the 12/13 patients, then the lower one-sided 90% confidence bound is at least 4.5%. Simultaneously, the upper one-sided 90% confidence bound is at least 38.6%. Thus, attaining 2 or more responses would be sufficient to demonstrate ruling out a 4% response rate and also be consistent with a desirable 25% rate or greater. If the response rate (PR + CR) in a cohort is identified as being adequate, consideration will be given to enrolling replicate cohorts as appropriate by amendment to evaluate additional patients independently from the initial 12/13 patients. It is expected that up to 5-6 HCC patients and 5-6 non-HCC can be enrolled within one year; thus, 2-3 years may be required to accrue 12 evaluable HCC patients and 13 evaluable non-HCC patients (25 total evaluable patients). To allow for a small number of inevaluable patients, the accrual ceiling will be set at 27 patients.
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as of amendment version date

The trial will no longer enroll any patients with HCC but will enroll only additional patients without HCC. There were 5 evaluable patients with HCC enrolled prior to December 2021, and up to 20 evaluable non-HCC patients will be enrolled based on the following: With 20 patients, an exact binomial test with a one-sided 0.10 significance level will have 82.3% power to detect the difference between an unacceptably low 5% response rate (PR + CR) and a desirable, targeted response rate (PR + CR) of 21%. In practice if there are at least 3 responses among the 20 patients, then the lower two-sided 90% exact mid-p Clopper-Pearson confidence bound is at least 5.2% (and the lower one sided 90% confidence bound is at least 6.9%).  
Simultaneously, the upper two-sided 90% confidence bound is at least 32.0% (and the upper onesided 90% confidence bound is 28.0%). Thus, attaining 3 or more responses would be sufficient to demonstrate ruling out a clinically not interesting 5% (that is, will not be worse than 5%) response rate and be consistent with a desirable 21% rate or greater. As of December 2021, there are 12 non-HCC patients enrolled. It is expected that the remaining 5 non-HCC patients can be enrolled in less than one year. With 5 HCC patients and 20 non-HCC patients, the total evaluable patients will be 25. To allow for a small number of inevaluable patients, the accrual ceiling will remain at 27.
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10.3 populations for analyses

All patients received treatments, regardless of drug types included in this study and had at least one re-staging will be evaluated for response (PR + CR).
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10.4 statistical analyses 10.4.1 general

The fraction of patients who experience a response will be reported along with confidence intervals.
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10.4.2 analysis of the primary

In each cohort, the fraction of patients who experience a response (PR + CR) will be reported along with 80% and 95% two-sided confidence intervals.
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10.4.3 analysis of the secondary

The safety of the treatment will be monitored, and any toxicities identified will be reported by type and grade. Overall survival will be calculated from the on-study date using the Kaplan-Meier method.
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10.4.4 safety analyses

Patients will be assessed for toxicity by reporting the grades of toxicity and the type of toxicity observed for all patients. 10.4.5 Baseline Descriptive Statistics Baseline demographic characteristics will be reported.
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10.4.7 exploratory analyses

The following objectives will result in descriptive or comparative analyses when adequate data exist to perform them:  
- To measure changes in immune cell population in the peripheral blood and tumors of patients treated on this study, including CD4+, CD8+, MDSCs, MAITS NKT cells, IFNgamma signaling pathway, perforin and granzyme.  
- To measure secreted cytokines in the peripheral blood to monitor the relative level change with the treatment
- To measure changes in bile acid in the peripheral blood of patients treated in this study.  
- To evaluate tadalafil and oral vancomycin pharmacokinetic level to monitor compliance and association between the level and response rate or immune parameters
- To evaluate TCR beta sequencing to analyze the T cell response from PBMC and tumor samples
- To assess gene and protein expression and metabolite profile from tumor to evaluate the association of the change immune cell signature, signaling pathways and metabolite with clinical outcome
- To measure changes in gut microbiome in the stool of patients treated on this study.  
- To evaluate the incidence of vancomycin-resistant enterococci (VRE) development during the trial.  
- To evaluate the relationship between the response rate, overall survival and immune parameters, gut microbiome, and bile acid component in patients treated on this study.
Any exploratory evaluations which generate quantitative measures will be done using descriptive statistics including confidence intervals when appropriate. Any statistical tests performed for evaluation of exploratory objectives will be done without formal adjustment for multiple comparisons, but in the context of the number of tests performed.
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11 collaborative agreement 11.1 cooperative

The CRADA for this protocol (03259) has been executed between Thoracic & GI Malignancies Branch, NCI, NIH and Bristol-Myers Squibb, the manufacturer of nivolumab.
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12 human subjects protections 12.1

This study was designed to include women and minorities but was not designed to measure differences of intervention effects. Males and females will be recruited with no preference to gender. No exclusion to this study will be based on race. Minorities will actively be recruited to participate.
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12.2 participation of children

