epigenetic priming for immune therapy
untitled h2

Protocol Number: CC\# 197520 Investigational Product(s): pembrolizumab, vorinostat, tamoxifen Version Number: 3.3 Version Date: 09/30/2021 IND Number: 144356 Principal Investigator (Sponsor-Investigator)  
Pamela N. Munster, MD  
  
  
E-mail:
Statistician Mi-Ok Kim, PhD
Revision History  
Version 3.3 09/30/2021 Version 3.2 04/15/2021 Version 3.1 01/28/2020 Version 3.0 07/23/2019 Version 2.1 06/26/2019  
Version 2.0 06/07/2019  
Version 1.0 02/12/2019 Version 00 07/01/2018  
Proprietary and Confidential The information in this document is considered privileged and confidential, and may not be disclosed to others except to the extent necessary to obtain Institutional Review Board approval and informed consent, or as required by federal and state laws. Persons to whom this information is disclosed should be informed that this information is privileged and confidential and that it should not be further disclosed.
epigenetic priming for immune therapy
protocol signature page

Protocol No.: CC\# 197520 **Version Date:** 09/30/2021  
1. I agree to follow this protocol version as approved by the UCSF Protocol Review Committee
(PRC), Institutional Review Board (IRB), and Data and Safety Monitoring Committee (DSMC).  
2. I will conduct the study in accordance with Good Clinical Practices (ICH-GCP) and the applicable IRB, ethical, federal, state, and local regulatory requirements.  
3. I certify that I, and the study staff, have received the required training to conduct this research protocol.  
4. I agree to maintain adequate and accurate records in accordance with IRB policies, federal, state, and local laws and regulations.
epigenetic priming for immune therapy
abstract

| Title                     | Epigenetic priming for Immune therapy in ER-positive breast cancer in  biomarker select population (Pembrolizumab, Vorinostat, Tamoxifen)                                                                                  |                                                       |
|---------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------|
| Study Description         | We propose a randomized two-arm trial, enrolling 65 patients with  hormone therapy-resistant breast cancer (HTR) to test the role of  epigenetic priming in a biomarker enriched patient population in HTR  breast cancer. |                                                       |
| Phase of Study            | Phase II                                                                                                                                                                                                                   |                                                       |
| Investigational           | Pembrolizumab, Vorinostat, Tamoxifen                                                                                                                                                                                       |                                                       |
| Products Study population | Pre- and postmenopausal women or men with stage IV ER+ breast  cancer with histological or cytological confirmation, 18 years or older                                                                                     |                                                       |
| Primary Objective         | To define the role of epigenetic immune priming in a biomarker enriched  ER+ breast cancer population on the basis of overall response rate  (ORR: CR+PR+SD at 24 weeks).                                                  |                                                       |
| Secondary                 | a)                                                                                                                                                                                                                         | To assess duration of response (DOR) 24-week landmark |
| Objectives                | progression-free survival (PFS:24)                                                                                                                                                                                         |                                                       |
| b)                        | Median PFS and overall survival (OS)                                                                                                                                                                                       |                                                       |
| c)                        | Tumor responses will also be calculated by Immune Related  Response-Criteria (irRC).                                                                                                                                       |                                                       |  
| Exploratory        | a)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | Evaluation of biomarker target threshold on response rate   |
|--------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------|
| Objectives         | (retrospective cut off of 20% vs. 10%)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |                                                             |
| b)                 | To assess the ratio of effector T cells: regulatory T cells in blood and  tumor biopsies pre- and post-therapy.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |                                                             |
| c)                 | To evaluate inflammatory T cell signature changes in blood and  tumor biopsies pre- and post-therapy.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |                                                             |
| d)                 | To evaluate changes in number of myeloid-derived suppressor cells  (MDSCs) in peripheral blood and tumor biopsies pre- and posttherapy.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |                                                             |
| e)                 | To evaluate changes in histone acetylation in peripheral blood cells  and tumor biopsies pre- and post-therapy                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |                                                             |
| f)                 | (Initial comparison to vorinostat-induced PD-1 in lymphocytes, PDL1 modulation                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |                                                             |
| g)                 | Nanostring and 10x sequencing and single cell immune phenotyping  (on stored tissue for successful arms only)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                                                             |
| h)                 | Impact of HDAC inhibition of response to pembrolizumab vs.  pembrolizumab in biomarker enriched population                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                             |
| Sample Size        | Arm A (pembro + vorinostat + TAM): A total of 35 patients will be  accrued. Arm B (pembro + TAM): A total of 30 patients will be accrued.  Total of 65 patients will be accrued.                                                                                                                                                                                                                                                                                                                                                                                                                                                    |                                                             |
| Duration of Study  | Patients may continue treatment until development of toxicity or until the                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                             |
| Treatment          | patient no longer experiences clinical benefit.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |                                                             |
| Duration of Follow | Patients will be followed for 30 days after end of study participation or                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |                                                             |
| up                 | removal from study, and serious adverse events will be collected for 30  days after the end of treatment should the patient be taken off therapy.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |                                                             |
| Unique Aspects of  | This is one of the first studies to look at the response of hormone                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |                                                             |
| this Study         | therapy-resistant breast cancer to epigenetic immune priming and the  combination of an HDAC inhibitor (vorinostat), an anti-estrogen  (tamoxifen) and a PD-1 inhibitor (pembrolizumab) in pre or  postmenopausal patients with ER+ advanced breast cancer with  progression on multiple prior therapies in a biomarker-enriched  population.  The goal of this study is to demonstrate that HDAC induced epigenetic  priming in a biomarker enriched study will increase the efficacy of  pembrolizumab with patient selection based on exhausted CD8+ cells in  blood or tumor in a small subset of (ER)+ breast cancer patients. |                                                             |
epigenetic priming for immune therapy
list of abbreviations

