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1 Abbreviated Title: anti-CTLA4 & ablative therapy CC Protocol \#: 13-C-0120 Version Date: 08/14/2017 Amendment: J  
| NCT Number: NCT01853618  Title: A Pilot Study of Tremelimumab - A Monoclonal Antibody against CTLA-4 - in  combination with Trans-Arterial Catheter Chemoembolization (TACE), Radiofrequency  Ablation (RFA), or Cryoablation in Subjects with Hepatocellular Carcinoma (HCC) or Biliary  Tract Carcinomas (BTC) NCI Principal Investigator: Tim Greten, MD A-F  Thoracic & GI Oncology Branch   National Cancer Institute   Building 10, Room 12N226   9000 Rockville Pike   Bethesda, MD 20892   240-760-6114   gretentf@mail.nih.gov  NIH Associate Investigators: Changqing Xie, MD, HB, NHLBI A-F  Brad Wood MD, DRD/CC/NIH A,B,E,F  William D. Figg, PharmD, GMB/CCR/NCI E-F  Suzanne Fioravanti, RN, OCD/CCR/NCI A,B,C,E,F  Melissa Walker, RN, OCD/CCR/NCI A,B,C,E,F  Seth Steinberg, PhD, BDMS, CCR, NCI E-F   Gregoire Yves Altan-Bonnet, PhD, CIP, NCIE-F    Referral Contact/  Study Coordinator: Suzanne Fioravanti, RN, OCD, CCR, NCI   Building 10, Room 12N226   9000 Rockville Pike   Bethesda, MD 20892   240-760-6113   fioravas@mail.nih.gov Non-NIH Associate Investigators: Firouzeh Korangy, PhD, Kelly Government Solutions E-F   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Ratnadeep Mukherjee, PhD, Fellow, CIP, NCIE-F
Investigators Roles: A. Obtain information by intervening or interacting with living individuals for research purposes B. Obtaining identifiable private information about living individuals C. Obtaining the voluntary informed consent of individuals to be subjects D. Makes decisions about subject eligibility E. Studying, interpreting, or analyzing identifiable private information or data/specimens for research purposes F. Studying, interpreting, or analyzing de-identified data or specimens for research purposes G. Some/all research activities performed outside NIH Investigational Agents:  
| Drug Name:    | Tremelimumab                                          |
|---------------|-------------------------------------------------------|
| IND Number:   | 117537                                                |
| Sponsor:      | Center for Cancer Research, National Cancer Institute |
| Manufacturer: | MedImmune, Inc.                                       |
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precis background

x Worldwide, hepatocellular carcinoma (HCC) is the fifth most common malignancy with a median survival of 6-9 months. For patients with advanced disease sorafenib is the only approved drug and this has limited benefit.  
x Tremelimumab is a monoclonal antibody against CTLA4. Anti-CTLA4 therapy has been shown to enhance anti-tumor immunity by blocking tumor-induced immune suppression of cytotoxic T cells.  
x Various tumor ablative procedures and techniques have been shown to result in immunogenic cell death and induction of a peripheral immune response. Both transarterial catheter chemoembolization (TACE) and radiofrequency ablation (RFA) have been shown to do this, as well as cryoablation and external beam radiation x The underlying hypothesis of this study is that the effect of anti-CTLA4 treatment can be enhanced by TACE or RFA in patients with advanced hepatocellular carcinoma. We will also evaluate this in the context of cryoablation and radiation in HCC and RFA in cholangiocarcinoma.  
Objective:
x To assess the safety and feasibility of combining Tremelimumab with trans-arterial catheter chemoembolization (TACE) radiofrequency ablation (RFA) or cryoablation in patients with advanced HCC.  
Eligibility:
x Histologically or cytologically confirmed diagnosis of HCC or biliary tract carcinoma
(cohort E).  
x Childs-Pugh A/B7 cirrhosis only is allowed. If patient does not have cirrhosis, this limitation does not apply.  
x Barcelona Clinic Liver Cancer (BCLC) Stage B and C patients.  
x Patients must have disease that is not amenable to potentially curative resection, radiofrequency ablation, or liver transplantation.
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design

x The proposed study is a pilot study of Tremelimumab - at a planned dose 10mg/kg/IV q 4 weekly x 6 doses and then 1 dose every 12 weeks - in patients with advanced HCC or cholangiocarcinoma treated with ablative therapy for 2 years or until disease progression (as per immune-related RECIST criteria) whichever comes first.  
x The main study population is Cohort A as described below and these patients will be treated with either RFA or TACE. The decision on which treatment to administer (i.e. RFA vs. TACE) will be made in consultation with the interventional radiologists. The reason for this is that there are technical considerations (size and location of lesion, main portal vein occlusion etc.) which would make one procedure preferable over the other.  
x We will also enroll an additional cohort of up to N=20 patients (Cohort B) with liverconfined disease for whom TACE is indicated as standard of care. These patients will receive TACE in combination with Tremelimumab using the same schema. They will not receive RFA.  
x For Cohort A (BCLC Stage C patients): TACE or radiofrequency ablation (RFA) will be performed once only.  
x For Cohort B (BCLC Stage B patients): TACE may be repeated (as per standard of care)
on months 3, 7, and 13, and q6 months thereafter (if indicated).  
x Cohort B will only be recruited following completion of the dose-escalation portion of Cohort A.  
x Dose escalation: Initially three patients will be treated at 3.5mg/kg (with subsequent dosing also at 3.5mg/kg) prior to enrolling to the10mg/kg dose level. If a single patient experiences dose-limiting toxicity (DLT) related to the combination of TACE/RFA and Tremelimumab this cohort will be expanded to N=6 as per a standard 3+3 design. If 3.5mg/kg is established to be safe we will proceed to the 10mg/kg dose level.  
x Cohorts A+B: Following dose escalation up to N=40 patients will be treated at 10mg/kg
(20 patients in Cohort A and 20 in Cohort B).  
x Subjects will no longer enroll onto Cohort C effective with amendment I. x Cohorts D-E: N=Up to 30 patients will be treated at 10mg/kg.  
- Evaluation period for Dose-limiting Toxicities (DLT) will extend for the first 8 weeks of the study. Restaging CT /MRI scan every 8 weeks to evaluate TTP in target lesion. (Both standard RECIST and Immune-related Response Criteria [irRC] will be employed).  
- Once safety is established for the initial concept of combining anti-CTLA4 with ablative procedures (RFA and TACE), we will similarly explore feasibility of tremelimumab at the same dose and schedule in 2 additional cohorts (denoted D-E) evaluating tremelimumab in combination with cryoablation in HCC (cohort D) and with RFA in a cohort of patients with biliary tract carcinoma (cohort E). For these additional cohorts the dose (10mg/kg) and schedule of tremelimumab will be as per cohorts A and B. The timing of the interventional procedure will also be identical.  
| TABLE OF CONTENTS                                                                                                                            |                                                                                                                                            |                    |
|----------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------|--------------------|
| Précis                                                                                                                                       |                                                                                                                                            |                    |
| TABLE OF CONTENTS.                                                                                                                           |                                                                                                                                            |                    |
| INTRODUCTION .                                                                                                                               |                                                                                                                                            |                    |
| 1                                                                                                                                            | 1.1                                                                                                                                        | Study Objectives . |
| 1.1.1                                                                                                                                        | Primary:                                                                                                                                   |                    |
| 1.1.2                                                                                                                                        | Secondary:                                                                                                                                 |                    |
| 1.1.3                                                                                                                                        | Exploratory: .                                                                                                                             |                    |
| 1.2                                                                                                                                          | Background and Rationale .                                                                                                                 |                    |
| 1.2.1                                                                                                                                        | HCC and the current therapeutic paradigm (radiofrequency ablation [RFA] trans-                                                             |                    |
| arterial catheter chemoembolization [TACE] and sorafenib)                                                                                    |                                                                                                                                            |                    |
| 1.2.2                                                                                                                                        | Recent advances in immune-based approaches in solid tumor malignancies........ 12                                                          |                    |
| 1.2.3                                                                                                                                        | Immune response to interventional radiological treatments ....................................... 13                                       |                    |
| 1.2.4                                                                                                                                        | T Tremelimumab                                                                                                                             |                    |
| 1.2.5                                                                                                                                        | Justification for dose and schedule ................................................................................................... 16 |                    |
| 1.2.6                                                                                                                                        | Rationale for Re-Induction .                                                                                                               |                    |
| 1.2.7                                                                                                                                        | Rationale for Viral Hepatitis Studies and Specifically for Hepatitis B Surface                                                             |                    |
| Antigen and Hepatitis B Immune Monitoring ............................................................................................... 17 |                                                                                                                                            |                    |
| 1.2.8                                                                                                                                        | Rationale for Amendment F.                                                                                                                 |                    |
| 1.2.9                                                                                                                                        | RATIONALE FOR Amendment I                                                                                                                  |                    |
| 1.2.10                                                                                                                                       | Rationale for Amendment J                                                                                                                  |                    |
| 2                                                                                                                                            | ELIGIBILITY ASSESSMENT AND ENROLLMENT.                                                                                                     |                    |
| 2.1                                                                                                                                          | Eligibility Criteria .                                                                                                                     |                    |
| 2.1.1                                                                                                                                        | Inclusion Criteria .                                                                                                                       |                    |
| 2.1.2                                                                                                                                        | Exclusion Criteria .                                                                                                                       |                    |
| 2.1.3                                                                                                                                        | Re-Induction Criteria (if applicable)                                                                                                      |                    |
| 2.1.4                                                                                                                                        | Inclusion of Women and Minorities ................................................................................................ 24      |                    |
| 2.1.5                                                                                                                                        | Recruitment Strategies .                                                                                                                   |                    |
| 2.2                                                                                                                                          | Screening evaluation .                                                                                                                     |                    |
| 2.2.1                                                                                                                                        | History and Physical Examination                                                                                                           |                    |
| 2.2.2                                                                                                                                        | Laboratory Evaluation                                                                                                                      |                    |
| 2.2.3                                                                                                                                        | Histologic confirmation (at any time point prior to commencement) ................... 24                                                   |                    |  
6 2.3 Baseline Studies .  
2.3.1 Imaging studies (obtained within 28 days prior to first dose) ..................................... 25 2.3.2 Laboratory evaluation (obtained within 72 hours prior to first dose) ...................... 25 2.3.3 Laboratory evaluation (obtained within 28 days prior to first dose) ...................... 25 2.3.4 History and physical exam with vital signs (obtained within 1 week prior to first dose).  
25 3  
| 2.3.5                                                                                                | Electrocardiogram (obtained within 28 days prior to first dose) .............................. 25               |
|------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------|
| 2.4                                                                                                  | Registration Procedures                                                                                         |
| STUDY IMPLEMENTATION .                                                                               |                                                                                                                 |
| 3.1                                                                                                  | Study Design .                                                                                                  |
| 3.2                                                                                                  | SCHEDULE .                                                                                                      |
| 3.3                                                                                                  | Cohort A                                                                                                        |
| 3.3.1                                                                                                | Definition .                                                                                                    |
| 3.3.2                                                                                                | T Treatment                                                                                                     |
| 3.3.3                                                                                                | Protocol Stopping Rules                                                                                         |
| 3.3.4                                                                                                | R-Induction                                                                                                     |
| 3.4                                                                                                  | Cohort B                                                                                                        |
| DEFINITION .                                                                                         |                                                                                                                 |
| 3.4.1                                                                                                |                                                                                                                 |
| 3.4.2                                                                                                | T Treatment                                                                                                     |
| 3.5                                                                                                  | Cohort C                                                                                                        |
| 3.6                                                                                                  | Cohorts D-E.                                                                                                    |
| Dose Limiting Toxicities                                                                             |                                                                                                                 |
| 3.7                                                                                                  | Definition of Dose-limiting Toxicities (DLTs): ............................................................. 30 |
| 3.7.1                                                                                                |                                                                                                                 |
| 3.7.2                                                                                                | Exclusions to Dose Limiting Toxicities                                                                          |
| 3.8                                                                                                  | D Drug Administration                                                                                           |
| Tremelimumab Drug Administration .                                                                   |                                                                                                                 |
| 3.8.1                                                                                                |                                                                                                                 |
| 3.8.2                                                                                                | Monitoring of Dose Administration                                                                               |
| 3.9                                                                                                  | Dosing Delays                                                                                                   |
| Dosing Delays/Dose Modifications: General Guidelines ............................................ 33 |                                                                                                                 |
| 3.9.1                                                                                                |                                                                                                                 |
| 3.9.2                                                                                                | Toxicities Mandating Permanent Discontinuation of Tremelimumab                                                  |
| 3.9.3                                                                                                | Specific immune-mediated adverse reactions                                                                      |
| Specific Procedures for TACE, RFA and Cryoablation                                                   |                                                                                                                 |
| 3.10                                                                                                 | Transarterial chemoembolization                                                                                 |
| 3.10.1                                                                                               |                                                                                                                 |  
| Version Date: 08/14/2017        |                                                                                                                       |                 |
|---------------------------------|-----------------------------------------------------------------------------------------------------------------------|-----------------|
| 3.10.2                          | Radiofrequency ablation                                                                                               |                 |
| 3.10.3                          | Cryoablation                                                                                                          |                 |
| 3.11                            | Study Calendar .                                                                                                      |                 |
| 3.12                            | Criteria for Removal from Protocol Therapy and Off Study Criteria ....................... 44                          |                 |
| 3.12.1                          | Criteria for removal from protocol therapy ........................................................................ 4 |                 |
| Criteria for Removal from Study |                                                                                                                       |                 |
| 3.12.2                          |                                                                                                                       |                 |
| 3.12.3                          | Off protocol therapy and Off Study Procedure                                                                          |                 |
| 4                               | CONCOMITANT MEDICATIONS/MEASURES                                                                                      |                 |
| S                               | BIOSPECIMEN COLLECTION                                                                                                |                 |
| 5.1                             | CORRELATIVE STUDIES FOR RESEARCH/PHARMACOKINETIC STUDIES... 45                                                        |                 |
| 5.2                             | NCI Correlative Studies                                                                                               |                 |
| 5.2.1                           | Immune Monitoring (All cohorts)                                                                                       |                 |
| 5.2.2                           | Liver autoantibody panel for autoimmune hepatitis (AIH) ........................................ 48                   |                 |
| 5.2.3                           | Hepatitis serology and viral load                                                                                     |                 |
| 5.2.4                           | MDSC functional assay                                                                                                 |                 |
| 5.2.5                           | Optional Tumor Biopsy                                                                                                 |                 |
| 5.2.6                           | T TCR Beta Sequencing                                                                                                 |                 |
| 5.3                             | MedImmune Planned Studies                                                                                             |                 |
| 5.3.1                           | Pharmacokinetics .                                                                                                    |                 |
| Immunogenicity .                |                                                                                                                       |                 |
| 5.3.2                           |                                                                                                                       |                 |
| 5.3.3                           | T CELL Activation/ICOS expression                                                                                     |                 |
| 5.3.4                           | Plasma-based assays for circulating receptors/ligands e.g. PDL1 ............................. 50                      |                 |
| 5.4                             | Sample Storage, Tracking and Disposition                                                                              |                 |
| 5.4.1                           | Sample Data Collection .                                                                                              |                 |
| 5.4.2                           | Sample Storage and Destruction                                                                                        |                 |
| 5.4.3                           | Protocol Completion/Sample Destruction .                                                                              |                 |
| DATA COLLECTION AND EVALUATION  |                                                                                                                       |                 |
| 6                               | 6.1                                                                                                                   | DATA COLLECTION |
| 6.2                             | DATA SHARING Plans .                                                                                                  |                 |
| 6.2.1                           | HUMAN DATA SHARING PLANS                                                                                              |                 |
| 6.3                             | Response Criteria                                                                                                     |                 |
| 6.3.1                           | Definitions                                                                                                           |                 |
| 6.3.2                           | Disease Parameters .                                                                                                  |                 |  
7 6.3.3 Methods for Evaluation of Measurable Disease .  
| 6.3.4                                              | Response Criteria      |
|----------------------------------------------------|------------------------|
| 6.3.5                                              | D Duration of Response |
| Modified immune-related response criteria (irRC) . |                        |
| Toxicity Criteria                                  |                        |  
6.4 6.5 7 SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN
59  
| 7.1                                      | DEFINITIONS                                                                                                                              |
|------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------|
| 7.1.1                                    | A Adverse Event                                                                                                                          |
| 7.1.2                                    | Suspected adverse reaction .                                                                                                             |
| Unexpected adverse reaction              |                                                                                                                                          |
| 7.1.3                                    |                                                                                                                                          |
| 7.1.4                                    | Serious                                                                                                                                  |
| 7.1.5                                    | Serious Adverse Event                                                                                                                    |
| 7.1.6                                    | Disability                                                                                                                               |
| Life-threatening adverse drug experience |                                                                                                                                          |
| 7.1.7                                    |                                                                                                                                          |
| 7.1.8                                    | Protocol Deviation (NIH Definition)                                                                                                      |
| 7.1.9                                    | Protocol Non-compliance (NIH Definition) ........................................................................... 60                  |
| 7.1.10                                   | Unanticipated Problem                                                                                                                    |
| 7.2                                      | NCI-IRB and Clinical Director Reporting ............................................................................................. 61 |
| 7.2.1                                    | NCI-IRB and NCI CD Expedited Reporting of Adverse Events , and Deaths...... 61                                                           |
| 7.2.2                                    | NCI-IRB Requirements for PI Reporting at Continuing Review .............................. 61                                             |
| 7.2.3                                    | NCI-IRB Reporting of IND Safety Reports.............................................................................. 62                 |
| 7.3                                      | IND Sponsor Reporting Criteria .                                                                                                         |
| 7.3.1                                    | Reporting Pregnancy                                                                                                                      |
| 7.4                                      | Safety Reporting criteria to the Pharmaceutical Collaborators .............................................. 63                          |
| 7.5                                      | DATA AND SAFETY Monitoring Plan                                                                                                          |
| 7.5.1                                    | Principal Investigator/Research Team ............................................................................................. 63    |
| 7.5.2                                    | Sponsor Monitoring Plan                                                                                                                  |
| STATISTICAL CONSIDERATIONS               |                                                                                                                                          |
| 8.1                                      | Cohorts A and B                                                                                                                          |
| 8.2                                      | Cohorts D- E                                                                                                                             |
| 8.3                                      | Secondary and Exploratory Analyses .                                                                                                     |
| Accrual                                  |                                                                                                                                          |
| 8.4                                      |                                                                                                                                          |  
8 8  
| 9      | COLLABORATIVE AGREEMENTS 66                                     |
|--------|-----------------------------------------------------------------|
| 9.1    | COOPERATIVE RESEARCH AND DEVELOPMENT AGREEMENT (CRADA) ... 66   |
| 9.2    | MATERIAL TRANSFER AGREEMENTS (MTA) 66                           |
| 10     | HUMAN SUBJECTS PROTECTIONS 66                                   |
| 10.1   | Rationale For Subject Selection 66                              |
| 10.2   | Participation of Children 67                                    |
| 10.3   | Participation of Subjects Unable to Give Consent 67             |
| 10.4   | Evaluation of Benefits and Risks/Discomforts 67                 |
| 10.4.1 | Risks of exposure to ionizing radiation 67                      |
| 10.4.2 | Risk of optional biopsy 67                                      |
| 10.4.3 | Other risks/benefits 67                                         |
| 10.5   | Risks/Benefits Analysis 67                                      |
| 10.6   | Consent and Assent Process and Documentation 68                 |
| 10.6.1 | Telephone re-consent 68                                         |
| 10.6.2 | Informed consent of non-English speaking subjects 68            |
| 11     | PHARMACEUTICAL INFORMATION 69                                   |
| 11.1   | Tremelimumab 69                                                 |
| 11.1.1 | Investigational Product Dose Preparation 69                     |
| 11.1.2 | Dose Calculation 70                                             |
| 11.1.3 | Investigational Product Inspection 70                           |
| 11.1.4 | Dose Preparation Steps 70                                       |
| 11.1.5 | Investigational Product Accountability 71                       |
| 11.1.6 | Reporting Product Complaints 71                                 |
| 12     | REFERENCES 73                                                   |
| 13     | APPENDICES 78                                                   |
| 13.1   | APPENDIX A: Performance Status Criteria 78                      |
| 13.2   | Appendix B: Child-Pugh classification system 79                 |
| 13.3   | Appendix C: Modified immune-related response criteria (irRC) 80 |
| 13.4   | Appendix D: MedImmune Sample Preparation Procedures 82          |
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1 introduction 1.1 study objectives