Children (younger than 18 years) will not be included in this protocol due to the limited data on nivolumab in children and the different biology of childhood malignancy.
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12.3 participation of subjects unable

Adults unable to consent are excluded from enrolling in the protocol. However, it is possible that subjects enrolled in the protocol may permanently lose the capacity to consent for themselves during the course of this study. If a subject loses the capacity to consent during the course of the study for reasons unrelated to protocol participation and is receiving direct benefit from the study therapy in the opinion of the PI, the subject may be retained on the study therapy with the permission of the alternate decision maker. If the permanent loss of capacity is due to toxicity from the study therapy or disease progression, the participant will be removed from study therapy as required per protocol, but may be retained on study to be followed for safety reasons and/or overall survival as described in the Study Calendar 3.4 The follow-up procedures involve non-invasive, low risk studies. The benefit of a safety follow up would outweigh the risks of such procedures. 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 a LAR, as needed. Please see section **12.6.1** for consent procedure.
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12.4 evaluation of benefits and

The study drug may help to control the disease. The results may help the investigators learn more about the disease and develop new treatments for patients with this disease.
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12.4.2 risks

The primary risk to patients participating in this research study is from the toxicity of nivolumab, oral vancomycin and tadalafil. All care will be taken to minimize study treatment side effects, but they can be unpredictable in nature and severity. Patients will be examined and evaluated prior to enrollment. All evaluations to monitor the treatment of patients will be recorded in the patient chart. If patients suffer any physical injury as a result of the participation in this study, immediate medical treatment is available at the Clinical Center, National Cancer Institute, Bethesda, Maryland. Although no compensation is available, any injury will be evaluated and treated in keeping with the benefits or care to which patients are entitled under applicable regulations. In all publications and presentations resulting from this trial, patients' anonymity will be protected to the maximum extent possible. Authorized personnel from the National Cancer Institute (NCI) and Food and Drug Administration (FDA) or other regulatory authorities may have access to research files in order to verify that patients' rights have been safeguarded. In addition, patient names will be given to the Central Registration to register and verify patients' eligibility.
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12.4.2.1 risk of biopsy

All care will be taken to minimize risks that may be incurred by tumor sampling. However, there are procedure-related risks (such as bleeding, infection and visceral injury) that will be explained fully during informed consent.
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12.4.2.2 risks of sedation

Biopsies will be done under sedation. Potential side effects of sedation include headache, nausea and drowsiness. These side effects usually go away quickly.
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12.4.2.3 risks of exposure to

The study will involve radiation from the following sources:
- Up to 6 CT scans per year for disease assessment  
- 2 CT scans for the collection of 2 mandatory biopsies
Subjects in this study may have up to 6 CT scans and 2 CT guided biopsies and be exposed to approximately 8.2 rem per year. This amount is more than would be expected from everyday background radiation. Being exposed to excess radiation can increase the risk of cancer. The risk of getting cancer from the radiation exposure in this study is 0.8 out of 100 (0.8%) and of getting a fatal cancer is 0.4 out of 100 (0.4%).
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12.4.2.4 risks of ct scans

In addition to the radiation risks discussed above, CT scans may include the risks of an allergic reaction to the contrast. Participants might experience hives, itching, headache, difficulty breathing, increased heartrate and swelling.
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12.4.2.5 blood collection risks

Risks of blood draws include pain and bruising in the area where the needle is placed, lightheadedness, and rarely, fainting. When large amounts of blood are collected, low red blood cell count (anemia) can develop.
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12.4.2.7 stool collection or rectal

There is no physical risk involved with stool or rectal swab collection.
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12.4.2.8 questionnaires risk

Questionnaires may contain questions that are sensitive in nature. The participants are asked to only answer questions they are comfortable with
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12.4.2.9 other risks

Risks include the possible occurrence of any of a range of side effects which are listed in the Consent Document or this protocol document. Frequent monitoring for adverse effects will help to minimize the risks associated with administration of the study agents.
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12.4.2.10 risk of losing data

This includes the risk that data obtained during this study, including data related to genotype, DNA sequencing or risk for disease tendency or trait can be released to members of the public, insurers, employers, or law enforcement agencies. Although there are no plans to release results to the patients, family members or health care providers, this risk will be included in the informed consent document
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12.4.2.11 non-physical risks of genetic

Anxiety and stress may arise as a result of the anticipation that unwanted information regarding disease related DNA sequencing or disease tendencies, or misattributed paternity. Patients will be clearly informed that the data related to DNA sequencing and genetic analysis is coded, investigational and will not be shared with patients, family members or health care providers.
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risk related to possibility that

This includes the risk that data related to genotype, DNA sequencing or risk for disease tendency or trait can be released to members of the public, insurers, employers, or law enforcement agencies. Although there are no plans to release results to the patients, family members or health care providers, this risk will be included in the informed consent document
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12.5 risks/benefits analysis for all