| AE      | adverse event                                       |
|---------|-----------------------------------------------------|
| ALP     | alkaline phosphatase                                |
| ALT     | alanine aminotransferase                            |
| AST     | aspartate aminotransferase                          |
| BUN     | blood urea nitrogen                                 |
| CBC     | complete blood cell (count)                         |
| CNS     | central nervous system                              |
| CR      | complete response                                   |
| CRF     | case report form                                    |
| CT      | computerized tomography                             |
| CTCAE   | Common Terminology Criteria for Adverse Events      |
| CTMS    | Clinical Trial Management System                    |
| DFS     | disease-free survival                               |
| DLT     | dose-limiting toxicity                              |
| DSMC    | Data and Safety Monitoring Committee                |
| DSMP    | Data and Safety Monitoring Plan                     |
| ECG/EKG | electrocardiogram                                   |
| ECOG    | Eastern Cooperative Oncology Group                  |
| FDA     | Food and Drug Administration                        |
| FDG     | Fluorodeoxyglucose                                  |
| FLC     | free light chain                                    |
| GCP     | Good Clinical Practice                              |
| GFR     | glomerular filtration rate                          |
| HBeAg   | hepatitis B "e" antigen                             |
| HBV     | hepatitis B virus                                   |
| HCV     | hepatitis C virus                                   |
| HDFCCC  | Helen Diller Family Comprehensive Cancer Center     |
| HIPAA   | Health Insurance Portability and Accountability Act |
| HIV     | human immunodeficiency virus                        |
| ICF     | informed consent form                               |
| ICH     | International Conference on Harmonization           |
| IDS     | Investigational Drug Services (UCSF)                |
| IND     | investigational new drug application                |
| IP      | investigational product                             |  
| IRB   | Institutional Review Board              |
|-------|-----------------------------------------|
| IV    | intravenous                             |
| LDH   | lactate dehydrogenase                   |
| MRI   | magnetic resonance imaging              |
| MTD   | maximum tolerated dose                  |
| NCI   | National Cancer Institute               |
| ORR   | overall response rate                   |
| PD    | disease progression                     |
| PK    | pharmacokinetics                        |
| PO    | Per os (by mouth, orally)               |
| PR    | partial response                        |
| PRC   | Protocol Review Committee (UCSF)        |
| SD    | stable disease                          |
| SGOT  | serum glutamic oxaloacetic transaminase |
| SGPT  | serum glutamic pyruvic transaminase     |
epigenetic priming for immune therapy
table of contents