To assess the safety and feasibility of combining Tremelimumab with trans-arterial catheter chemoembolization (TACE) or radiofrequency ablation (RFA) in patients with advanced HCC.
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1.1.2 secondary

To evaluate clinical indicators of efficacy (response rate, time to tumor progression, overall survival) in patients with advanced HCC undergoing TACE or radiofrequency ablation (RFA) in combination with Tremelimumab.
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1.1.3 exploratory

1.1.3.1 To evaluate changes in immune parameters as well as pharmacokinetics in the peripheral blood of patients with advanced HCC undergoing TACE or radiofrequency ablation (RFA) in combination with Tremelimumab 1.1.3.2 Following amendment F we will evaluate safety and feasibility of combining Tremelimumab with cryoablation in patients with advanced HCC and radiofrequency ablation (RFA) in advanced intrahepatic cholangiocarcinoma.  
1.1.3.3 To evaluate changes in immune parameters as well as pharmacokinetics in the peripheral blood of patients with advanced HCC or intrahepatic cholangiocarcinoma undergoing TACE, radiofrequency ablation (RFA) or cryoablation in combination with Tremelimumab.
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1.2 background and rationale

1.2.1 HCC and the current therapeutic paradigm (radiofrequency ablation [RFA] trans-arterial catheter chemoembolization [TACE] and sorafenib)
Worldwide, hepatocellular carcinoma (HCC) is the fifth most common malignancy with a median survival of 6-9 months1. The therapeutic paradigm - including the possible interventions and their indications - is outlined below. The approach to management has traditionally comprised of loco-regional strategies: surgery (partial resection or transplantation) or interventional radiologic procedures, such as chemoembolization or ablative techniques. Recently, sorafenib has been added to this paradigm and it is the only systemic drug therapy which has demonstrated a survival benefit in modern randomized studies.  
| Intervention        | Indication                 | Potentially curative   |
|---------------------|----------------------------|------------------------|
| Partial Hepatectomy | Limited liver disease with | Yes                    |  
| god reserve                   |                                |     |
|-------------------------------|--------------------------------|-----|
| Transplantation               | 1 lesion <5cm or 3 < 3cm       | Yes |
| RFA                           | Lesion to be ablated at least  | Yes |
| lcm and not greater than 5cm. |                                |     |
| TACE                          | Liver confined; no portal vein | No  |
| thrombosis                    |                                |     |
| Sorafenib                     | Metastatic disease             | No  |  
Version Date: 08/14/2017 The most widely used staging system in HCC is the Barcelona Clinic Liver Cancer (BCLC)
Staging system, the major advantage of which is that in addition to staging patients it also directs management 2 . The BCLC staging system is shown in Figure 1 below.  
  
  
For the proposed study we will not include patients for whom surgical resection or transplantation is a possibility. We will also exclude patients with isolated lesions for whom l l 12 RFA alone is possible, given the curative potential of that intervention. The target population will be those with advanced disease for whom the only available interventions are TACE or sorafenib. 1.2.2 Recent advances in immune-based approaches in solid tumor malignancies The past two years have seen progress for immune-based approaches in solid tumor malignancies, with FDA approvals for these approaches in prostate cancer and melanoma3-5. In melanoma, anti-CTLA4 therapy has been shown to demonstrate a median survival benefit in two separate phase 3 studies, both of which were associated with long-term disease control in approximately one-fifth of patients. More recently, anti-PD1 therapy has demonstrated a similar degree of clinical activity not only in melanoma and kidney cancer but also in lung cancer, a disease type previously thought to be refractory to an immune approach. Appreciation of the role in developing tumors of immune-evasion has also been evidenced by its inclusion as one of the
(updated) hallmarks of cancer6.  
Both anti-PD1 and anti-CTLA4 therapy enhance anti-tumor immunity by blocking tumor induced immune suppression of cytotoxic T cells and therefore exaggerating the immune activation that must first occur of its own accord and is thought to be the result of tumor neoantigens formed as a tumor progresses. The role of CTLA4 in inhibiting T-cell activation is represented in the schematic in Figure 2 below. CTLA4-mediated immune checkpoint is induced in T cells at the time of their initial response to antigen. The level of CTLA4 induction depends on the amplitude of the initial T cell receptor (TCR)-mediated signaling. After the TCR is triggered by antigen encounter, CTLA4 is transported to the cell surface. The stronger the stimulation through the TCR (and CD28), the greater the amount of CTLA4 that is deposited on the T cell surface. Therefore, CTLA4 functions as a signal dampener to maintain a consistent level of T cell activation in the face of widely varying concentrations and affinities of ligand for the TCR.  
  
Whilst these studies demonstrate the potential for anti-CTLA4 (or anti PD1) therapy to enhance the anti-tumor immune response already in process, it clearly does not work in the majority of patients. The question is whether the initial immune activation stage can be enhanced. The potential for this was intriguingly demonstrated in a recent publication by Postow et al who reported a case of the so-called abscopal effect in a patient with melanoma treated with ipilimumab and radiotherapy8. Temporal associations were noted between tumor shrinkage and antibody responses to the cancer-testis antigen NY-ESO-1 in addition to changes in peripheralblood immune cells, and increases in antibody responses to other antigens after a patient being treated with anti-CTLA4 was then treated with external beam radiotherapy. The enhanced effect from anti-CTLA4 seen after radiation appears to have been due to a boost effect of tumor cell death and immune stimulation following deliverance of a tumor antigen load. It is this enhanced effect we wish to study in the context of interventional procedures for HCC.
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1.2.3 immune response to interventional

The aim of our proposed study is to enhance the antitumor immune response which has been shown to occur following interventional radiological treatments. The attractive aspect of this approach - especially in a disease as prevalent as HCC - is that it has the potential for enhancing the effect of treatments (both RFA and TACE) which have already been approved, have demonstrated survival benefit in randomized studies and are commonly performed throughout the community. Following RFA and TACE necrotic tumor results in the release of tumor antigens which are taken up by antigen-presenting cells (mainly dendritic cells) and which activate a tumor-specific immune response9. Ablated tumor tissue has been shown to promote dendritic cell maturation (and resultant T-cell stimulatory properties)10,11. This antigen release is potentially significant because, although ablative procedures are very effective in eradicating visible lesions, a tumor-specific immune response may prevent recurrent disease in addition to treating distant metastases. In other words, RFA and TACE have the potential to turn a patient's tumor into an endogenous vaccine. Several studies have documented an increase in peripheral antitumor immunity following interventional radiological procedures, predominantly RFA10,12-15. This has been demonstrated both in animal models and in human studies12,14,15. Ayaru et al. evaluated the immune response in HCC patients (N=10) undergoing TACE16. They found that AFP-specific CD4-T cell responses were significantly expanded during (p < 0.0001) and after embolisation (p < 0.002). The development of higher frequencies of AFP-specific CD4 T cells after treatment were significantly associated with the induction of >50% necrosis of tumor and an improved clinical outcome (p < 0.007). Mizukoshi et al. measured cytotoxic T cell response to tumor associated antigens (TAA) in HCC patients undergoing treatment17. They measured the frequency of TAAspecific T cells before and after HCC treatment by ELISPOT assay in 12 cases who received TACE, RFA or chemotherapy. The frequency of TAA-specific T cells was found to be increased in all patients. Furthermore, incubation of T cells with CTLA-4 antibodies resulted in an increase of the number of TAA-specific T cells. Dromi et al. evaluated the influence of subtotal RFA on tumor-specific immune response in a murine urothelial carcinoma (MB49) tumor model18. RFA
resulted in enhanced systemic antitumor T-cell immune responses and tumor regression that was associated with increased dendritic cell infiltration into the remaining viable, non-ablated tumor. Hansler et al. assessed the specific cytotoxic T cell response in patients with malignant liver tumors (both metastatic colon cancer and HCC) which were treated with RFA. Significant tumor-specific cytotoxic T-cell stimulation was seen, with a dramatically increased tumor specific cytolytic activity of CD8(+) T cells against autologous liver and tumor lysate after RFA12. Hiroishi et al. analyzed tumor-specific CD8(+) T-cell responses in twenty patients with HCC undergoing loco-regional therapy (either RFA or TACE)13. They found that the CD8(+) T-
cell response was increased and, on multivariate analysis, the magnitude of the immune response was the only significant prognostic factor for a prolonged tumor-free interval (hazard ratio 0.342, P = 0.022).  
Despite this, only a few cases of spontaneous remission of metastases following RFA - the socalled abscopal effect - have been reported, presumably because this immune response by itself is too weak to be clinically significant19-21. A number of studies have tried to boost this antitumor immune response following ablation by combining with an immunomodulatory agent:
Waitz et al. provided preclinical proof-of-concept for this in a mouse model of prostate cancer using CTLA-4 blockade to augment the immune response from cryoablation22. The combination therapy resulted in >80% of intratumoral CD8+ T cells being antigen-specific. The effect on secondary responses was assessed by injection of the secondary tumor on the opposite flank of the mouse on Day 2 following ablation. The growth of secondary tumors was found to be unaffected by cryoablation alone, but the combination treatment (anti-CTLA4 + RFA) was sufficient to slow growth or trigger rejection. In addition, secondary tumors were highly infiltrated by CD4+ T cells and CD8+ T cells, and there was a significant increase in the ratio of intratumoral T effector cells to T regulatory cells, compared with either RFA or anti-CTLA4 alone. Johnson et al. sought to enhance the antitumor effect of RFA by combining it with huKSIL2 immunocytokine [tumor-specific monoclonal antibody fused to interleukin-2 (IL2)] in mice bearing CT26-KS colon adenocarcinoma. Mice were treated with RFA, huKS-IL2 via intratumoral injection, or combination therapy. Treatment of mice bearing s.c. tumors with RFA and huKS-IL2 resulted in significantly greater tumor growth suppression and enhanced survival compared with mice treated with either RFA or huKS-IL2 alone. When sub-therapeutic regimens of RFA or huKS-IL2 were used, tumors progressed in all treated mice. In contrast, the combination of RFA and immunocytokine resulted in complete tumor resolution in 50% of mice. Treatment of a tumor with RFA and intratumoral huKS-IL2 also showed antitumor effects against a distant untreated tumor. Tumor-free mice after treatment with RFA and huKS-IL2 showed immunologic memory based on their ability to reject subsequent challenges of CT26-KS and the more aggressive parental CT26 tumors. Flow cytometry analysis of tumor-reactive T cells from mice with complete tumor resolution showed that treatment with RFA and huKS-IL2 resulted in a greater proportion of cytokine-producing CD4 T cells and CD8 T cells compared with mice treated with RFA or huKS-IL2 alone. den Brok et al. evaluated combination treatment of RFA plus TLR9 stimulation in B16 tumors and showed that the combination resulted in far more potent antitumor immune responses than either treatment modality alone23.
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1.2.4 tremelimumab tremelimumab (formerly cp-675206)