It is possible that treatment on this protocol may reduce tumor burden or lessen symptoms caused by the cancer. While treatment on this protocol may not individually benefit subjects, the knowledge gained from this study may help others in the future who have gastric cancer. Potential risks include the possible occurrence of any of a range of side effects listed. The risks and benefits of participation for adults who become unable to consent, are no different than those described for patients who are less vulnerable
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12.6 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. Whether in person or remote, the privacy of the subject will be maintained. Consenting investigators (and participant, when in person) will be located in a private area (e.g., clinic consult room). When consent is conducted remotely, the participant 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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12.6.1 consent process for adults

For participants addressed in section **12.3**, an LAR will be identified consistent with Policy 403 and informed consent obtained from the LAR, as described in Section 12.6.
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13 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 study participants. If the study is prematurely terminated or suspended, the Principal Investigator (PI) will promptly inform study participants, the Institutional Review Board (IRB), and sponsor and will provide the reason(s) for the termination or suspension. Study participants will be contacted, as applicable, and be informed of changes to study visit schedule. Circumstances that may warrant termination or suspension include, but are not limited to:  
Determination of unexpected, significant, or unacceptable risk to participants Demonstration of efficacy that would warrant stopping Insufficient compliance to protocol requirements Data that are not sufficiently complete and/or evaluable Determination that the primary endpoint has been met Determination of futility
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, and satisfy the sponsor, IRB and as applicable, Food and Drug Administration (FDA).
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13.2 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 Harmonisation 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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13.3 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 NCI 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.
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13.4 confidentiality and privacy

Participant confidentiality and privacy is strictly held in trust by the participating investigators, their staff, and the sponsor. 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 each 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 transmitted to and 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(s) 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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14 pharmaceutical information 14.1 nivolumab

Bristol Myers Squibb will supply investigational nivolumab. Only 100mg, 10mg/mL nivolumab vials will be provided for this study. There are 5 vials per carton.
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14.1.2 toxicity

Nivolumab is most commonly associated with immune-related adverse reactions. Most of these, including severe reactions, resolved following initiation of appropriate medical therapy or withdrawal of nivolumab. Refer to investigator brochure for detailed toxicity information.
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14.1.3 formulation and preparation

Nivolumab Injection is a clear to opalescent, colorless to pale yellow liquid; light (few) particulates may be present. The drug product is a sterile, nonpyrogenic, single-use, isotonic aqueous solution formulated in sodium citrate, sodium chloride, mannitol, diethylenetriamine pentacetic acid (pentetic acid) and polysorbate 80 (Tween® 80), pH 6.0. Nivolumab injection can be infused undiluted (10 mg/mL) or diluted with 0.9% Sodium Chloride Injection, USP or 5% Dextrose, USP to concentrations no less than 1 mg/mL.
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14.1.4 stability and storage

Store intact vials at 2°C to 8°C (36°F to 46°F); do not freeze. Protect from light. Do not shake. Shelf-life surveillance of the intact vials is ongoing.  
Store the prepared infusion solution at room temperature or refrigerated. Do not freeze solutions prepared for infusion. Refer to investigator brochure for detailed stability information.
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14.1.5 administration procedures

Intravenous infusion 30 to 60 minutes. Do not administer as an IV push or bolus injection. Administer through a 0.2 micron to 1.2-micron pore size, low-protein binding in-line filter.
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14.2 tadalafil 14.2.1 source

Tadalafil tablets will be provided by the NIH Clinical Center Pharmacy according to standard pharmacy procedures.
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14.2.2 description

Tadalafil is a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5). Tadalafil has the empirical formula C22H19N3O4 representing a molecular weight of 389.41. The chemical designation is pyrazino [1′,2′:1,6] pyrido[3,4-b] indole-1,4-dione, 6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a hexahydro-2-methyl-, (6R,12aR)-. It is a crystalline solid that is practically insoluble in water and very slightly soluble in ethanol.
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14.2.3 how supplied

Tadalafil tablets are available in 2.5 mg, 5 mg, 10 mg and 20 mg. Excipient information may vary depending on manufacturer (particularly for generics). Consult specific product labeling for detailed information. Appearance of the generic drug product may differ depending on the manufacturer and patients will be notified of this difference.
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14.2.4 toxicity

Very common (in 100 people receiving tadalafil, 11 may have):
headaches Common risks (in 100 people receiving tadalafil, more than 2 and up to 10 may have):
stomach upset, pain in belly, diarrhea,  
nausea, reflux disease,
muscle or back pain, pain in arms and legs,  
flushing (redness or warmth of the face, neck, or chest),
flu-like symptoms (such as stuffy nose, sneezing, or sore throat),
infection, cough, Rare risks (in 100 people receiving tadalafil, 1 or fewer may have):
fatigue, face swelling, chest pain, heart problems, low blood pressure, elevated liver enzyme in the blood dry mouth, hemorrhoids, dizziness, luck of sleep, rash, blurred vision, changes in color vision, eye pain, swelling around eyes abnormal ejaculation a painful or prolonged erection lasting 4 or more hours; sudden decreased vision (including permanent blindness, in one or both eyes); a sudden decrease or loss of hearing, sometimes with ringing in the ears and dizziness
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14.3 oral vancomycin 14.3.1 source