| Table of Contents                                                                                                                      |                                                                                                                                                     |
|----------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------|
| Protocol Signature Page                                                                                                                |                                                                                                                                                     |
| Abstract                                                                                                                               | 3                                                                                                                                                   |
| List of Abbreviations                                                                                                                  |                                                                                                                                                     |
| Table of Contents                                                                                                                      |                                                                                                                                                     |
| 1                                                                                                                                      | I                                                                                                                                                   |
| Background on Indication ...........................................................................................................1( |                                                                                                                                                     |
| 1.1                                                                                                                                    | Introduction to breast cancer ........................................................................................1(                            |
| 1.1.1                                                                                                                                  |                                                                                                                                                     |
| 1.1.2                                                                                                                                  | Hormone-sensitive breast cancer..................................................................................1(                                 |
| 1.1.3                                                                                                                                  | Therapy resistance in breast cancer .................................................................................1(                             |
| 1.2                                                                                                                                    | Background on the Investigational Products and Associated Known Toxicities.11                                                                       |
| 1.2.1                                                                                                                                  | Pembrolizumab/MK-3475.......................................................................................................11                      |
| 1.2.2                                                                                                                                  | Suberoylanilide hydroxamic acid (Vorinostat, SAHA) ........................................12                                                       |
| 1.2.3                                                                                                                                  | Tamoxifen ..................................................................................................................................17      |
| 1.2.4                                                                                                                                  | Histone Deacetylases (HDAC) and their inhibitors ...............................................17                                                  |
| 1.3                                                                                                                                    | Rationale for the Proposed Study .                                                                                                                  |
| 1.3.1                                                                                                                                  | RATIONALE                                                                                                                                           |
| 1.4                                                                                                                                    | Rationale for the Dose Selection/Regimen ........................................................................10                                 |
| 1.5                                                                                                                                    | Correlative Studies .............................................................................................................................10 |
| 2                                                                                                                                      | Study Objectives                                                                                                                                    |
| 2.1                                                                                                                                    | Hypothesis .                                                                                                                                        |
| 2.2                                                                                                                                    | Primary Objective and Endpoints...........................................................................................17                        |
| 2.3                                                                                                                                    | Secondary Objective(s) and Endpoint(s)                                                                                                              |
| 2.4                                                                                                                                    | Exploratory (Correlative) Objectives .......................................................................................18                      |
| 3                                                                                                                                      | Study Design .                                                                                                                                      |
| 3.1                                                                                                                                    | Characteristics                                                                                                                                     |
| 3.2                                                                                                                                    | Sample Size                                                                                                                                         |
| 3.3                                                                                                                                    | Eligibility Criteria                                                                                                                                |
| 3.3.1                                                                                                                                  | Inclusion Criteria .                                                                                                                                |
| 3.3.2                                                                                                                                  | Exclusion Criteria                                                                                                                                  |
| Inclusion and Recruitment of Women and Minorities ..................................................21                                 |                                                                                                                                                     |
| 3.4                                                                                                                                    |                                                                                                                                                     |
| 3.5                                                                                                                                    | Duration of Treatment                                                                                                                               |
| 3.6                                                                                                                                    | Duration of Follow Up                                                                                                                               |
| 3.7                                                                                                                                    | Randomization Procedures .                                                                                                                          |
| 3.8                                                                                                                                    | Primary Completion .                                                                                                                                |
| 3.9                                                                                                                                    | Study Completion .                                                                                                                                  |
| 4                                                                                                                                      | Investigational Products .                                                                                                                          |
| 4.1                                                                                                                                    | Description, Supply, and Storage of Investigational Products..................................21                                                    |
| 4.1.1                                                                                                                                  | Investigational Product: Pembrolizumab (MK-3475)............................................2:                                                      |
| 4.1.2                                                                                                                                  | Investigational Product: Vorinostat.                                                                                                                |  
Table of Contents  
| 4.1.3                                                                                                                                                                                                                                                                                               | Investigational Product: Tamoxifen 25                                       |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------|
| 4.2                                                                                                                                                                                                                                                                                                 | Accountability Records for Investigational Product(s) 26                    |
| 4.3                                                                                                                                                                                                                                                                                                 | Ordering Investigational Product(s) 26                                      |
| 5                                                                                                                                                                                                                                                                                                   | Treatment Plan 26                                                           |