Upon T cell activation, CTLA-4 expression acts to dampen immune responses by CTLA-4 relocation to the cell surface in order to modulate and eventually switch off T cell activation. Tremelimumab blocks the inhibitory effect of CTLA-4, and therefore enhances T cell activation. The binding of CTLA-4 to its target ligands (B7.1 [CD80] and B7.2 [CD86]) provides a negative regulatory signal, which limits T cell activation. Blockade of B7 binding to CTLA-4 by antiCTLA-4 antibodies also results in markedly enhanced T cell activation and antitumor activity in animal models, including killing of established murine solid tumors and induction of protective antitumor immunity. Anti-CTLA-4 therapy has recently been shown to be a validated approach to cancer treatment by the approval of the mAb ipilimumab in 2011 for the treatment of patients with metastatic melanoma, based on 2 Phase III studies that demonstrated a significant improvement on OS in the first- and second-line settings. In general, tumor response rates to anti-CTLA-4 therapy are low, approximately 10%, but the durable response or stable disease seen in a proportion of patients can lead to a significant prolongation of OS. In a large, single-arm Phase II Tremelimumab trial in patients with advanced refractory and/or relapsed melanoma, objective responses (primary endpoint) following Tremelimumab 15 mg/kg Q3M were observed in 16 of SDWLHQWV&,>@5HVSRQVHVZHUHGXUDEOHSUHVHQWDWPRQWKV from enrollment) in all 16 responders. A Phase III, open-label, randomized study comparing Tremelimumab 15 mg/kg Q3M (Arm A) to either dacarbazine or temozolomide (Arm B) in patients with advanced melanoma was terminated following a pre-specified interim futility analysis. At the time of database lock, the median OS (primary endpoint) was 12.58 months in Arm A and 10.71 months in Arm B (HR=1.1416, p=0.1272).  
The efficacy data for tremelimumab are consistent with those of the related - and FDAapproved- anti-CTLA-4 antibody ipilimumab: tumor response rates are generally low
(approximately 10%) but the responses observed are generally durable. The ipilimumab melanoma data clearly demonstrate that a small proportion of patients with an objective response and a small impact on PFS rates can lead to a significant prolongation of OS, and support development of this class of agent in other tumors. Although Phase II and Phase III studies of tremelimumab in metastatic melanoma failed to meet the primary endpoints of response rate and OS, respectively, the data clearly indicate activity of tremelimumab in melanoma, with response rates and median OS similar to those observed in the ipilimumab trials. However, in contrast to tremelimumab, there are no safety data for the use of ipilimumab in HCC.
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1.2.4.1 clinical experience of tremelimumab

Tremelimumab has been evaluated in a number of clinical studies - and over 1000 patients - and demonstrated manageable toxicities24,25.  
The efficacy data for Tremelimumab are consistent with those of the related anti-CTLA-4 antibody ipilimumab: tumor response rates are generally low (approximately 10%) but the responses observed are generally durable. The ipilimumab melanoma data clearly demonstrate that a small proportion of patients with an objective response and a small impact on PFS rates can lead to a significant prolongation of OS, and support development of this class of agent in other tumors. Although Phase II and Phase III studies of Tremelimumab in metastatic melanoma failed to meet the primary endpoints of response rate and OS, respectively, the data clearly indicate activity of Tremelimumab in melanoma, with response rates and median OS similar to those observed in the ipilimumab trials. The AE profile of Tremelimumab is consistent with that of ipilimumab, and with the pharmacology of the target. To date, no tumor type or stage appears to be associated with unique AEs (except for vitiligo, which appears to be confined to patients with melanoma). Events reported at a frequency oIDQGDVVHVVHGE\WKHLQYHVWLJDWRUDVUHODWHGWRWUHDWPHQWOLVWHGLQ descending order of frequency) were diarrhea, rash, pruritus, fatigue, nausea, vomiting, anorexia, headache, abdominal pain, and colitis. 1.2.4.2 Experience in Hepatitis/HCC Tremelimumab has recently been shown to be safe in a study in HCC. Twenty-one patients with HCV-related HCC were treated with Tremelimumab intravenously at a dose of 15 mg/kg every 90 days for two cycles. Toxicity was manageable with reported treatment-related adverse events among 80% of patients. Grade 3 or higher adverse events included 1 case of pruritus, 1 case of purpura, and 5 cases of elevated transaminases. There was preliminary evidence of efficacy. In an intention-to-treat analysis, investigators observed a median overall survival of 7.5 months and time to progression of 6.4 months as well as a reduction of hepatitis C virus (HCV) in the patients' blood, which was also accompanied with objective enhancements of antiviral immunity. A significant and progressive decline in serum HCV viral load was observed (median values: baseline 3.78x10e5 copies/ml vs. day 120 3.02x10e4 copies/ml, P = .02; vs. day 210 1.69x10e3 copies/ml, P = .04).
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1.2.5 justification for dose and

There is safety data from ~70 patients treated with 10 mg/kg Q4 weeks and currently an ongoing study is collecting additional data on this dose and schedule. The proposed dose and schedule is informed by safety and efficacy data on Tremelimumab, and by data showing a relationship between exposure and survival in the advanced melanoma studies with Tremelimumab. The PK of Tremelimumab has been characterized in various clinical studies with more than 1,000 patients treated with Tremelimumab to date. Tremelimumab has been administered at doses ranging from 3 to 15 mg/kg Q4W or Q12W. The PK of Tremelimumab was linear and doseproportional. The disposition of Tremelimumab exhibits biphasic elimination, with a long terminal phase half-life of ~20 days. In an early Phase 1/2 study (A3671002; Camacho et al, 2009), patients with melanoma in the Phase 2 portion of the study were randomly assigned to Tremelimumab 15 mg/kg Q12W (n = 46) or Tremelimumab 10 mg/kg Q4W (n = 44). The overall AE frequency between the 2 schedules was comparable, albeit with fewer Grade 3-4 AEs (13%) in the less frequent dosing regimen (15 mg/kg Q12W) compared to the Q4W regimen (27%). The difference in Grade 3-4 AEs was largely due to differences in the incidence of *UDGHGLDUUKHDYV. 9%). However, all SAEs were manageable and reversible when appropriate intervention was applied. Efficacy results of this study showed similar response rates in each arm (10% for 10 mg/kg Q4W, 9% for 15 mg/kg Q12W) with no significant difference in OS.  
However, a retrospective analysis of data from Phase 2 and Phase 3 melanoma studies with15 mg/kg Q12W Tremelimumab showed an improvement in the OS outcome for patients who achieved a higher exposure as measured by the area under the concentration-time curve from time 0 to 90 days (AUC90). Furthermore, pharmacokinetic simulations indicate that following Tremelimumab at 10 mg/kg Q4W, approximately 90% patients are expected to be above the target concentration aȝJP/) compared to ~50% with 15 mg/kg every Q12W dosing regimen. Based on these data, MedImmune now proposes using a Tremelimumab dosing regimen of 10 mg/kg Q4W both for this study and across all other studies.
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1.2.6 rationale for re-induction

Re-treatment with Anti-CTLA-4 agent provide durable response or stable disease in patients with melanoma who initially respond to treatment but later progressed26,27 with some subjects achieved better overall response after re-induction4. No new types of toxicities observed after reinduction with safety profile consistent with induction profile.26,27 These findings support retreatment with immune checkpoint inhibitors after initial disease control.  
RFA or TACE are modalities inducing localized cell death and enhancing antitumor immune response28,29. To date it is unknown what type of treatment, RFA vs TACE is more immunogenic. By allowing the same patient to undergo TACE and RFA we will produce different immune stimulation and possibly enhance antitumor immune response. In addition, benefit of re-treatment with TACE or RFA is to provide a similar immune boost as revaccination treatment in cancer vaccine clinical trials. Combining TACE and RFA in HCC treatments has been atttemted in single centers small studies showing an improvement in OS without significant increase in side effects30,31. Larger randomized clinical trial of 189 patiens resulted in improvement in OS and recurrence free survival in RFA-Tace group relative to RFA group alone whithout significant differences in toxicity profiles.32 Findings from these studies confrim tolerability of combination. For definite conclusions on efficacy of combined interventional management larger clinical trials are needed. 1.2.7 Rationale for Viral Hepatitis Studies and Specifically for Hepatitis B Surface Antigen and Hepatitis B Immune Monitoring Anti-HCV specific immune responses as well as a reduction in Hepatitis C viral load have been described in in patients with chronic HCV infection and HCC (decline in serum HCV viral load from median values: basal 3.78x10e5 copies/ml to median values on day 120 of 3.02x10e4 copies/ml, p=0.02; vs. day 210 1.69x10e3 copies/ml, p=0.04); in addition three patients demonstrated a complete viral response for the duration of the follow-up33.  
The antiviral effect of CTLA-4 blockade in patients with chronic HBV infection is unknown. Hepatitis e antigen (HBeAg) is a marker of viral replication and infectivity that is usually associated with and active liver disease. Hepatitis B surface antigen (HBsAg) is a protein on the surface of the hepatitis B virus. Its presence identifies patients with chronic infection. CTLA-4 blockade in cynomolgus macaques enhanced antibody response to hepatitis B surface antigen
(HBsAg)34. Interestingly, polymorphisms of CTLA-4 have been associated with chronic hepatitis B and C infection35,36. The mechanisms behind these events imply an existing link between CTLA-4 activity and hepatic viral pathogenesis that should be exploited in details for therapeutic benefit. The population of HCC patients with chronic hepatitis B virus (HBV) infection will provide an exceptional occasion to simultaneously test the anti-tumoral, immune and antiviral effect of tremelimumab.
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1.2.8 rationale for amendment f

The purpose of amendment F is to add 3 additional patient cohorts (denoted C, D and E) of N=10 patients each to evaluate additional globally used and potentially immunomodulative procedures (cryoablation and external beam radiation) in patients with HCC. We would also like to evaluate RFA plus tremelimumab in biliary tract carcinoma. The justification for each of these cohorts is as follows: Cohort C: SBRT plus tremelimumab in HCC (*removed, effective with amendment I*). Several studies have documented an increase in peripheral antitumor immunity following radiation. It may even be the case that an intact immune system is critical for RT to exert its maximal antitumor effect. This was suggested by a mouse model of melanoma in which single-fraction RT slowed the growth of small tumors in immune-competent but not immune-deficient mice37.  
The same authors also showed that in an animal model of breast cancer ablative RT of a primary tumor prevented the growth of metastatic tumor colonies in the lung, an effect that was dependent on the presence of CD8+ T cells. Other animal models have likewise displayed augmentation of systemic antitumor immunity following local RT38-40. The underlying mechanism appears to be that RT-induced necrosis results in the exposure of tumor antigens, thereby increasing the pool of intracellular peptides for cross-presentation41. Radiation has been shown to augment MHC-I expression by tumors, which is critical for antigen recognition by cognate CD8+ TCRs and which is known to be diminished in tumors as one of their escape mechanisms42. Tumor antigen processing and presentation on MHC-I molecules is dependent on expression of a protein called highmobility group box 1 (HMGB-1), a "danger signal" which binds toll-like receptor 4 (TLR4) on dendritic cells. In a pivotal study Apetoh et al. demonstrated that RT causes dying tumor cells to release HMGB-143. Intriguingly these authors also reported that in breast cancer patients undergoing radiation and chemotherapy the presence of a polymorphism in TLR4 - and by implication a less immunogenic drug-induced cell death - was associated with an inferior prognosis. Similarly, in esophageal cancer patients preoperative chemoradiation has been shown to increase cancer-specific T cell responses and serum levels of HMGB-1, the latter of which correlated with overall survival44.  
Despite this, only a few cases of spontaneous decrease of metastases following radiation - the so-called abscopal effect - have been reported. This is presumably because the immune response by itself is too weak to be clinically significant. A number of studies have tried to boost this antitumor immune response following ablation by combining with an immunomodulatory agent:
Dewan et al. evaluated RT in combination with anti-CTLA-4 antibody in two separate mouse models of breast and colorectal carcinoma45. The authors found that the combination of antiCTLA4 and RT achieved enhanced tumor response at the primary site (compared to either modality alone). in addition to an abscopal effect. (Interestingly this only occurred in fractionated versus single-dose RT). The frequency of CD8+ T cells showing tumor-specific IFN-gamma production was proportional to the absopal effect. Demaria et al. tested the combination of RT with CTLA-4 blockade in a breast cancer model (4T1) known to be poorly immunogenic46. Anti-CTLA4 alone did not have any effect on primary tumor growth or survival.  
RT was able to delay the growth of the primary irradiated tumor, but in the absence of antiCTLA4 survival was similar to that of control mice. In contrast, mice treated with RT + antiCTLA4 had a statistically significant survival advantage. The increased survival correlated with inhibition of lung metastases formation and required CD8+. Administering radiation therapy to the liver is not standard or conventional. With newer techniques however, this has been shown to be a safe strategy and to have palliative benefit in an incurable population. For example, Dr. Citrin - one of our associate investigators - has previously conducted a study employing hepatic radiation in colorectal cancer47. In that study patients received a total radiation dose of 32 Gy to sites of metastatic disease in the liver, delivered in 8-Gy courses (4 separate 2-Gy fractions) beginning one day after each vaccine boost (days 22–25, 36–39, 50–53, 64–67). The treatment was well tolerated, with the most common toxicity being a transient dermatologic reaction as a result of the vaccine used in the study. There ZHUHQRJUDGHWR[LFLWLHVDWWULEXWDEOHWRWKHWUHDWPHQWDQGVSHFLILFDOO\WKHUHZHUHQRJUDGH hepatic toxicities. Other additional reports have documented the safety of SBRT to the liver at doses/schedules of 60 Gy/10 Fr and 50 Gy/4 Fr48; 36 to 60 Gy in 6 to 15 Gy per fraction (HCC,
cholangio and colorectal cancer)49; single doses of 17 to 30 Gy (median dose 24 Gy)50; median dose per fraction and total dose of 3.6 Gy (2.0-13.0 Gy) and 55 Gy (30-80 Gy) in N=26patients with colorectal cancer51.  
Cohort D: Cryoablation and tremelimumab in HCC. Whilst RFA is the more traditional ablative strategy in HCC there is a growing use of cryoablation globally. In addition there is a growing body of data which suggests that it may be more effective52. At a minimum it appears to be equivalent in terms of safety and efficacy, as recently shown in a recent multicenter randomized study comparing the outcomes of percutaneous cryoablation with RFA for the treatment of HCC52. Three hundred and sixty patients with Child-Pugh class A or B cirrhosis and one or two
+&&OHVLRQVFPWUHDWPHQWQDwYHZLWKRXWPHWDVWDVLVZHUHUDQGRPO\DVVLJQHGWRFU\RDEODWLRQ (n=180) or RFA (n=180). The primary end-points were local tumor progression at 3 years after treatment, and safety. Local tumor progression rates at 1, 2, and 3 years were 3%, 7%, and 7% for cryoablation and 9%, 11%, and 11% for RFA, respectively (P=0.043). For lesions >3 cm in diameter, local tumor progression rate was significantly lower in cryoablation group versus RFA group (7.7% vs 18.2%, P=0.041). The 1-, 3-, and 5-year overall survival rates were 97%, 67% and 40%, for cryoablation and 97%, 66%, and 38% for RFA, respectively (P=0.747). The 1-, 3-, and 5-year tumor-free survival rates were 89%, 54%, and 35% in cryoablation group and 84%, 50%, and 34% in RFA group, respectively (P=0.628). Major complications occurred in seven patients (3.9%) following cryoablation and in six patients (3.3%) following RFA (P=0.776). The conclusions of this study were that cryoablation appeared to be just as safe and feasible as radiofrequency or microwave ablation in HCC and is very commonly used across the world. Intriguingly - for our purposes - there is evidence that cryoablation may be better in terms of immune stimulation compared to other ablative strategies53.  
Cohort E: RFA in biliary tract carcinoma: Biliary tract carcinoma (BTC) is the second most common hepatobiliary cancer. For the majority of patients who present with or develop metastatic/ unresectable disease the modest standard therapy comprises Gemcitabine in combination with cisplatin based on the ABC-02 trial which demonstrated a median overall survival benefit of 11.7 v 8.1 months (P<0.001) in favor of cisplatin–gemcitabine compared to gemcitabine alone54. There is no standard second-line treatment for biliary tract carcinomas.  
The most compelling argument in favor of testing immune-based strategies (and anti-PD1 therapy in particular) in this disease is that chronic inflammation is the most common etiologic factor in the development of BTC. Recent studies have linked the increasing incidence of chronic hepatitis C virus infection with intrahepatic cholangiocarcinoma55, which may explain the increasing incidence of the intrahepatic subtype 56. In biliary tract cancer, the prevalence and prognostic relevance of infiltrating T-lymphocytes has been documented. Goeppert et al. evaluated immune cell infiltration in 375 cases of biliary tract cancer obtained following surgical resection57. Approximately half the patients had intraepithelial tumor infiltrating T cells (both CD4 and CD8) and this was associated with better survival. Further evidence of the importance of immune activation/suppression, this time specific to the PD1/PD-L1 axis, was provided by Ye et al. who evaluated the expression of B7-H1(PD-L1) and its receptor PD-1 in N=31 surgically resected cholangiocarcinoma tissues and the corresponding cancer adjacent tissues58. Expression of PD-L1 and PD-1 was found to be up-regulated in cholangiocarcinoma tissues compared with the cancer adjacent tissues. Tumor-related PD-L1 expression was significantly correlated with both tumor differentiation and pTNM stage and was inversely correlated with CD8+ tumorinfiltrating lymphocytes. Similarly, Nakakubo et al. investigated the significance of tumorinfiltrating immune cells in 110 surgically resected biliary tract cancer (gallbladder) specimens59.  
They found high levels of CD4+ T cell (51.1%), CD8+ T cell (37.8%), NL cell (33.3%), and dendritic cell (48.9%) infiltration. CD4+ and CD8+ T cell infiltration correlated with decreasing tumour invasion, and high numbers of infiltrating DCs correlated with decreasing lymph-node tumour metastasis. Furthermore, increased infiltration of CD4+ and CD8+ T cells and DCs exhibited a significant correlation with prolonged survival. Because of these data, and coupled to the data summarized earlier for immune stimulation following RFA, we wish to treat a cohort of biliary tract carcinoma patients.
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1.2.9 rationale for amendment i