Vancomycin is an antibacterial drug that inhibits bacterial cell wall synthesis by blocking glycopeptide polymerization through binding tightly to D-alanyl-D-alanine portion of cell wall precursor. Vancomycin capsules will be provided by the NIH Clinical Center Pharmacy according to standard pharmacy procedures
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14.3.3 description

Vancomycin CAPSULES for oral administration contain chromatographically purified vancomycin hydrochloride, a tricyclic glycopeptide antibiotic derived from Amycolatopsis orientalis (formerly *Nocardia orientalis*), which has the chemical formula C66H75Cl2N9O24•HCl. The molecular weight of vancomycin hydrochloride is 1485.73; 500 mg of the base is equivalent to 0.34 mmol. The capsules contain vancomycin hydrochloride equivalent to 125 mg (0.08 mmol) or 250 mg (0.17 mmol) vancomycin. The capsules also contain F D & C Blue No. 2, gelatin, iron oxide, polyethylene glycol, titanium dioxide, and other inactive ingredients.
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14.3.4 how supplied

Vancomycin capsules are available in 125 mg and 250 mg strength. Excipient information may vary depending on the manufacturer (particularly for generics). Consult specific product labeling for detailed information. Appearance of the generic drug product may differ depending on the manufacturer and patients will be notified of this difference.
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14.3.6 storage

Store at controlled room temperature, 59° to 86°F (15° to 30°C).
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16 appendices 16.1 appendix a

| Grade   | Descriptions                                                                                                                                                                                    |
|---------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 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.                                                                                                                                                                                           |  
ECOG Performance Status Scale
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16.2 appendix b patient's medication

Cycle___________________ Patient's ID ___________________ INSTRUCTIONS TO THE PATIENT:
1. Complete one form for each cycle of treatment 2. You will take vancomycin 125 mg every 6 hours a day for 3 weeks on and 1 week off 3. You will take tadalafil 10 mg tadalafil daily. 4. Record the date, the number of tablets that you took, and when you took them. 5. If you have any comments or notice any side effects, please record them in the comments column.  
| 6.                   | Please bring this form and your bottles (even it is empty) when you come for your clinic visits.   |            |                  |          |
|----------------------|----------------------------------------------------------------------------------------------------|------------|------------------|----------|
| Day                  | Date                                                                                               | Tadalafil, | Vancomycin, time | Comments |
| time                 |                                                                                                    |            |                  |          |
| 1 2 3 4 5 6 7 8 9 10 |                                                                                                    |            |                  |          |  
| Day   | Date   | Tadalafil,   | Vancomycin, time   | Comments   |
|-------|--------|--------------|--------------------|------------|
| time  |        |              |                    |            |
| ll    |        |              |                    |            |
| 12    |        |              |                    |            |
| 13    |        |              |                    |            |
| 14    |        |              |                    |            |
| 15    |        |              |                    |            |
| 16    |        |              |                    |            |
| 17    |        |              |                    |            |
| 18    |        |              |                    |            |
| 19    |        |              |                    |            |
| 20    |        |              |                    |            |
| 21    |        |              |                    |            |
| 22    |        |              |                    |            |
| 23    |        |              |                    |            |
| 24    |        |              |                    |            |
| 25    |        |              |                    |            |
| 26    |        |              |                    |            |  
  
16.3 Appendix C: Management Algorithms For Endocrinopathy, Gastrointestinal, Hepatic, Neurological, Pulmonary, Renal, And Skin Adverse Events
untitled h1
gi adverse event management algorithm

Rule out non-inflammatory causes. If non-inflammatory cause is identified, treat accordingly and continue I-O therapy.  
  
sustained clinical improvement is observed. Lower bioavailability of oral corticosteroids should be taken into account when switching to the tequivalent dose of oral corticosteroids.  
  