The purpose of amendment I is to remove Cohort C. Given a range of factors - the number of variables in this study, the finding of interesting activity with ablative methods (commonly used in HCC) and the relative infrequency of SBRT in this disease - it was decided both scientifically and for patient safety reasons to focus and refine our research efforts. At this point we will not enroll to Cohort C but preferentially enroll HCC patients to either cohort B of this protocol or our new protocol (16-C-0135). Should we decide to evaluate SBRT in combination with immune modulating therapy in this population in the future it will only be done after an IRB amendment.
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1.2.10 rationale for amendment j

The purpose of amendment J is to stop further tremelimumab treatment beyond 2 years if there is no evidence of disease progression. Currently patients would continue to receive tremelimumab treatment every 12 weeks until disease progression. The justification for this is as follows: 1) Two-year-duration of immune checkpoint inhibitor treatment is adequate for disease treatment and control: early trials in melanoma allowed treatment until progression; then, 2-year duration was the maximal adopted by majority of clinical trials 60, 61, 62 and approved regimens. Such study design does not prevent patients from benefiting the most from immune checkpoint inhibitor treatment 63. Effectively, the discontinuation of immune checkpoint inhibitor after a predetermined duration is not associated with an increased risk of acquired resistances such as that described with cytotoxic chemotherapy 63. Few reports have examined the outcome in patients who achieved a response and stopped therapy after 2 to 3 years 64. Among them, 61
(64%) stopped treatment after complete response and maintained their remission. Median duration of treatment was 23 months. Only 2 of the 61 patients who stopped treatment after complete response experienced disease progression. Many of those who did not achieve a complete response and whose disease progressed were able to respond to further immunotherapy 64. The existence of the "tail on the curve" of survival in melanoma, and now other cancers, suggests that a subset of patients who discontinue treatment with one of these agents experience durable responses after therapy and do not need to be treated until progression. Presumably, the patient's immune system has been engaged and is still monitoring and effectively suppressing the cancer. Nevertheless, re-challenging the tumor with the same immune checkpoint inhibitor even after a long stable period demonstrated a greater response and lesser adverse events 65.  
Thus, indefinite treatment may not be truly necessary, and a duration of not less than 1 year and not more than 2 years seems reasonable.  
2) Prolonged immune checkpoint inhibitor exposure has greater risks for potentially lifethreatening toxicities: despite important clinical benefits, immune checkpoint inhibition is associated with a unique spectrum of immune-related adverse events. Serious side effects are observed in less than 10% of patients, and typically emerge an average of 6 to 12 weeks after the start of treatment 66, 67. The risk of side effects increases with higher doses, and overall seems to increase with cumulative exposure. Moreover, as immunotherapies are being tested in combination, side effects tend to be additive. For example, the more common adverse effects are related to GI inflammation, which can be as minor as slight irritation or as severe as colitis, potentially even leading to perforation of the bowel. Those potentially life-threatening toxicities in the context of prolonged treatment may outweigh the benefits of indefinite immune checkpoint inhibitor therapy. Given above discussed justifications, it is decided both scientifically and for patient safety reasons to focus and refine our research efforts. At this point we will discontinue tremelimumab administration after 2 years if patient is experiencing responding to the treatment over 2 years, including stable disease, partial or complete remission.
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2 eligibility assessment and enrollment

2.1.1 Inclusion Criteria 2.1.1.1 Patients must have histopathological confirmation of hepatocellular carcinoma (HCC) or
(Cohort E only) biliary tract carcinoma (BTC) by the Laboratory of Pathology of the NCI prior to entering this study OR histopathological confirmation of carcinoma in the setting of clinical and radiological characteristics which, together with the pathology, are highly suggestive of a diagnosis of HCC (or biliary tract carcinoma in Cohort E). Fibrolammelar variant is also allowed. For cohort E, the term BTC includes intra- or extrahepatic cholangiocarcinoma, gallbladder cancer or ampullary cancer, as long as there is an intrahepatic component amenable to RFA.  
2.1.1.2 Patients must have disease that is not amenable to potentially curative resection, transplantation or ablation. For Cohorts A and D patients must have progressed on, been intolerant to, or refused prior sorafenib therapy. Cohort E patients must have received at least one line of chemotherapy for BTC.  
2.1.1.3 Disease must be technically amenable to transhepatic arterial chemoembolization
(TACE), radiofrequency ablation (RFA), or cryoablation. Each case will be discussed at GI tumor board with interventional radiology. Patients must have evaluable disease.  
2.1.1.4 If liver cirrhosis is present, patient must have a Child-Pugh A/B7 classification (see Appendix B section **12.2** ).  
2.1.1.5 Age >18 years 2.1.1.6 Life expectancy of greater than 3 months. 2.1.1.7 ECOG performance status 0-2 (see Appendix A section **12.1**) 2.1.1.8 Patients must have normal organ and marrow function as defined below:  
| leukocytes                | >3,000/mcL                               |
|---------------------------|------------------------------------------|
| absolute neutrophil count | > 1,000/mcL                              |
| platelets                 | •,000/mcL                                          |
| total bilirubin           | If cirrhosis present: Part of Child Pugh |  
| requirement  ,IQRFLUUKRVLV%LOLVKRXOGEH" xULN                                                                                                                                                                                                                                                |    |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----|
| Serum albumin                                                                                                                                                                                                                                  | If cirrhosis present: Part of Child Pugh  requirement  ,IQRFLUUKRVLVDOEXPLQVKRXOGEH• 2.5g/dl    |
| Patients are eligible with ALT or AST up to 5 x ULN.  creatinine <1.5X institution upper limit of  normal  OR  creatinine clearance >45 mL/min/1.73 m2 , as calculated  below, for patients with creatinine  levels above institutional normal |    |  
2.1.1.9 Patients must have recovered from any acute toxicity related to prior therapy, including surgery. Toxicity should be  grade 1 or returned to baseline.  
2.1.1.10 Patients must not have other invasive malignancies within the past 5 years (with the exception of non-melanoma skin cancers, non-invasive bladder cancer or localized prostate cancer for whom systemic therapy is not required).  
2.1.1.11 Patient must be able to understand and willing to sign a written informed consent document.  
2.1.2 Exclusion Criteria
2.1.2.1 Patients who have had standard of care chemotherapy, large field radiotherapy, or major surgery must wait 2 weeks prior to entering the study. For recent experimental therapies a 28 day period of time must elapse before treatment.  
2.1.2.2 Patients who have undergone prior liver transplantation are ineligible.
2.1.2.3 Patients with known brain metastases will be excluded from this clinical trial because of their poor prognosis and because they often develop progressive neurologic dysfunction that would confound the evaluation of neurologic and other adverse events.  
2.1.2.4 Uncontrolled intercurrent illness including, but not limited to, ongoing or active systemic infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia (excluding insignificant sinus bradycardia and sinus tachycardia) or psychiatric illness/social situations that would limit compliance with study requirements.
2.1.2.5 History of chronic autoimmune disease (e.g., Addison's disease, multiple sclerosis, Graves' disease, Hashimoto's thyroiditis, rheumatoid arthritis, hypophysitis, etc.) with symptomatic disease within the 3 years before randomization. Note: Active vitiligo or a history of vitiligo will not be a basis for exclusion.  
2.1.2.6 Dementia or significantly altered mental status that would prohibit the understanding or rendering of Information and Consent and compliance with the requirements of the protocol.  
2.1.2.7 Diverticulitis (either active or history of) within the past 2 years. Note that diverticulosis is permitted.  
2.1.2.8 Active or history of inflammatory bowel disease (colitis, Crohn's), irritable bowel disease, celiac disease, or other serious, chronic, gastrointestinal conditions associated with diarrhea. Active or history of systemic lupus erythematosus or Wegener's granulomatosis.  
2.1.2.9 Currently receiving immunosuppressive doses of steroids or other immunosuppressive medications (inhaled and topical steroids are permitted)
2.1.2.10 History of sarcoidosis syndrome 2.1.2.11 Patients should not be vaccinated with live attenuated vaccines within 1 month of starting Tremelimumab treatment.  
2.1.2.12 Has a known history of Human Immunodeficiency Virus (HIV) (HIV 1/2 antibodies) 2.1.2.13 HIV-positive patients receiving anti-retroviral therapy are excluded from this study due to the possibility of pharmacokinetic interactions between antiretroviral medications and Tremelimumab. HIV positive patients not receiving antiretroviral therapy are excluded due to the possibility that Tremelimumab may worsen their condition and the likelihood that the underlying condition may obscure the attribution of adverse events.  
2.1.2.14 History of hypersensitivity reaction to human or mouse antibody products. 2.1.2.15 Pregnancy and breast feeding are exclusion factors. The effects of Tremelimumab on the developing human fetus are unknown. Enrolled patients must agree to use adequate contraception (hormonal or barrier method of birth control; abstinence) prior to study entry, the duration of study participation and 3 months after the end of the treatment. Should a woman become pregnant or suspect she is pregnant while she or her partner is participating in this study, she should inform her treating physician immediately.  
2.1.2.16 Patients with unhealed surgical wounds for more than 30 days.
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2.1.3 re-induction criteria (if applicable)

2.1.3.1 Subjects must have entered the Maintenance Phase and are under ongoing maintenance treatment or Subjects who stopped maintenance treatment because of a CR or Subjects with an initial PR or CR or at least 3 months of SD on tumor assessment and who subsequently have a confirmed and documented disease progression (per immune-related RECIST criteria).  
2.1.3.2 Patients are excluded from re-induction if they have experienced any related Doselimiting toxicities as described in Section 3.6, delayed dosing beyond 35 days due to Tremelimumab-related AEs, or have been taken off treatment due to toxicity as described in Section 3.8.3.  
24  
2.1.3.3 Patients who have progressed on initial therapy will not be considered for re-induction treatment.  
2.1.3.4 Tremelimumab Dose: Patients will be treated at the same dose of Tremelimumab as they previously received. For patients on Dose Level 1 who had already satisfied criteria for escalation to 10mg/kg they will be re-treated at 10mg/kg.  
2.1.3.5 The patient must - at the time of re-induction - satisfy all the eligibility criteria as set out in Sections 2.1.1 & **2.1.2**.  
2.1.3.6 The patient must be discussed at GI Tumor Board, NCI and suitability for the interventional procedure (TACE or RFA) re-affirmed.
Eligible patients will follow the same treatment criteria, evaluation plan and schedule as was previously described for the main part of the study, and doses may be delayed or modified as described in Section 3.8. There will be no limit to the number of additional re-inductions a patient may receive if the same efficacy and safety criteria described above are met, until intolerable toxicity or progressive disease necessitates ending the re-induction phase. 2.1.4 Inclusion of Women and Minorities Men and women of all races and ethnic groups are eligible for this trial. 2.1.5 Recruitment Strategies The study will be posted on the CCR website and on clinicaltrials.gov. 2.2 SCREENING EVALUATION 2.2.1 History and Physical Examination Complete history (including prior hormone use) and physical examination (including height, weight, vital signs, EKG, and performance status) will be conducted prior to starting study drug. 2.2.2 Laboratory Evaluation  
x Hematological profile: CBC with differential and platelet count, PT, INR, aPTT,
fibrinogen.  
x Biochemical profile: electrolytes, BUN, creatinine, AST, ALT, total bilirubin (including direct and indirect), calcium, phosphorus, albumin, magnesium, uric acid, amylase.  
x Serum or urine pregnancy test for female participants of childbearing age (in the absence of prior hysterectomy). Test to be performed within 72 hours prior to initiating treatment.  
2.2.3 Histologic confirmation (at any time point prior to commencement)
A block or unstained slides of primary or metastatic tumor tissue will be required from each participant to confirm diagnosis with analysis being performed by the Laboratory of Pathology, NIH. As stated in the inclusion criteria above, histopathological confirmation of carcinoma may be sufficient. Fibrolammelar variant is also allowed.
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2.3 baseline studies

2.3.1 Imaging studies (obtained within 28 days prior to first dose)
x CT scan of chest, abdomen and pelvis And/or  
x MRI liver
2.3.2 Laboratory evaluation (obtained within 72 hours prior to first dose) If laboratory tests are done within 72 hours of Cycle 1 Day 1 they do not need to be repeated on Day 1.  
x Hematological profile: CBC with differential and platelet count, PT, INR, aPTT,
fibrinogen.  
x Biochemical profile: electrolytes, BUN, creatinine, AST, ALT, total bilirubin (including direct and indirect), calcium, phosphorus, albumin, magnesium, uric acid, amylase.  
x Tumor marker profile: aFP or Ca19.9 (obtained within 7 days prior to first dose)
2.3.3 Laboratory evaluation (obtained within 28 days prior to first dose)
x Thyroid function tests (TSH, T3, T4) x Hepatitis B and/or C viral load and serology x HLA-A2 phenotype 2.3.4 History and physical exam with vital signs (obtained within 1 week prior to first dose). 2.3.5 Electrocardiogram (obtained within 28 days prior to first dose)
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2.4 registration procedures

Authorized staff must register an eligible candidate with NCI Central Registration Office (CRO) within 24 hours of signing consent. A registration Eligibility Checklist from the web site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) must be completed and f sent via encrypted email to: NCI Central Registration Office ncicentralregistration-l@mail.nih.gov. After confirmation of eligibility at Central Registration Office, CRO staff will call pharmacy to advise them of the acceptance of the patient on the protocol prior to the release of any investigational agents. Verification of Registration will be forwarded electronically via e-mail to the research team. A recorder is available during non-working hours.
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3 study implementation 3.1 study

- The proposed study is a pilot study of Tremelimumab - at a planned dose 10mg/kg/IV q 4 weekly - in patients with advanced HCC treated with either TACE or RFA. The initial study population is Cohort A as described below and these patients will be treated with either RFA or TACE. The decision on which treatment to administer (i.e. RFA vs. TACE) will be made in consultation with the interventional radiologists. The reason for this is that there are technical considerations (size and location of lesion, main portal vein occlusion etc.) which would make one procedure preferable over the other.  
| Cohort       | Population        | Immune stimulative   | Dose level   | Planned N   |
|--------------|-------------------|----------------------|--------------|-------------|
| procedure    | (Trem)            |                      |              |             |
| A            | Advanced HCC,     | RFA/TACE             | 3.5mg/kg     | 3-6         |
| BCLC Stage C | 10mg/kg           | 20                   |              |             |
| B            | Intermediate HCC, | TACE                 | 10mg/kg      | 20          |
| BCLC B       |                   |                      |              |             |  
- Transarterial chemoembolization is percutaneous procedure performed under general anesthesia or conscious sedation. (See **3.9.1** for details)  
- Patients undergoing trans-arterial chemoembolization, RFAor cryoablation are premedicated with analgesics, antibiotics and anti-emetics per clinical standards. For details regarding each procedure please see 3.9.  
- We will also enroll an additional cohort of N=20 patients (Cohort B) with liver-confined disease for whom TACE is indicated as standard of care. These patients will receive TACE in combination with Tremelimumab using the same schema, although for these patients TACE may be repeated (as per standard of care). They will not receive RFA.  
- With protocol amendment F, it was proposed that once cohort A has demonstrated safety and feasibility for the combination of ablative treatment and tremelimumab in HCC, we will add 3 additional cohorts (C-E) to the study. However, with amendment I we have revised the study design to remove Cohort C, so only 2 cohorts, D and E, remain. For these cohorts, the schedule of administration for tremelimumab and the timing of the immune-stimulative procedure is identical to cohorts A and B, as summarized below.  
| Cohort   | Population               | Immune  stimulative  procedure   | Dose level   | Planned N   |
|----------|--------------------------|----------------------------------|--------------|-------------|
| (Trem)   |                          |                                  |              |             |
| D        | Advanced HCC, BCLC Stage | Cryoablation                     | 10mg/kg      | 10          |
| C        |                          |                                  |              |             |  
| RFA                | 10mg/kg   | 20   |
|--------------------|-----------|------|
| cholangiocarcinoma |           |      |
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3.2 schedule

- Day 1: Tremelimumab IV every 4 weeks x 6 doses and then every 12 weeks for 2 years or until disease progression whichever comes first.  
- Day 36 [+/- 96 hours]: RFA or TACE.  
- Restaging CT /MRI scan every 8 weeks to evaluate TTP in target lesion. (Both standard RECIST and Immune-related Response Criteria [irRC] will be employed).  
- Dose: (Cohort A only): Initially three to six patients will be treated at 3.5mg/kg (with subsequent dosing also at 3.5mg/kg) prior to enrolling to the10mg/kg dose level. If a single patient experiences dose-limiting toxicity (DLT) related to the combination this cohort will be expanded to N=6 as per a standard 3+3 design. If 3.5mg/kg is established to be safe we will proceed to the 10mg/kg dose level. N=20 patients in cohort A will be treated at 10mg/kg.  
- For Cohort A (BCLC Stage C patients): TACE or radiofrequency ablation (RFA) will be performed once only.  
- For Cohort B (BCLC Stage B patients): TACE may be repeated (as per standard of care)
on months 3, 7, and 13, and q6 months thereafter (if indicated).  
- Cohort B will only be recruited following completion of the dose-escalation portion of Cohort A.
For Cohort E (BTC patients, status post prior chemotherapy with intrahepatic disease) RFA will be performed once only, also on Day 36 (+/- 96hrs).
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3.3 cohort a

3.3.1 Definition The definition of Cohort A is as follows:  
Cohort A (N=23-26):
x Advanced stage HCC, BCLC group C
x Patients must have progressed on, been intolerant of prior sorafenib therapy. x Child Pugh A/B7 x No available surgical option (i.e. transplantation or hepatic resection) or curative ablative option.  
x Disease amenable to either TACE or RFA
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3.3.2 treatment

Three patients will be treated at 3.5mg/kg (with subsequent dosing also at 3.5mg/kg i.e. no intrapatient dose escalation) prior to enrolling to the10 mg/kg dose level. If a single patient experiences dose-limiting toxicity (DLT) related to the combination of TACE/RFA and Tremelimumab this cohort will be expanded to N=6 as per a standard 3+3 design. If 3.5mg/kg is established to be safe we will proceed to the 10 mg/kg dose level. N=20 patients will be treated at 10 mg/kg to evaluate the feasibility endpoint, provided no more than 2 of 3-6 patients have a DLT at this level.  
| Dose level   | Tremelimumab dose (mg/kg)   | N   |
|--------------|-----------------------------|-----|
| 1            | 3.5                         | 3-6 |
| 2            | 10                          | 20  |  
- The 3.5 mg/kg cohort will have at least 3 patients to evaluate for toxicity to establish MTD for the combination of Tremelimumab with either TACE or RFA. Three patients will be treated at this dose level. The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting. If none of the three patients at the 3.5 mg/kg dose level experiences dose-limiting toxicity (DLT), accrual to DL2 will proceed to the 10mg/kg cohort. If one of three patients treated at a dose level experiences DLT that is considered to be at least possibly related to the combination of Tremelimumab with either TACE or RFA, then three more patients will be enrolled at that same level. If the incidence of DLT among those six patients is one in six, then we will proceed to 10 mg/kg. If two or more of the six patients experience DLT, then the MTD is considered to have been exceeded.  
- Evaluation period for Dose-limiting Toxicities (DLT) will extend for the first 8 weeks of the study.  
- Following completion of the dose escalation portion of the study Tremelimumab will be administered at the MTD at the same schedule. A total of 20 patients will be treated in cohort A at the MTD, not including those patients treated at the lower doses.  
- For patients on Dose Level 1 who have completed at least 16 weeks of protocol therapy, we will consider dose escalation to 10mg/kg if criteria for continuance of therapy are met and the patient does not have >Grade 2 toxicity attributable to tremelimumab.
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3.3.3 protocol stopping rules

This study will utilize the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 for grading systemic toxicity. For safety reasons, the protocol will be temporarily halted pending discussions with the NCI IRB and Sponsor regarding necessary amendment for either of the following events:
x One occurrence of grade 5 toxicity by the NCI-CTCAE version 4.0 attributable to the treatment regimen.  
x Two occurrences of grade 4 toxicity by the NCI-CTCAE version 4.0 attributable to the treatment regimen.
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3.3.4 re-induction

Patients who entered the maintenance phase and have confirmed PD (per immune-related RECIST criteria) during the maintenance treatment may be eligible to receive re-induction therapy using Tremelimumab at the assigned dose and schedule: i.e. 10mg/kg monthly for 6 months followed by maintenance 3-monthly Tremelimumab. Patients who stopped maintenance treatment because of a CR and subsequently had confirmed PD (per immune-related RECIST criteria) are eligible to receive re-induction as well. Subjects with an initial PR or CR or SD for at least 3 months and subsequently have PD are also eligible for re-induction. As part of reinduction they will also undergo the repeat ablative procedure on Day 36 (+/- 96hrs). This may be either an RFA or TACE depending on the opinion of the interventional radiologist. To be eligible for re-induction patients must satisfy the criteria stated in section **2.1.3**.
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3.3.4.1 tumor biopsies

If a patient opts to undergo re-induction therapy we would like to perform pre- and post-reinduction tumor biopsies to better understand the role of emerging resistance to anti-CTLA4 therapy. These would occur prior to re-induction (< 2 weeks prior to starting) and then during the interventional radiologic procedure.
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cohort b (n=20)

x HCC, Barcelona Clinic Liver Cancer (BCLC) Stage B;  
x Child Pugh A/B7; x No available surgical (i.e. transplantation or hepatic resection) or curative ablative option.  
x Disease amenable to TACE
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3.4.2 treatment

The treatment schema for both cohorts is identical with the exception of the fact that in cohort B TACE may be repeated (as per standard of care) on months 3, 7, and 13, and q6 months thereafter (if indicated). Patients in Cohort B will only be recruited following completion of the dose-escalation portion of Cohort A and will be treated at the dose level which has been determined to be safe in that cohort.
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3.5 cohort c

This cohort, designed to evaluate tremelimumab in combination with external beam radiation was removed with amendment I.
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3.6 cohorts d-e

Once safety is established for the initial concept of combining anti-CTLA4 with ablative procedures (RFA and TACE), we will similarly explore feasibility of tremelimumab at the same dose and schedule in 2 additional cohorts (denoted D-E) evaluating tremelimumab in combination with cryoablation in HCC (cohort D) and with RFA in a cohort of patients with biliary tract carcinoma (cohort E).  
- For these additional cohorts, the dose (10mg/kg) and schedule of tremelimumab will be as per cohorts A and B. The timing of the interventional procedure will also be identical.  
- Cohort Definition: Cohort D will have the same definition as Cohort A above:
Advanced stage HCC, BCLC group C Patients must have progressed on, been intolerant of prior sorafenib therapy. Child Pugh A/B7 No available surgical option (i.e. transplantation or hepatic resection) or curative ablative option.  
Disease amenable to cryoablation
- Cohort E will contain BTC patients as specified in the eligibility criteria.  
- For cohorts D-E no dose escalation will occur. All patients will receive tremelimumab at 10mg/kg at the same schedule established as safe and feasible in Cohort A of the study. However, protocol stopping rules will apply as outlined in Section 3.3.
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3.7 dose limiting toxicities 3.7.1

$'/7LVGHILQHGDVD*UDGHDGYHUVHGUXJUHDFWLRQ$'5DFFRUGLQJWRWKH1&,-CTCAE v4.0, that is possibly, probably, or definitely related to the combination of Tremelimumab with either TACE or RFA, occurring during the DLT evaluation period except for any of the following outlined in section **3.6.2**. ADRs are defined in this trial as any AEs suspected to be related to Tremelimumab by the investigator. Evaluation period for Dose-limiting Toxicities (DLT) will extend for the first 8 weeks of the study. Treatment-related SAE will be ascribed as related to drug except where a clear relationship to the underlying disease or recognized co-morbidities is evident. For this trial, the MTD is defined as the highest dose where < 2 of 6 subjects experience a DLT.  
3.7.2 Exclusions to Dose Limiting Toxicities x Grade 3 infusion-related reaction resolving within 6 hours and controlled with medical management.  
x Any Grade 3/4 non-hem toxicity that occurs in the first 10 days after RFA or TACE and which is an expected toxicity of the procedure in the opinion of the PI. These symptoms include, but are not limited to, fevers/rigors, pain, fatigue and nausea.  
x Any grade 3 immune-mediated adverse event (including but not limited to Grade 3 diarrhea, Grade 3 skin toxicity, or Grade 3 liver enzyme elevation) that improves to *UDGH 2 within 1 week of onset with supportive care (which may include systemic corticosteroids).  
x Nausea and vomiting Grade 3 will only be considered dose-limiting if it is refractory to anti-emetic therapy and unable to be corrected to Grade 1 or less within 48 hours.  
x Grade 3 rise in creatinine, not corrected to Grade 1 or less after 2 liters of intravenous fluids within 24 hours, will be considered dose limiting.  
x Single laboratory values out of normal range that are unlikely related to trial treatment according to the investigator, do not have any clinical correlate, and resolve within 7 days with adequate medical management.  
x 7UDQVLHQWKRXUV*UDGHIDWLJXHORFDOUHDFWLRQVKHDGDFKHQDXVHDHPHVLVWKDW
UHVROYHVWR*UDGH
x Grade 4 elevation in transaminase elevation that improves to *UDGHwithin 10 days of onset with supportive care.  
x Grade 3 endocrinopathy that is managed with or without systemic corticosteroid therapy and/or hormone replacement therapy and the following criteria are met:
The subject's hormone levels are within normal limits The subject is asymptomatic x Grade 3 inflammatory reaction attributed to a local antitumor response (eg, inflammatory reaction at sites of metastatic disease, lymph nodes, etc.)
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3.8 drug administration 3.8.1 tremelimumab

The first day of dosing is considered Day 1. Each dose of investigational product should be administered using the following guidelines:  
1) Investigational product must be administered at room temperature (25°C) by controlled infusion at a rate of 250 mL/hr via an infusion pump into a peripheral vein. Prior to the start of the infusion, ensure that the bag contents are at room temperature to avoid an infusion reaction due to the administration of the solution at low temperatures.  
2) Investigational product must not be administered via IV push or bolus but as a slow IV
infusion. The entire content of each IV bag will be infused using an infusion pump.  
3) The infusion lines should be attached only at time of use. Lines used for infusion during dose administration will need to be equipped with 0.22 or 0.2 ȝPLQ-line filters.  
4) If there are no requirements to slow, interrupt, or permanently stop the infusion, the anticipated infusion time to deliver each dose (250 mL) is anticipated to be approximately 60 minutes.
The duration of the investigational product administration will be recorded. 3.8.2 Monitoring of Dose Administration Vital signs will be collected before investigational product infusion and at the completion of the infusion. As with any antibody, allergic reactions to dose administration are possible. Therefore, appropriate drugs and medical equipment to treat acute anaphylactic reactions must be immediately available, and study personnel must be trained to recognize and treat anaphylaxis, as per local institutional guidelines.
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3.9 dosing delays