Pulmonary Adverse Event Management Algorithm Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue I-O therapy. Evaluate with  
  
  
Hepatic Adverse Event Management Algorithm Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue I-O therapy. Consider imaging for  
  
equivalent dose of oral corticosteroids.  
Endocrinopathy Adverse Event Management Algorithm Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue I-O therapy. Consider visual field testing, endocrinology consultation, and imaging.  
  
  
sustained clinical improvement is observed. Lower bioavailability of oral corticosteroids should be taken into account when switching to the equivalent dose of oral corticosteroids.
untitled h1
skin adverse event management algorithm

Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue I-O therapy.  
  
sustained clinical improvement is observed. Lower bioevailability of oral corticosteroids should be taken into account when switching to the equivalent dose of oral corticosteroids.  
*Refer to NCI CTCAE v5 for term-specific grading criteria.  
°If SJS/TEN is suspected, withhold I-O therapy and refer patient for specialized care for assessment and treatment. If SJS or TEN is diagnosed, permanently discontinue I-O therapy.
neurological adverse event management algorithm
skin adverse event management algorithm

Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue I-O therapy.  
  
  
once sustained clinical improvement is observed. Lower bioavailability of oral corticosteroids should be taken into account when  
| Infusion-related Reactions Severity Grade of  the Event (CTCAE  5.0 Dose Modifications   | Toxicity Management                                                                                                                                                                                                                                                                                                                                                         |                                                                                                                                                                                                                                                                                                                          |
|------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Any Grade                                                                                | General Guidance                                                                                                                                                                                                                                                                                                                                                            | For Any Grade: − Manage per institutional standard at the discretion of  investigator. − Monitor patients for signs and symptoms of infusionrelated reactions (e.g., fever and/or shaking chills,  flushing and/or itching, alterations in heart rate and  blood pressure, dyspnoea or chest discomfort, or skin  rashes) and anaphylaxis (e.g., generalized urticaria,  angioedema, wheezing, hypotension, or tachycardia).                                                                                                                                                                                                                                                                                                                          |
| Grade 1 or 2                                                                             | For Grade 1:  The infusion rate of study drug/study  regimen may be decreased by 50% or  temporarily interrupted until resolution of  the event. For Grade 2:  The infusion rate of study drug/study  regimen may be decreased 50% or  temporarily interrupted until resolution of  the event.  Subsequent infusions must be given at  50% of the initial infusion rate. | For Grade 1 or 2: − Acetaminophen and/or antihistamines may be  administered per institutional standard at the discretion  of the investigator. − Consider premedication per institutional standard prior  to subsequent doses. − Steroids should not be used for routine premedication of  Grade ≤2 infusion reactions. |
| Grade 3 or 4                                                                             | For Grade 3 or 4:  Permanently discontinue study  drug/study regimen.                                                                                                                                                                                                                                                                                                      | For Grade 3 or 4: − Manage severe infusion-related reactions per  institutional standards (e.g., IM epinephrine, followed  by IV diphenhydramine and ranitidine, and IV  glucocorticoid).                                                                                                                                |
neurological adverse event management algorithm
16.4 appendix d infusion-related reaction

| CTCAE - Common Terminology Criteria for Adverse Events; IM - Intramuscular; IV Intravenous; NCI - National Cancer Institute. Non-immune-mediated Reactions Severity Grade of the  Event (CTCAE 5.0) Dose Modifications Toxicity  Management Any Grade  Note: Dose modifications are not required for AEs not deemed to  be related to study treatment (i.e., events due to underlying  disease) or for laboratory abnormalities not deemed to be  clinically significant. Grade 1  No dose modifications. Grade 2  Hold study drug/study regimen until resolution to ≤Grade 1 or  baseline. Grade 3  Hold study drug/study regimen until resolution to ≤Grade 1 or  baseline.  For AEs that downgrade to ≤Grade 2 within 7 days or resolve to  ≤Grade 1 or baseline within 14 days, resume study drug/study  regimen administration. Otherwise, discontinue study drug/study  regimen. Grade 4  Discontinue study drug/study regimen (Note: For Grade 4 labs,  decision to discontinue should be based on accompanying clinical  signs/symptoms, the Investigator's clinical judgment, and  consultation with the Sponsor.). Treat accordingly,  as per institutional  standard. AE - Adverse event; CTCAE - Common Terminology Criteria for Adverse Events; NCI - National Cancer Institute.   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
neurological adverse event management algorithm
16.5 appendix e modified immune-related

This new classification is based on the recent learning from clinical studies with cancer immunotherapies that even if some new lesions appear at the beginning of a treatment or if the total tumor burden does not increase substantially, tumor regressions or stabilizations might still occur later. The irRC were created using bi-dimensional measurements (as previously widely used in the World Health Organization criteria). For this trial, the concepts of the irRC are combined with RECIST 1.1 to come up with the modified irRC. For modified irRC, only target and measurable lesions are taken into account. In contrast to the RECIST 1.1 criteria, the modified irRC criteria (a) require confirmation of both progression and response by imaging at 6 weeks after initial imaging and (b) do not necessarily score the appearance of new lesions as progressive disease if the sum of lesion diameters of target lesions (minimum of 10 mm per lesion, maximum of 5 target lesions, maximum of 2 per organ) and measurable new lesions does not increase by ≥ 20%. The same method of assessment and the same technique should be used to characterize each identified and reported target lesion(s) at baseline, during the trial, and at the end of trial visit. All measurements should be recorded in metric notation. The modified irRC based on RECIST 1.1 are displayed below.
neurological adverse event management algorithm
overall responses derived from changes