The following broad guidelines for dose delays apply to both of the planned dosages of Tremelimumab and are dependent on the clinical and laboratory assessment on the day of dosing. No dose reductions will be employed. Patients will either proceed or forgo treatment.  
Dosing may be delayed up to 14 days to allow recovery from treatment-related toxicity. For specific immune-mediated adverse reactions see Section **3.8.3** which will take precedence over this broad management guideline.  
| Condition                                                                                                      | Management                                                                                                                                                                                                                                                                                                                               |
|----------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Onset of any toxicity                                                                                          | Rule out alternative etiology  In case of doubt, investigator should consult with study medical  monitor promptly                                                                                                                                                                                                                        |
| NCI CTCAE Grade 1                                                                                              | Provide symptomatic treatment  Possible topical steroids if applicable                                                                                                                                                                                                                                                                   |
| NCI CTCAE Grade 2                                                                                              | Provide symptomatic treatment  In the case of an immune-mediated adverse event do not give  scheduled dose; dosing may be resumed once symptoms are  resolved.  Consider oral or IV steroids at the onset of symptoms. Taper  steroid if symptoms improve.                                                                               |
| NCI CTCAE Grade 3                                                                                              | Start high-dose IV steroids at the onset of the symptoms   Provide symptomatic treatment                                                                                                                                                                                                                                                 |
| NCI CTCAE Grade 4                                                                                              | Start high-dose IV steroids at the onset of the symptoms  Provide symptomatic treatment  Permanent discontinuation a  of Tremelimumab for all NCI  CTCAE Grade 4 events (unless specific exemption stated  elsewhere in protocol)                                                                                                        |
| Steroid refractory toxicity (no  improvement after 5 days on highdose IV steroids) or relapse after  reducing high-dose steroids                                                                                                                | Continue symptomatic treatment and steroids  b  5 mg/kg IV for GI toxicities unless  Possible infliximab  contraindicated [consult with GI specialist]. Caution: rule out  bowel perforation and refer to label before using infliximab  For subjects with increased AST/ALT, or total bilirubin levels,  consider mycophenolate mofetil |
| ALT = alanine transaminase; AST = aspartate transaminase; GI = gastrointestinal; IV = intravenous; NCI CTCAE = |                                                                                                                                                                                                                                                                                                                                          |  
ALT = alanine transaminase; AST = aspartate transaminase; GI = gastrointestinal; IV = intravenous; NCI CTCAE = National Cancer Institute Common Terminology Criteria for Adverse Events.  
a Subjects will not receive any subsequent dose, but will remain on study and follow the other procedures required from the study (e.g., follow-up procedures, disease assessment scans, blood sample collections). b Use will result in discontinuation of treatment.  
During the study, subjects may require immunosuppressive medications such as steroids for management of underlying disease, treatment-related toxicity, or unrelated conditions. If symptoms UHVROYHGWR1&,&7&$(*UDGHTremelimumab dosing may be resumed during steroid taper. Subjects with adrenal insufficiency may take daily prednisone or equivalent therapy for their endocrinopathy while receiving Tremelimumab treatment. Topical and inhaled steroids in standard doses are allowed.
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3.9.1 dosing delays/dose modifications general

Treatment with Tremelimumab need not be interrupted, although for chronic low grade toxicity causing significant detrimental effect in patient's well-being the PI may elect to delay/omit dosing at his discretion, in particular in the case of a suspected immune-mediated adverse event. 3.9.1.2 Grade 3 Hematologic or Non-Hematologic Toxicity Hold Tremelimumab and re-HYDOXDWHXQWLOWR[LFLW\LPSURYHVWRJUDGHRUSUH-treatment baseline. Treatment will be discontinued in patients who experience grade 3 non-hematologic toxicities felt to be drug-related. 3.9.1.3 Grade 4 Non-Hematologic Toxicity Patients with clinical treatment-related grade 4 non-hematologic toxicity (except pulmonary embolism without significant hypoxia and hemodynamic instability) will be taken off treatment permanently. Unacceptable toxicities that have not resolved at time of "off treatment" must be followed until stabilization or resolution, at which time they will continue in follow up for survival. 3.9.2 Toxicities Mandating Permanent Discontinuation of Tremelimumab As per the Investigator's brochure the following criteria will result in permanent discontinuation of tremelimumab. 3.9.3 Specific immune-mediated adverse reactions Tremelimumab can result in severe and fatal immune-mediated adverse reactions due to T-cell activation and proliferation. These immune-mediated reactions may involve any organ system, however, the most common severe immune-mediated adverse reactions are enterocolitis, hepatitis, dermatitis (including toxic epidermal necrolysis), neuropathy, and endocrinopathy. The majority of these immune-mediated reactions initially manifest during treatment, however, a minority may occur weeks to months after discontinuation of Tremelimumab. Tremelimumab will be discontinued and systemic high-dose corticosteroid therapy initiated for severe immunemediated reactions.  
Patients will be assessed for signs and symptoms of enterocolitis, dermatitis, neuropathy and endocrinopathy and evaluate clinical chemistries including liver function tests and thyroid function tests at baseline and before each dose.  
The following are anticipated immune-mediated adverse reactions for which specific guidance and management is outlined below. 3.9.3.1 Gastrointestinal
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3.9.3.2 liver (immune-mediated hepatitis)

Tremelimumab can result in severe and fatal inflammation of the liver most commonly manifested as elevation of transaminases and hyperbilirubinemia. Liver enzymes and liver function tests (hepatic transaminase and bilirubin levels) will be evaluated and patients assessed for signs and symptoms of hepatitis before each dose of Tremelimumab. In patients who experience AST or ALT > 5.0 ULN and/or baseline or total bilirubin > 3.0 ULN liver biopsy should be considered as per standard of care investigation for automimmune hepatitis.  
| Symptoms   | Immediate action   | Subsequent management                                                                                                                                                                    |
|------------|--------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Moderate  ‡$67RU$/7!WR ULN  and/or baseline (if this was  abnormal).  ‡7RWDOELOLUXELQ!WR ULN            | Withhold Tremelimumab ‡5XOHRXWLQIHFWLRXVRU malignant causes  ‡,QFUHDVHIUHTXHQF\RIOLYHU function test monitoring until  resolution                    | Symptoms resolve:  Resume Tremelimumab if  liver function tests are 2.5  ULN  or return to baseline and  bilirubin level is 1.5 ULN or  returns to baseline  IF AST OR ALT  CONTINUES TO |  
| ELEVATE TO >5.0 ULN  OR  TOTAL BILIRUBIN >3.0  ULN,  See management for 'severe'   |
|------------------------------------------------------------------------------------|  
| Symptoms   | Immediate action   | Subsequent management                                                                                                                                                                                                                                                                               |
|------------|--------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Severe  ‡$67RU$/7!8/1 and/or  ‡7RWDOELOLUXELQ!8/1            | Hold Tremelimumab and reevaluate until toxicity  LPSURYHVWR"JUDGHRUSUHtreatment baseline. Treatment  will be discontinued in  patients who experience grade  3 non-hematologic toxicities  that do not resolve to grade 1  or baseline within 4 weeks.  ‡Increase frequency of liver  function  test monitoring until  resolution  ‡Administer systemic  corticosteroid of  1 to 2 mg/kg/day of  prednisone  or equivalent.  Consider liver biopsy as per  standard of care investigation  for autoimmune hepatitis                    | Symptoms resolve:  When liver function tests  show sustained improvement  or return to baseline, initiate  corticosteroid tapering and  continue to taper over 1 month  Consider alternative  immunosuppressive therapy  Symptoms Ongoing >1  week: Consider alternative  immunosuppressive therapy |  
3.9.3.3 Skin: Immune-mediated dermatitis  
| Symptoms                                                       | Immediate action                                                                                                              | Subsequent management                                                                                                                                                                                                          |
|----------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Moderate  Non-localized rash (diffuse,  <50%  of skin surface) | Withhold Tremelimumab Administer topical or systemic  corticosteroids if there is no  improvement of symptoms  within  1 week | Symptoms resolve:  Resume Tremelimumab if  dermatitis resolves or  improves to  mild (localized) symptoms  and systemic steroid dose is  10 mg prednisone equivalent  or less  If symptoms worsen see  management for 'severe' |  
| Symptoms                                                                                                                                                                         | Immediate action                                                                                                             | Subsequent management                                                                                                       |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------|
| Severe  Stevens-Johnson syndrome,  toxic epidermal necrolysis, or  rash complicated by full  thickness dermal  ulceration, or necrotic,  bullous, or hemorrhagic  manifestations | Permanently d/c  Tremelimumab  Administer systemic  corticosteroid  therapy of 1 to 2 mg/kg/day of  prednisone or equivalent | Symptoms resolve:  When dermatitis is controlled,  corticosteroid tapering  should occur over a period of  at least 1 month |  
3.9.3.4 Central/Peripheral nervous system  
| Symptoms                                                                                                   | Immediate action                                                  | Subsequent management                                                                                                                 |
|------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------|
| Moderate  Moderate symptoms,  clinically  detectable with no impact on  activities  of daily living (ADLs) | Withhold Tremelimumab Introduce appropriate medical  intervention | Symptoms resolve:  Resume Tremelimumab when  symptoms resolve or return  to baseline  If symptoms worsen see  management for 'severe' |  
| Symptoms                                                      | Immediate action   | Subsequent management                                                     |
|---------------------------------------------------------------|--------------------|---------------------------------------------------------------------------|
| Severe  Severe symptoms (impact on  ADLs) or life threatening | Permanently d/c  Tremelimumab  Institute appropriate medical  intervention  ‡Consider the use of systemic  corticosteroid of 1 to 2  mg/kg/day of  prednisone or equivalent                    | Permanently d/c  Tremelimumab  Continue appropriate medical  intervention |  
3.9.3.5 Endocrine system  
Symptoms Immediate action Subsequent management Moderate Signs and/or symptoms of dysfunction Endocrinopathies requiring hormone  
| Moderate  Signs and/or symptoms of  dysfunction  ‡Endocrinopathies requiring  hormone   | Withhold Tremelimumab Evaluate endocrine function  ‡Consider radiographic  pituitary gland  imaging   |
|---|---|  
| Symptoms resolve:  Resume Tremelimumab when  Patient is stable and symptoms  are resolved or return  to baseline   |
|--------------------------------------------------------------------------------------------------------------------|  
| replacement or medical  intervention  ‡Adverse reactions requiring  hospitalization, urgent medical  intervention, or interfering  with  activities of daily living  (including  adrenal crisis)   | ‡Continue to assess as  indicated  ‡Withhold Tremelimumab in  symptomatic  patients  ‡Administer systemic  corticosteroid  therapy of 1 to 2 mg/kg/day of  prednisone or equivalent  ‡Initiate appropriate  hormone-replacement therapy   | ‡Patient is stable on hormonereplacement therapy  (as indicated)  ‡Patient is receiving "7.5 mg  prednisone or equivalent  per day   |
|---|---|---|
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3.10 specific procedures for tace

Is percutaneous procedure performed under general anesthesia or conscious sedation. Access is obtained from the common femoral artery and selective catheterization of the superior mesenteric and celiac arteries is performed to define anatomy and hepatic arterial supply. Superselective catheterization of hepatic artery branches feeding the tumors is then performed followed by infusion of a chemotherapeutic mixture. Trans-arterial chemoembolization will be performed with drug eluding beads (DEB). In this case, 100mg of doxorubicin are loaded into 100-250 micron beads. The DEB are mixed with contrast and infused into the hepatic artery branches supplying the tumor. Patients undergoing trans-arterial chemoembolization are pre-medicated with analgesics, antibiotics and anti-emetics per clinical standards.
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3.10.2 radiofrequency ablation

Thermal ablation is a minimally invasive image guided procedure performed under general anesthesia or conscious sedation. Radiofrequency or microwave ablations are performed; the ablation zone includes the tumor and a safety margin of surrounding tissue. Thermal ablation therapy will be administered according to the manufacturer's instructions of the device. The probe chosen varies depending on the type of thermal ablation, size of the tumor. The ablation probe will be inserted into the lesion, under image guidance. After confirmation of appropriate positioning, the lesion will be heated typically for 10-15 minutes for RFA and 5-10 minutes for microwave, according to manufacturer's guidelines. The time of ablation will depend on thermal ablation technique (RF vs. microwave) as well as the size of the ablation zone desired. The ablation time might also vary depending on proximity of critical structures, patient-specific anatomy or issues. Depending on ablation zone size, several probes might be required with several heating times to achieve complete coverage of the desired ablation zone. Balloon catheter may be used in cases where the tumor is adjacent to a hepatic vein to avoid "heat sync" where the ablation zone is cooled by the blood inflow. This catheter is typically inserted from the neck vein and is then placed into a hepatic vein. Hydrodissection may also be used in cases where the tumor is adjacent to a critical structure such as the heart or colon. These organs can sustain damage from heating, therefore a catheter is inserted into the space separating them and the tumor and fluid (dextrose 5%) is administered during the ablation.
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3.10.3 cryoablation