| Measurable Response                                                                                        | Non-Measurable Response   | Overall Response   |        |
|------------------------------------------------------------------------------------------------------------|---------------------------|--------------------|--------|
| Using Modified irRC                                                                                        |                           |                    |        |
| Index and New, Measurable                                                                                  | Non-Index Lesions         | New, Non-          |        |
| Lesions (Tumor Burden)¹                                                                                    | Measurable Lesions        | irCR²              |        |
| Decrease 100%                                                                                              | Absent                    | Absent             |        |
| Decrease 100%                                                                                              | Stable                    | Any                | irPR 2 |
| Decrease 100%                                                                                              | Unequivocal progression   | Any                | irPR²  |
| Decrease ≥ 30%                                                                                             | Absent / Stable           | irPR²              |        |
| Any                                                                                                        |                           |                    |        |
| Decrease ≥ 30%                                                                                             | Unequivocal progression   | Any                | irPR²  |
| Decrease < 30% to increase <                                                                               | Absent / Stable           | Any                | irSD   |
| 20%                                                                                                        |                           |                    |        |
| Decrease < 30% to increase <                                                                               | Unequivocal progression   | Any                | irSD   |
| 20%                                                                                                        |                           |                    |        |
| Increase ≥ 20%                                                                                             | Any                       | Any                | irPD   |
| - Decreases assessed relative to baseline                                                                  |                           |                    |        |
| F Assuming that the response (irCR and irPR) and progression (irPD) are confirmed by a second, consecutive |                           |                    |        |  
assessment at least 4 weeks apart (normally it should be done 6 weeks apart).  
| Points assigned   |                 |                 |           |
|-------------------|-----------------|-----------------|-----------|
| Parameter         | 1               | 3               |           |
| 2                 |                 |                 |           |
| Ascites           | Absent          | Slight          | Moderate  |
| Bilirubin         | <2 mg/dL        | 2-3 mg/dL       | > mg/dL   |
| ( <34.2           | (34.2 to 51.3   | (>51.3          |           |
| micromol/liter)   | micromol/liter) | micromol/liter) |           |
| Albumin           | >.3.5 g/dL (35  | 2.8-3.5 g/dL    | <2.8 g/dL |
| (28 to 35         |                 |                 |           |
| g/liter)          | ( <28 g/liter)  |                 |           |
| g/liter)          |                 |                 |           |
| Prothrombin time  |                 |                 |           |
| Seconds over      | <4              | 4-6             | >6        |
| control           |                 |                 |           |
| INR               | <1.7            | 1.7-2.3         | >2.3      |
| Encephalopathy    | None            | Grade 1-2       | Grade 3-4 |
neurological adverse event management algorithm
16.6 appendix f child-pugh classification

Modified Child-Pugh classification of the severity of liver disease according to the degree of ascites, the plasma concentrations of bilirubin and albumin, the prothrombin time, and the degree of encephalopathy. A total score of 56 is considered grade A (well-compensated disease); 7-9 is grade B (significant functional compromise); and 10-15 is grade C (decompensated disease). These grades correlate with one- and two-year patient survival: grade A - 100 and 85 percent; grade B - 80 and 60 percent; and grade C - 45 and 35 percent.
neurological adverse event management algorithm
16.7 appendix g pregnancy form

| Pregnancy Surveillance Form Part I                        |                        |                    |            |                 |                      |
|-----------------------------------------------------------|------------------------|--------------------|------------|-----------------|----------------------|
| t Bristol-Myers Squibb Company (Antepartum Information)   |                        |                    |            |                 |                      |
| CARES Number; (BMS only) Local Country Number; (BMS only) |                        |                    |            |                 |                      |
| P ATIENT I DENTIFIER :                                    |                        |                    |            |                 |                      |
| ..                                                        | i                      | 1 1 1              | xl         | in the state of | a control of the pro |
| -                                                         |                        |                    |            |                 |                      |
| ucccc                                                     |                        |                    |            |                 |                      |
| P REGNANCY                                                | Dose                   | n                  | PERIOD(S)  |                 |                      |
| MEDICATION NAME                                           | 0 m x '                | of drug            | ONCOLOGY   | S TART AND      |                      |
| RELATED TO                                                | AND                    |                    |            |                 |                      |
| AND INDICATION                                            | EXPO-
SURE***                        | DRUGS ONLY         | STOP DATES |                 |                      |
| MEDICATION?"                                              | UNITS                  | D D M  M  M  Y   Y |            |                 |                      |
| 1.                                                        | CTCLE ®;               |                    |            |                 |                      |
| INDICATION                                                | IMULATIVE
SOE VATH UNITS                        |                    |            |                 |                      |
| D MOTESML                                                 | □ PATERN               | - NOT RELATED      |            |                 |                      |
| OR                                                        | on Classic             |                    |            |                 |                      |
| □ NON-STUDY FOR                                           | - Stude                |                    |            |                 |                      |
| 2.                                                        | CYCLE #:               |                    |            |                 |                      |
| INDICATION                                                | OUMULATIVE
DOEE VATH UNITS                        |                    |            |                 |                      |
| I MATERNAL OR                                             | - PATERN               | □ NOT RELATION     | ROR DOONG  |                 |                      |
| □ NON-STUDY LOR                                           | □ R ELATED             |                    |            |                 |                      |
| 3.                                                        | CACLE $                |                    |            |                 |                      |
| INDICATION                                                | OUNULATIVE
DOSE VATH UNITS                        |                    |            |                 |                      |
| - MATERIAL PATERIAL                                       | □ NOT REATE            | OR I ON            |            |                 |                      |
| - R ELATED                                                |                        |                    |            |                 |                      |
| 4 [                                                       | CICLE #:               | 1                  |            |                 |                      |
| INDICATION                                                | ologically the control |                    |            |                 |                      |
| □ M A T A                                                 | PATERN
INSTUDY                        | - NOT RELATED      |            |                 |                      |
| I NON-STUDY OR                                            | OR IN ONDONG           |                    |            |                 |                      |
| CYCLE 0:                                                  |                        |                    |            |                 |                      |
| 5.                                                        |                        |                    |            |                 |                      |
| INDICATION                                                | owners
Dose with units                        |                    |            |                 |                      |
| I M I T E P I A L I O R I                                 | [ P ATERN              | □ NOT RELATE
□ RELATED                    |            |                 |                      |
| l                                                         | or                     | __                 |            |                 |                      |
| □ NON-STUDY LOR                                           | [ Stude                |                    |            |                 |                      |
| 6.                                                        | CYCLE IS:              |                    |            |                 |                      |
| INDICATION                                                | OUNULATIVE
DOEE VATH UNITE                        |                    |            |                 |                      |
| D MODAL OR                                                | □ Paterna
□ Study                        | □ NOT RELATED      | or movena  |                 |                      |
| □ NON-STUDY LOR                                           | □ R ELATED             |                    |            |                 |                      |
| 7.                                                        | CTCLE *:               |                    |            |                 |                      |
| INDING I                                                  | CUNULATIVE
DOSE VATH UNITS                        |                    |            |                 |                      |
| D MATERNIL OR OD PATERNIL
D NON-STUDY OR OD STUDY                                                           | - NOT RELATED
- RELATIO                        | OR IN ONCOM        |            |                 |                      |
| * M ANDATORY FOR ALL STUDIES                              |                        |                    |            |                 |                      |
| *ROUTE:                                                   |                        |                    |            |                 |                      |  
2 = I NTRAVENOUS
1 = ORAL
**Period(s) of drug exposure: (include all that apply)
0 = Price to conce 4 = L AROR & DELIVERY
3 = Subcutaneous 4 = OTHER
2 = 2 ND TRIMESTES
5 = U NINOVAL
28 March 2011 CT SOP 108 FRM 01.v03 Page _
Alterations to this form are not permitted unless otherwise specified in the governing Procedural Document.  
Original - Bristol-Myers Squibb; Copy - Retained by Investigator  
  