The freeze zone of cryoablation effects depend upon the probe size and configuration as well as the nadir of the freeze cycle (dependent upon the specific cryogen). Two cryoablation systems are commercially available and FDA cleared for human use (Endocare / Healthtronics and Galil) Both use helium and argon gases to induce lethal temperatures locally. The systems require argon and helium gases which produce low temperatures upon phase change from gas to liquid. The cryotherapy equipment will be an argon-helium gas system using 1.6mm or 2.4 mm cryoprobes (Endocare, Inc., Irvine, CA) or 0.049 inches = 18 gauge = 1.3 mm cryoprobes (Galil). The ice-ball diameters for these probes may vary according to the tissue and blood supply, but their freeze lengths should be comparable to the RFA system (10-30 mm). Iceball diameters refer to the outer 0°C margin, but cytotoxic temperatures (e.g., < -20 to -40 °C)
are known to occur 3-5 mm behind visualized ice margins. A 10- 20 minute freeze, followed by a 5-10 minute thaw, and a 10-20 minute re-freeze produce visible ice and necrosis by rapid and slow mechanisms (dehydration and vascular stasis). The specific times depend upon the temperatures and potentially the temperatures of any remote thermometry used to asse4ss temperature at the margin of a freeze. Rapid intracellular ice formation causes irreversible cell death. Cell membrane dysfunction occurs below -10 degrees C, leading to intracellular ice formation, disrupting organelles. During thawing, osmolar shifts cause cellular swelling and rupture. Apoptosis and vascular stasis may also play a role in cell death by freezing. The probes are placed into the tumor(s) by ultrasound, CT or MRI guidance. Up to 8 simultaneous probes may be placed sequentially for simultaneous treatment (different from current monopolar RFA). 1 % lidocaine is applied to the subcutaneous tissues down to the organ of interest. The helium and argon gases are hooked up to the system, and the cryotherapy machine and software are relatively automatic at this point with the freeze, thaw, refreeze cycles as described above. The process is monitored with ultrasound and CT scans as clinically required for standard operating procedures described by the manufacturers.  
| FUn                                |                                                                |                                                   |                            |      |    |    |      |    |
|------------------------------------|----------------------------------------------------------------|---------------------------------------------------|----------------------------|------|----|----|------|----|
| Off  Treat                         | ment l                                                         | X                                                 | X                          | X  X | X  | X  |      |    |
| years or  until  PD                |                                                                |                                                   |                            |      |    |    |      |    |
| Every 12                           | weeks  for 2                                                   | X                                                 | X                          | X    | X  | X  | X  X | X  |
| 8 (day 36)  1  22  15  8  1        | 22  15  8  1  22  15  8  1  22  15  8  1  22  15  8  1  22  15 | X X X X X X X X X X X X X X X X X X X X X X X X X |                            |      |    |    |      |    |
| Cycle 6                            | X                                                              | X                                                 | X                          | X    | X  | X  | X    |    |
| Cycle 5                            | X                                                              | X                                                 | X                          | X    | X  | X  | X    |    |
| Cycle 4                            | X                                                              | X                                                 | X                          | X    | X  | X  | X    |    |
| Cycle 3                            | X                                                              | X                                                 | X                          | X    | X  | X  | X    |    |
| Cycle 2                            | X                                                              |                                                   |                            |      |    |    |      |    |
| X                                  | X                                                              | X                                                 | X                          | X    | X  | X  |      |    |
| Tremelimumaba X                    | X X                                                            |                                                   |                            |      |    |    |      |    |
| X                                  | X X                                                            | X X                                               | X X                        | X X  |    |    |      |    |
| Informed consent X  Demographics X | X                                                              |                                                   |                            |      |    |    |      |    |
| Medical history  and concomitant   | meds                                                           |                                                   |                            |      |    |    |      |    |
| RFA/TACE/  /Cryoablation b         | Physical exam                                                  |                                                   |                            |      |    |    |      |    |
| Adverse event                      | evaluation                                                     | CBC                                               | w/differential,c Platelets |      |    |    |      |    |
| Performance  Status                |                                                                |                                                   |                            |      |    |    |      |    |
| Vital signsi                       | Weight                                                         |                                                   |                            |      |    |    |      |    |
| Height                             |                                                                |                                                   |                            |      |    |    |      |    |
| Day                                |                                                                |                                                   |                            |      |    |    |      |    |  
Version Date: 08/14/2017 3.11 STUDY CALENDAR Prestudy Cycle 1  
40  
| X                                                 | X                                                                  | X                                            | X               |                    |    |    |    |    |    |
|---------------------------------------------------|--------------------------------------------------------------------|----------------------------------------------|-----------------|--------------------|----|----|----|----|----|
| Xm                                                |                                                                    |                                              |                 |                    |    |    |    |    |    |
| X                                                 | X  X                                                               | X                                            | X               | X                  | X  | X  | X  | X  | X  |
| X X X X X X X X X X X X X X X X X X X X X X X X X | Serum chemistryc X X X X X X X X X X X X X X X X X X X X X X X X X |                                              |                 |                    |    |    |    |    |    |
| X                                                 | X                                                                  | X                                            |                 |                    |    |    |    |    |    |
| 41                                                | X                                                                  | X                                            | X               | X                  |    |    |    |    |    |
| X                                                 | X                                                                  | X                                            | X               | X                  | X  | X  | X  | X  |    |
| X                                                 | X                                                                  | X                                            | X               | X                  | X  | X  | X  | X  | X  |
| Abbreviated Title: anti-CTLA4 & ablative therapy  | X                                                                  | X X                                          |                 |                    |    |    |    |    |    |
| X                                                 | X                                                                  | X                                            | X               | X                  | X  | X  | X  |    |    |
|                                                   |                                                                    |                                              |                 |                    |    |    |    |    |    |
| X X X                                             | X                                                                  |                                              |                 |                    |    |    |    |    |    |
| Version Date: 08/14/2017                          |                                                                    |                                              |                 |                    |    |    |    |    |    |
| X                                                 | X                                                                  |                                              |                 |                    |    |    |    |    |    |
| X                                                 |                                                                    |                                              |                 |                    |    |    |    |    |    |
| X                                                 |                                                                    |                                              |                 |                    |    |    |    |    |    |
| Thyroid Panelc X                                  | X                                                                  | X                                            | X  X            | X                  | X  | X  | X  |    |    |
| MDSC functional                                   | assay  Hepatitis serology  and viral load                          |                                              |                 |                    |    |    |    |    |    |
| Serum aFP or Ca  19.9, hepatitis  serologyd       | Pharmacokinetic                                                    | studiesg                                     | Immunogenicityh |                    |    |    |    |    |    |
| PT, INR, PTT,c                                    | Tumor biopsyf,m                                                    | T cell                                       | activation/ICOS | expression  (PBMC) |    |    |    |    |    |
| Tumor-specific  Į)3UHVSRQVHV                                                   |                                                                    |                                              |                 |                    |    |    |    |    |    |
| HLA-A2  Phenotyping                               | Liver                                                              | autoantibody  panel  (autoimmune  hepatitis) |                 |                    |    |    |    |    |    |
| Immune                                            | monitoringj                                                        |                                              |                 |                    |    |    |    |    |    |
| Restaging  radiologic  Evaluatione                |                                                                    |                                              |                 |                    |    |    |    |    |    |
| ECG                                               |                                                                    |                                              |                 |                    |    |    |    |    |    |  
| X                        |                                                                                   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                                                                                                    |                                                                                                                                                                                                                                                                   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
|--------------------------|-----------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Version Date: 08/14/2017 | a: Tremelimumab: Dose as assigned  b: RFA/TACE/cryoablation on day 35 +/- 96 hrs. | c: Acute, hepatic and mineral panels, uric acid and amylase. If laboratory tests done within 72 hours of Cycle 1 Day 1 they do not need to be  repeated on Day 1.Thyroid Panel and Hepatitis Serologies do not need to be repeated on Day 1 if done within the last 28 days. For all labs a '+/-  48hrs' window applies, with the exception of those needed to determine proceeding with treatment. Labs may be performed outside of NIH. Once  patients are in the every 12 week phase, weekly labs only need to be completed after their dose of tremelimumab for the 28 day period during the  12 weeks. | d: For Hepatitis B: HBV viral load; Anti-HBc Antibody, Anti-HBe Antibody, HBeAg and quantitative HepB Ag, HBV specific immune response.  For Hepatitis C: HCV viral load, anti-HCV Antibody titer. | e: Restaging CT /MRI scan every 8 weeks to evaluate TTP in target lesion. Post cycle 6 restaging will occur every 8 weeks.  f: An optional tumor biopsy may be performed at baseline and at the time of the ablative therapy for analysis of immune infiltration. | g: pre-infusion and immediately post infusion. Day 8 and 15 for local patients only (optional research blood).  h: pre-infusion.  i: Vitals may be done +/- 30 minutes at required timepoints. If patients are local or still post op procedure.  j: Immune monitoring +/- 7 days.  k: As indicated in section 9.3 , all subjects will be offered the opportunity to complete an NIH Advance Directive form. This should be done  preferably at baseline but can be done at any time during the study as long as the capacity to do so is retained. The completion of the form is  strongly recommended, but is not required. | 1: The off treatment visits/labs will occur if patient is agreeable to coming back and physically able to return for the visit. This visit should occur  around day 30 post the last dose of study drug. If toxicities cause discontinuation of therapy, patients will be followed until resolution  of toxicity to at least Grade 1. If the patient cannot return to the Clinical Center for this visit, a request will be made to collect required clinical  labs from a local physician or laboratory. If this is not possible, patients may be assessed by telephone for symptoms |
| X  X X X X X X           | X                                                                                 | Annual telephone  contact                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |                                                                                                                                                                                                    |                                                                                                                                                                                                                                                                   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
| Plasma-based             | assays for  circulating  receptors/ligands  e.g. PDL1                             | NIH Advance  Directive Formk                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |                                                                                                                                                                                                    |                                                                                                                                                                                                                                                                   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |  
42  
Version Date: 08/14/2017 m: An optional tumor biopsy may be obtained 2 weeks prior starting re-induction treatment.n: Follow-up will be annual telephone contact to assess survival status. Every attempt will be made to contact patient/subject including: contacting referring physician, contacting emergency contact patient identified on admission, checking SSDI (Social Security Death Index)
43
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3.12 criteria for removal from

3.12.1 Criteria for removal from protocol therapy Prior to removal from the study, effort must be made to have all subjects complete a safety visit approximately 30 days following the last dose of study therapy In the absence of treatment delays due to adverse events, treatment may continue until one of the following criteria applies:
x Disease progression (per immune-related response criteria)  
x Completion of 2 years' protocol therapy with tremelimumab x Intercurrent illness that prevents further administration of treatment x Unacceptable adverse event(s) x Participant requests to be withdrawn from active therapy x Investigator discretion x Positive pregnancy test x Delayed recovery from toxicity that prevents re-treatment in d 28 days of scheduled therapy x Completed end of treatment safety visit 3.12.2 Criteria for Removal from Study x Death x Investigator discretion x Participant requests to be withdrawn from study x PI decision to close the study
3.12.3 Off protocol therapy and Off Study Procedure Authorized staff must notify Central Registration Office (CRO) when a subject is taken off protocol therapy and when a subject is taken off-study. A Participant Status Updates Form from the web site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must be completed and sent via encrypted email to: NCI Central Registration Office ncicentralregistration-l@mail.nih.gov.
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4 concomitant medications/measures

All routine and appropriate supportive care (including blood products) will be provided during this study, as clinically indicated, and in accordance with the standard of care practices. Clinical judgment should be utilized in the treatment of any AE experienced by the patient. Information on all concomitant medications, administered blood products, as well as interventions occurring during the study must be recorded on the patient's eCRF.
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5 biospecimen collection 5.1 correlative

The correlative studies which we wish to perform are outlined below and summarized in the table. A description of each test including a brief statement of rationale and processing information is made below.  
| Test/assay                                                                                                                               | Volume blood                                                                                                                  | Type of     | Collection point   | Location of              |
|------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------|-------------|--------------------|--------------------------|
| (approx)                                                                                                                                 | tube                                                                                                                          | (+/- 48hrs) | specimen analysis  |                          |
| Immunemonitoring                                                                                                                                          | 120mls (for                                                                                                                   | EDTA        | C1D1, C2D1,        |                          |
| PBMC)                                                                                                                                    | C2D8 (D36) (or  corresponding  day+7 after  TACE/RFA/  cryoablation will  be optional)  C3D1, C4D1,  every 12 weeks  until PD | Figg Lab    |                    |                          |
| 5-10mls (for                                                                                                                             | EDTA                                                                                                                          |             |                    |                          |
| serum)                                                                                                                                   |                                                                                                                               |             |                    |                          |
| PK                                                                                                                                       | 3mls                                                                                                                          | SST         | C1D1 (preinfusion and  immediately postinfusion*), C1D8  (local only),  C1D15 (local  only),  C2D1 (preinfusion and  immediately postinfusion*),  C3D1 (preinfusion and  immediately postinfusion*), and  End-of-treatment  (EOT)                    | MedImmune/Intertek  Alta |
| *Immediately  post-infusion PK  samples should  be collected  within  approximately 15  minutes. Dose 1: pre-infusion,  immediately post |                                                                                                                               |             |                    |                          |  
| Test/assay                                  | Volume blood                          | Type of              | Collection point                                                             | Location of         |
|---------------------------------------------|---------------------------------------|----------------------|------------------------------------------------------------------------------|---------------------|
| (approx)                                    | tube                                  | (+/- 48hrs)          | specimen analysis                                                            |                     |
| infusion, C1D8  (local only), and  C1D15 (local  only)  Dose 2 and  thereafter: preinfusion,  immediately post  infusion  EOT                                             |                                       |                      |                                                                              |                     |
| Immunogenicity                              | 4mls                                  | SST                  | C1D1 (preinfusion), C2D1  (pre-infusion),  C3D1 (preinfusion), C4D1,  Every 3 months,  End-of-treatment  (EOT)                                                                              | MedImmune/PPD       |
| ANA                                         | 4mls                                  | SST                  | Baseline and                                                                 | CC Department of    |
| C4D1                                        | Laboratory Medicine  (DLM)            |                      |                                                                              |                     |
| AMA &  Liver/kidney  microsomal  antibody   | 4 mL                                  | SST                  | Baseline and                                                                 | CC DLM will send to |
| C4D1                                        | Mayo Labs                             |                      |                                                                              |                     |
| Tumor-specific  responses (e.g.  Į)3        | 20mls                                 | EDTA                 | C1D1, C2D1,  C3D1, C4D1,  every 12 weeks  until PD                           | Greten Lab          |
| CFSE-proliferation                          | 5mls                                  | EDTA                 | C1D1, C2D1,                                                                  |                     |
| T cells                                     | C3D1, C4D1,  every 12 weeks  until PD | Greten Lab           |                                                                              |                     |
| MDSC functional                             | 20mls                                 | EDTA                 | C1D1, C2D1,                                                                  |                     |
| assay                                       | C3D1, C4D1,  every 12 weeks  until PD | Greten Lab           |                                                                              |                     |
| Hepatitis B & C                             | 8mls                                  | SST                  | Baseline and q28                                                             | CC Department of    |
| viral load                                  | days.                                 | Transfusion Medicine |                                                                              |                     |
| T cell  activation/ICOS  expression  (PBMC) | 10 mL                                 | EDTA                 | Baseline (both  screening and  preinfusion C1D1  if possible),  C1D15, C2D1, | MedImmune/Covance   |  
| Test/assay                                                          | Volume blood                | Type of     | Collection point   | Location of   |
|---------------------------------------------------------------------|-----------------------------|-------------|--------------------|---------------|
| (approx)                                                            | tube                        | (+/- 48hrs) | specimen analysis  |               |
| C3D1, C4D1,  every 12 weeks  until PD                               |                             |             |                    |               |
| Plasma-based  assays for  circulating  receptors/ligands  e.g. PDL1 | 6mls                        | EDTA        | Baseline, preinfusion every  cycle C1D1,  C2D1, C2D8  (D36), C3D1,  C3D8 (if seen in  clinic), D85,  every 12 weeks  until PD and  EOT.                    | MedImmune     |
| Optional tumor                                                      | NA                          | NA          | Baseline and at    |               |
| biopsy                                                              | time of ablative  procedure | Pathology   |                    |               |
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5.2 nci correlative studies 5.2.1

We will analyze PBMC for quantitative and functional changes of effector cells as well as analyze sera for cytokines and chemokines. 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, (vi) MDSC: frequency + functional assay, (vii) selected cytokines in serum, and (viii) the detection of HCC-associated antigens using ELISPOT assay. Patients will undergo blood sampling (c.120mls blood) on the time points outlined in the table (+/- 48 hrs). Blood will initially be sent to the Figg laboratory for barcoding and processing. On certain occasions the blood may also be brought to the Greten lab for processing and analysis. For Dr. Figg's Blood Processing Core (BPC): Please e-mail Julie Barnes at Julie.barnes@nih.gov and Paula Carter pcartera@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred).
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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 Julie Barnes by e-mail or at 240-760-6044..  
PBMC will be isolated by Ficoll density centrifugation. Aliquots of 1 x 107 PBMC/tube will be individually frozen - after initial handling and processing at the Figg laboratory. 5.2.2 Liver autoantibody panel for autoimmune hepatitis (AIH) AIH is a chronic disorder characterized by progressive hepatocellular loss and cell-mediated immunologic attack. Histologic inflammation is present and is usually accompanied by fibrosis, which can progress to cirrhosis and liver failure. AIH accounts for 11% to 23% of chronic liver disease in North America and about 6% of liver transplants in the United States. The AIH Diagnostic Panel includes tests for actin (smooth muscle) antibody, antinuclear antibodies (ANAs), and liver/kidney microsome antibody (LKM-1). ANAs and actin antibody are associated with type 1 AIH, the most common form in the United States, while LKM-1 antibody is associated with type 2 AIH, more commonly found in Europe and in some South American countries. The panel also includes mitochondrial antibody, which can help differentiate AIH from PBC. Given that autoimmune hepatitis is a potential complication of immune checkpoint inhibition we will perform this panel of autoimmune antibodies to investigate this. Samples will be collected at baseline and on D85 approximately. These antibody titers are exploratory and will not be used to guide therapy in the absence of clinical correlation. 5.2.3 Hepatitis serology and viral load As mentioned in the background section the phase II study of Tremelimumab in HCC showed significant anti-HCV immune response with progressive decline in serum HCV viral load (median values: basal 3.78x10e5 copies/ml vs. day 120 3.02x10e4 copies/ml, p=0.02; vs. day 210 1.69x10e3 copies/ml, p=0.04). Therefore, for all viral hepatitis patients we will measure viral load and antibody titers at baseline and every 30 days. For patients with chronic HBV infection, in addition to the studies already described, we plan to:
- determine HBsAg concentrations in serum
- determine HBeAg (in HBeAg positive patients)
- determine HBsAg fractions (small, middle, large HBsAg)  
- determine HBV specific Tcell responses
These studies will be performed on samples obtained at baseline and every 28 days (collection point +/-48 hours). Samples with be processed by CC Department of Laboratory medicine. For HBsAg and Hepatitis B specific immune response, samples will be processed by the Department of Gastroenterology, Hannover Medical School, Germany. With Amendment E the NCI Thoracic and GI Oncology Branch will release coded serum samples collected in conventional "CryoTubes" in association with this protocol to:
Heiner Wedemeyer Department of Gastroenterology Hannover Medical School Carl Neuberg Strasse 1 30625 Hannover Germany for HBsAg and Hepatitis B specific immune response, as will be specified in NCI Material Transfer Agreement \#38376-14. Specimens will be labeled with the study identifier only and will be shipped to the address above, either on dry ice or at ambient temperatures as required by the type of sample to be analyzed. Tumor-VSHFLILFĮ)3UHVSRQVHV
Frequency of AFP-specific CD4+ T cells will be analyzed using the interferon (IFN)-[gamma]
cytokine secretion assay as previously described68. In brief, PBMCs will be resuspended in medium cultured in duplicate with the following peptides: AFP137-145 (PLFQVPEPV), AFP249-258 (KVNFTEIQKL), and AFP364-373 (QLAVSVILRV) or an irrelevant control peptide (SIINFEKL).  
Recombinant interleukin-2 (25 IU/mL) will be added on day 2 of culture. After 7 days, PBMCs will be re-stimulated with the same peptide and incubated for 5 hours at 37°C and IFN-[gamma]
secretion by CD4+ T cells analyzed by IFN-[gamma] capture assay. Live gating on CD4+ T cells will be performed until up to 100,000 events were acquired. Only AFP responses >0.1% of CD4+
T cells will be considered positive.
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5.2.4 mdsc functional assay

Freshly isolated PBMC will be labeled with CSFE and stimulated with CD2/CD3/CD28 beads in the presence/absence of L-NMMA and NorNOHA. In control experiments CD14 depleted PBMC will be stimulated under the same conditions. Proliferative response of T cells will be assessed by FACS analysis (Serafini et al. Journal of Experimental Medicine (2006) 203: 2691).
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5.2.5 optional tumor biopsy

An optional tumor biopsy may be performed at baseline and at the time of the ablative therapy for analysis of immune infiltration. Tumor Tissue will be processed by the Department of Pathology, NCI. NIH (Dr. David Kleiner). Two core biopsies will be attempted. For each specimen obtained the core will be divided in two parts for Surgical Pathology and frozen preservation. If for some reason only one core is able to be obtained, the core will be divided, with half submitted to Surgical Pathology and half used for PD studies.
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1) formalin-fixed.

i. The half fixed in 10% formalin will be submitted to Surgical Pathology, CCR/NCI (Bldg 10, 2N212).  
ii. The specimens will have routine H&E stains made as well as 5 additional unstained sections.
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2) frozen-preservation

i. Two 1.5 ml cryogenic vials (obtained from Greten lab) will be labeled with the patient's name, accession number (HP\#) and date using a waterproof sharpie.  
ii. The isotherm flask (Greten lab) will be filled with liquid nitrogen on the morning of the procedure and will be available together with the cryogenic vials for pick up from there when radiology page the contact person to collect the specimens.  
iii. Once the biopsy is ready, the half-core to be cryopreserved will be transferred into an empty 1.5-mL cryogenic vial with the use of sterile, pre-chilled (in dry ice) disposable tweezers.  
iv. The vial with specimen will be immediately dropped into liquid nitrogen contained in an isotherm flask.
v. The frozen half will be transferred in the isotherm flask to the protocol-specified location for that particular analysis.  
5.2.6 TCR Beta Sequencing With Amendment H the NCI Thoracic and GI Oncology Branch will release coded tumor and PBMC samples collected in association with this protocol to:
Lara Gruye Adaptive Biotechnologies 1551 Eastlake Ave E \#200, Seattle, WA 98102
(855) 466-8667 For TCR (cell receptor) gene usage may be quantitated in samples using conventional sequencing techniques of the T cell receptor variable region of the beta chain as will be specified in NCI Material Transfer Agreement \#**pending**. Fewer than 100 genes will be analyzed.
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5.3 medimmune planned studies

All samples will be processed and cryopreserved in the Figg laboratory. Transfer in batch to Medimmune/vendor facilities for further analysis will occur at a later date. Please also see Appendix D: MedImmune Sample Preparation Procedures.
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5.3.1 pharmacokinetics

The time points for PK sampling are described in the table in section 5 above. A validated enzyme-linked immunosorbent assay (ELISA) will be used for the quantitative determination of Tremelimumab in human serum.
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5.3.2 immunogenicity

The time points for the assessment of anti-Tremelimumab antibodies are described in the table in section 5 above. A validated electrochemiluminescence assay (ECLA) using a Meso Scale Discovery (MSD) platform will be used for the detection of anti-drug antibodies against Tremelimumab in human serum.
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5.3.3 t cell activation/icos expression

The number and subsets of T cells as well as other immune cells will be evaluated in PBMC by flow cytometry. The activation status of T cells will also be assessed in the same study. Whole blood samples will be collected pre-infusion at baseline (screening and D1), D8, D29, D57 and D85. Samples will be processed to PBMC and stored frozen until time of analysis. Additionally, absolute lymphocyte count at baseline and in response to Tremelimumab treatment will be evaluated for any relationship with treatment outcome. 5.3.4 Plasma-based assays for circulating receptors/ligands e.g. PDL1 Plasma samples are to be collected at the time points listed directly into plastic 6 ml lavender vacutainer tube with EDTA as anticoagulant. 2-3 ml of obtained plasma are to be aliquoted (1 ml each) and frozen. Samples may be analyzed for circulating levels of soluble factors such as CRP, cytokines, and chemokines. They may include but are not limited to soluble CTLA-4, soluble PD-L1, soluble B7.1/B7.2, soluble IL-6R, vascular endothelial growth factor, fibroblast growth factor, IL-1 IL-2, IL-4, IL-6, IL-8, IL-10, cancer biomarkers (alpha fetoprotein, carcinoembryonic antigen, cancer antigen 125, prostate specific antigen, soluble mesothelinrelated protein [SMRP]), granzyme B, IFN, C-X-C motif chemokine 10 (CXCL10), suppressor of cytokine signaling 3 (SOCS3), a proliferation inducing ligand, B-cell activating factor, insulin-like growth factor (IGF)-1, IGF-2, and autoantibodies to host and tumor antigens and explore their association with Tremelimumab treatment and clinical outcome.
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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 Dr. Figg's lab for processing and storage until they are distributed to Dr.  
Greten's lab, Dr. Altan-Bonnet's lab or MedImmune for sample analysis as described in the protocol. Samples will not be sent outside NIH without IRB notification and an executed MTA. All specimens obtained in the protocol are used as defined in the protocol. Any specimens that are remaining at the completion of the protocol will be stored in the conditions described below. The study will remain open so long as sample or data analysis continues. 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. The PI will report any loss or destruction of samples to the NCI IRB as soon as he is made aware of such loss. 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.
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5.4.1 sample data collection

All samples sent to the Blood Processing Core (BPC) will be barcoded, with data entered and stored in the LABrador (aka LabSamples) utilized by the BPC. This is a secure program, with access to LABrador limited to defined Figg lab personnel, who are issued individual user accounts. Installation of LABrador is limited to computers specified by Dr. Figg. These computers all have a password restricted login screen. All Figg lab personnel with access to patient information annually complete the NIH online Protection of Human Subjects course. LABrador creates a unique barcode ID for every sample and sample box, which cannot be traced back to patients without LABrador access. The data recorded for each sample includes the patient ID, name, trial name/protocol number, time drawn, cycle time point, dose, material type, as well as box and freezer location. Patient demographics associated with the clinical center patient number are provided in the system. For each sample, there are notes associated with the processing method (delay in sample processing, storage conditions on the ward, etc.).
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5.4.2 sample storage and destruction

Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -80qC
according to stability requirements. These freezers are located onsite in the BPC and offsite at NCI Frederick Central Repository Services in Frederick, MD. Visitors to the laboratory are required to be accompanied by laboratory staff at all times. Access to stored clinical samples is restricted. Samples will be stored until requested by a researcher named on the protocol. All requests are monitored and tracked in LABrador. All researchers are required to sign a form stating that the samples are only to be used for research purposes associated with this trial (as per the IRB approved protocol) and that any unused samples must be returned to the BPC. It is the responsibility of the NCI Principal Investigator to ensure that the samples requested are being used in a manner consistent with IRB approval.
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5.4.3 protocol completion/sample destruction

x The PI will report destroyed samples to the IRB if samples become unsalvageable because of environmental factors (ex. broken freezer or lack of dry ice in a shipping container) or if a patient withdraws consent. Samples will also be reported as lost if they are lost in transit between facilities or misplaced by a researcher. Freezer problems, lost samples or other problems associated with samples will also be reported to the IRB, the NCI Clinical Director, and the office of the CCR, NCI.
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6 data collection and evaluation

The PI will be responsible for overseeing entry of data into an in-house password protected electronic system (C3D) 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. All data obtained during the conduct of the protocol will be kept in secure network drives or in approved alternative sites that comply with NIH security standards. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant. End of study procedures: Data will be stored according to HHS, FDA, and NIH Intramural Records Retention Schedule regulations as applicable. Loss or destruction of data: Should we become aware that a major breach in our plan to protect subject confidentiality and trial data has occurred, the IRB will be notified. 6.2 **DATA SHARING PLANS** 6.2.1 HUMAN DATA SHARING PLANS Human data generated in this research will be shared for future research as follows: De-identified data in an NIH-funded or approved public repository Identified data in BTRIS Data will be shared through: An NIH-funded or approved public repository: clinicaltrials.gov BTRIS Publication and/or public presentations. Data will be shared at the time of publication or shortly thereafter.
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6.3 response criteria

For the purposes of this study, patients should be re-evaluated for response every 8 weeks. In addition to a baseline scan, confirmatory scans should also be obtained 4-8 weeks following initial documentation of objective response. 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) [*Eur J Ca* 45:228-247, 2009]. 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 definitions 6.3.1.1 evaluable for

All patients will be evaluable for toxicity from the time of their first treatment with Tremelimumab. 6.3.1.2 Evaluable for objective response Only those patients who have measurable disease present at baseline, have received at least one cycle of therapy, and have had their disease re-evaluated will be considered evaluable for response. These patients will have their response classified according to the definitions stated below. (Note: Patients who exhibit objective disease progression prior to the end of cycle 1 will also be considered evaluable.)
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6.3.2 disease parameters 6.3.2.1 measurable

Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as >20 mm by chest x-ray, as >10 mm with CT scan, or >10 mm with calipers by clinical exam. All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters).
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6.3.2.2 malignant lymph nodes

To be considered pathologically enlarged and measurable, a lymph node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). At baseline and in follow-up, only the short axis will be measured and followed. 6.3.2.3 Non-measurable disease All other lesions (or sites of disease), including small lesions (longest diameter <10 mm or SDWKRORJLFDOO\PSKQRGHVZLWKWRPPVKRUWD[LVDUHFRQVLGHUHGQRQ-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by CT or MRI), are considered as non-measurable. Note: Cystic lesions that meet the criteria for radiographically defined simple cysts should not be considered as malignant lesions (neither measurable nor non-measurable) since they are, by definition, simple cysts. 'Cystic lesions' thought to represent cystic metastases can be considered as measurable lesions, if they meet the definition of measurability described above. However, if non-cystic lesions are present in the same patient, these are preferred for selection as target lesions.
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6.3.2.4 target lesions

All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, should be identified as **target lesions** and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. It may be the case that, on occasion, the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected. A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease.
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6.3.2.5 non-target lesions

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

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

Clinical lesions will only be considered measurable when they are superficial (e.g., skin nodules and palpable lymph nodes) and t10 mm diameter as assessed using calipers (e.g., skin nodules).  
In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended.
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6.3.3.2 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. 6.3.3.3 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.
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6.3.3.4 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 in the course of 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.
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6.3.3.5 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.
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6.3.3.6 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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6.3.4 response criteria

6.3.4.1 Evaluation of Target Lesions Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum 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. 6.3.4.2 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). 6.3.4.3 Evaluation of Best Overall Response The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria.  
| Target    | Non-Target             | New     | Overall                                           | Best Overall Response when   |
|-----------|------------------------|---------|---------------------------------------------------|------------------------------|
| Lesions   | Lesions                | Lesions | Response                                          | Confirmation is Required*    |
| CR        | CR                     | No      | CR                                                | >4 wks. Confirmation**       |
| CR        | Non                        |         |                                                   |                              |
| CR/Non-PD | No                     | PR      |                                                   |                              |
| CR        | Not                    | No      | PR                                                |                              |
| evaluated | >4 wks. Confirmation** |         |                                                   |                              |
| PR        | Non                        |         |                                                   |                              |
| CR/NonPD/not  evaluated           | No                     | PR      |                                                   |                              |
| SD        | Non                        |         |                                                   |                              |
| CR/Non           | No                     | SD      | Documented at least once >4  wks. from baseline** |                              |  
| Target                                                                                                                                                                                                                                                                                                                              | Non-Target                                                                                                           | New     | Overall               | Best Overall Response when   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------|---------|-----------------------|------------------------------|
| Lesions                                                                                                                                                                                                                                                                                                                             | Lesions                                                                                                              | Lesions | Response              | Confirmation is Required*    |
| PD/not  evaluated                                                                                                                                                                                                                                                                                                                   |                                                                                                                      |         |                       |                              |
| PD                                                                                                                                                                                                                                                                                                                                  | Any                                                                                                                  | Yes or  | PD                    |                              |
| No                                                                                                                                                                                                                                                                                                                                  |                                                                                                                      |         |                       |                              |
| Any                                                                                                                                                                                                                                                                                                                                 | PD***                                                                                                                | Yes or  | no prior SD, PR or CR |                              |
| PD                                                                                                                                                                                                                                                                                                                                  |                                                                                                                      |         |                       |                              |
| No                                                                                                                                                                                                                                                                                                                                  |                                                                                                                      |         |                       |                              |
| 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. |                                                                                                                      |         |                       |                              |  
| 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-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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6.3.5 duration of response 6.3.5.1

The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented. 6.3.5.2 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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6.4 modified immune-related response criteria

Modified immune-related response criteria (irRC) 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 irRC. Please refer to Appendix C section **12.3** for further details.
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6.5 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 4.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 4.0. A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov). All appropriate treatment areas should have access to a copy of the CTEP Active Version of CTCAE.