| B Bristol-Myers Squibb Company (Antepartum Information)                                                    |                                                                    |                                  |                             |     |      |
|------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------|----------------------------------|-----------------------------|-----|------|
| P ATIENT I DENTIFIER :                                                                                     | CARES N UMBER : (BMS ONLY )  L OCAL C OUNTRY N UMBER : (BMS ONLY ) |                                  |                             |     |      |
| [ Long Common Common Long Common C                                                                         | ..                                                                 | ting the control of              |                             |     |      |
| P P RENATAL DIAGNOSTIC B ASE                                                                               | T EST RESULTS                                                      | normal range                     |                             |     |      |
| DATE                                                                                                       |                                                                    |                                  |                             |     |      |
| TESTING                                                                                                    | LINE                                                               | D DEMEMEY Y                      | UNITS                       | Low | HIGH |
| -                                                                                                          |                                                                    |                                  |                             |     |      |
| -                                                                                                          |                                                                    |                                  |                             |     |      |
| L                                                                                                          |                                                                    |                                  |                             |     |      |
| DESCRIBE RESULTS IN DETAIL, IF APPLICABLE:                                                                 |                                                                    |                                  |                             |     |      |
| REPORTER INFORMATION:                                                                                      | ] BMS STUDY                                                        | [                                | [[                          |     |      |
| INVESTIGATOR                                                                                               | TUDY SPONSOR                                                       |                                  |                             |     |      |
| *Qualification:  (Complete only if "other" is checked)                                                     |                                                                    |                                  |                             |     |      |
| P P HYSICIAN                                                                                               | [                                                                  | N N URSE /N URSE PRACTITIONER    | - OTHER HEALTH PROFESSIONAL |     |      |
| [                                                                                                          | [ A TTORNEY                                                        | - O THER NON-HEALTH PROFESSIONAL |                             |     |      |
| Person completing the form ( if different from I nvestigator/S ponsor) :                                   | DATE                                                               |                                  |                             |     |      |
| Printed name                                                                                               |                                                                    |                                  |                             |     |      |
| S S IGNATURE                                                                                               | D                                                                  | DEMIC MICROVI                    |                             |     |      |
| I NSTITUTION / ORGANIZATION :                                                                              |                                                                    |                                  |                             |     |      |
| STREET ADDRESS:                                                                                            | CITY:                                                              | STATE/PROVINCE:                  |                             |     |      |
| POST CODE:                                                                                                 | COUNTRY:                                                           | 'HONE NUMBER:                    |                             |     |      |
| INVESTIGATOR/SPONSOR/OTHER                                                                                 | LAST NAME                                                          | M M IDOLE
INITIAL                                  |                             |     |      |
| F F IRST NAME                                                                                              |                                                                    |                                  |                             |     |      |
| S IGNATURE :                                                                                               | DATE                                                               |                                  |                             |     |      |
| D MEMOM                                                                                                    |                                                                    |                                  |                             |     |      |
| 28 March 2011 CT SOP 108 FRM 01.v03                                                                        | Page ___ of                                                        |                                  |                             |     |      |
| Aterations to this form are not permitted unless otherwise specified in the governing Procedural Document. |                                                                    |                                  |                             |     |      |
| Original - Bristol-Myers Squibb; Copy - Retained by Investigator                                           |                                                                    |                                  |                             |     |      |  
  
| C AUSALITY (MANDATORY POR STUDIES)                                                                  |
|-----------------------------------------------------------------------------------------------------|
| IN THE INVESTIGATOR'S OPINION, WAS THE CEFECTAVEDICAL PROBLEM RELATED TO MEDICATION UNDER STUDY ? ; |
| IE FELATED, PLEASE COMMENT ON SPECIFIC EVENT(S) AND MEDICATION(S) BELOW:                            |
| IE NOT RELATED, INDICATE VMAT THE DEFECTAVEDICAL FROM LEM VAS ATTROUTED TO                          |  
1 NOT RELATED
□ R R R ATED
28 March 2011 CT SOP 108 FRM 01.v03 Page       of Alterations to this form are not permitted unless otherwise specified in the governing Procedural Document.  
Original - Bristol-Myers Squibb; Copy - Retained by Investigator
16.8 appendix h food diary
16.7 appendix g pregnancy form

| FOODS AND
BEVERAGES                        |       |      |        |                      |                    |
|------------------------|-------|------|--------|----------------------|--------------------|
| 19C0033                | PLACE | TIME | amount | COMPLETE DESCRIPTION | REMEWER'S CONNENTS |
| NAME :                 |       |      |        |                      |                    |
| Day of Week:           |       |      |        |                      |                    |
| Date:                  |       |      |        |                      |                    |
| Is this a typical day? |       |      |        |                      |                    |
| __                     |       |      |        |                      |                    |
| I no, give reason:     |       |      |        |                      |                    |
| Daytime                |       |      |        |                      |                    |
| phone:                 |       |      |        |                      |                    |
| Birthdate of patient   |       |      |        |                      |                    |
| /                      | /     |      |        |                      |                    |
| Age of patient         |       |      |        |                      |                    |
| __                     |       |      |        |                      |                    |
| Male Female            |       |      |        |                      |                    |
| Study ID number:       |       |      |        |                      |                    |
| C1D0                   |       |      |        |                      |                    |
| C1D10                  |       |      |        |                      |                    |
| RDHT reviewer:         |       |      |        |                      |                    |
16.9 appendix i mixed meal
nivolumab tadalifil & vancomycin 19c0033

| Amount                                                                                  |                                         |            |          |           |                  |
|-----------------------------------------------------------------------------------------|-----------------------------------------|------------|----------|-----------|------------------|
| Consumed:                                                                               | Mixed meal test for bile acids in blood |            |          |           |                  |
| (Time 0 is prior to the drink, all other time points are after completion of the drink) |                                         |            |          |           |                  |
| Date:                                                                                   | Clock used:                             |            |          |           |                  |
| Baseline:                                                                               | C1D10:                                  |            |          |           |                  |
| Time                                                                                    | Schedule                                | Actual     |          |           |                  |
| Interval                                                                                | Time                                    | Time       | Comments | Signature | Reason if missed |
| (Minutes)                                                                               | (Military)                              | (Military) |          |           |                  |
| eeee                                                                                    |                                         |            |          |           |                  |  
5 ml Sodium Heparin green top tube (GTT) at every time interval Page Dr. Figg's lab at 102-11964 for specimen pickup