| 5.1                                                                                                                                                                                                                                                                                                 | Dosage and Administration 26                                                |
| 5.2                                                                                                                                                                                                                                                                                                 | Dose Modifications and Dosing Delays 28                                     |
| 5.2.1                                                                                                                                                                                                                                                                                               | Dose Modifications and Dosing Delays Tables for Specific Adverse Events     |
| for Pembrolizumab 31 5.2.2 Dose Modification and toxicity management for immune-related AEs  associated with pembrolizumab 32 5.2.3 Dose Modification and Toxicity Management of Infusion-Reactions Related  to Pembrolizumab 39 5.2.4 Second Course 41 5.2.5 Monitoring and Toxicity Management 42 |                                                                             |
| 5.3                                                                                                                                                                                                                                                                                                 | Stopping Rules 42                                                           |
| 5.4                                                                                                                                                                                                                                                                                                 | Discontinuation and Withdrawal 43                                           |
| 5.4.1                                                                                                                                                                                                                                                                                               | Supportive Care Guidelines for Pneumonitis 44                               |
| 6                                                                                                                                                                                                                                                                                                   | Study Procedures and Schedule of Events 45                                  |
| 6.1                                                                                                                                                                                                                                                                                                 | Study Calendar 46                                                           |
| 6.2                                                                                                                                                                                                                                                                                                 | Schedule of Procedures and Observations 48                                  |
| 6.2.1                                                                                                                                                                                                                                                                                               | Prescreening Period 48                                                      |
| 6.3                                                                                                                                                                                                                                                                                                 | Participant Registration 48                                                 |
| 6.3.1                                                                                                                                                                                                                                                                                               | Pretreatment Period 48                                                      |
| 6.3.2                                                                                                                                                                                                                                                                                               | Treatment Period 49                                                         |
| 6.3.3                                                                                                                                                                                                                                                                                               | Survival Follow-up 51                                                       |
| 6.4                                                                                                                                                                                                                                                                                                 | Correlative Studies 52                                                      |
| 6.4.1                                                                                                                                                                                                                                                                                               | Histone Acetylation in peripheral blood mononuclear cells 52                |
| 6.4.2                                                                                                                                                                                                                                                                                               | Evaluation of vorinostat effects on tumor tissue acetylation and changes in |
| estrogen receptor expression 52 6.4.3 Logistics on Immune modulation 52                                                                                                                                                                                                                             |                                                                             |
| 6.5                                                                                                                                                                                                                                                                                                 | Use of Concurrent/Concomitant Medications 52                                |
| 6.5.1                                                                                                                                                                                                                                                                                               | Acceptable Concomitant Medications 52                                       |
| 6.5.2                                                                                                                                                                                                                                                                                               | Rescue Medications & Supportive Care 53                                     |
| 6.6                                                                                                                                                                                                                                                                                                 | Prohibited Medications 54                                                   |
| 7                                                                                                                                                                                                                                                                                                   | Reporting and Documentation of Results 55                                   |
| 7.1                                                                                                                                                                                                                                                                                                 | Evaluation of Efficacy: Antitumor Effect - Solid Tumors 55                  |
| 7.1.1                                                                                                                                                                                                                                                                                               | Definitions 55                                                              |
| 7.1.2                                                                                                                                                                                                                                                                                               | Disease Parameters 56                                                       |
| 7.1.3                                                                                                                                                                                                                                                                                               | Methods for Evaluation of Measurable Disease 57                             |
| 7.1.4                                                                                                                                                                                                                                                                                               | Response Criteria 58                                                        |
| 7.2                                                                                                                                                                                                                                                                                                 | Evaluation of Safety 60                                                     |
| 7.3                                                                                                                                                                                                                                                                                                 | Definitions of Adverse Events 60                                            |
| 7.3.1                                                                                                                                                                                                                                                                                               | Adverse Event 60                                                            |  
Table of Contents  
| Table of Contents 7.3.2 Adverse Reaction 60 7.3.3 Suspected Adverse Reaction 60 7.3.4 Recording of Adverse Events 62 7.3.5 Follow-up of Adverse Events 63 7.3.6 Adverse Events Reporting 63 7.3.7 Definition of an Overdose for this Protocol and Reporting of Overdose to  the Study PI and Merck 64 7.3.8 Adverse Events Monitoring 65 7.3.9 Expedited Reporting 65 8 Statistical Considerations and Evaluation of Results 67 8.1 Sample Size Considerations 67 8.1.1 Sample Size and Power Estimate 67 8.1.2 Randomization 68 8.1.3 Stratification Factors 68 8.1.4 Accrual Estimates 68 8.2 Interim Analyses and Stopping Rules 68 8.3 Analyses Plans 68 8.3.1 Analysis Population 68 8.3.2 Primary Analysis (or Analysis of Primary Endpoints) 68 8.3.3 Secondary Analysis (or Analysis of Secondary Endpoints) 69 8.3.4 Exploratory/Correlative Analysis/Assessments 69 9 Study Management 71 9.1 Pre-study Documentation 71 9.2 Institutional Review Board Approval 71 9.3 Informed Consent 71 9.4 Changes in the Protocol 72 9.5 Handling and Documentation of Clinical Supplies 72 9.6 Case Report Forms (CRFs) 72 9.7 Oversight and Monitoring Plan 72 9.8 Record Keeping and Record Retention 73 10 References 74 Appendix 1 Performance Status Criteria 78 Appendix 2 Data and Safety Monitoring Plan for a Phase II or III Institutional Study 79 Appendix 3 Prohibited Medications List 82   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
epigenetic priming for immune therapy
1 introduction 1.1 background on

Breast cancer is one of the most common cancers in the western hemisphere and the second most common cause of cancer death in women1. Endocrine therapy (including ovarian ablation, estrogen administration, and more recently, antiestrogen administration) has been a major component of antitumor therapy in breast cancer for more than a century2. Hormone-sensitive metastatic breast cancer remains a therapeutic challenge despite the many recent advances in therapy as these tumors are frequently less sensitive to chemotherapy3. Barring a few exceptions, cure is not achieved in this disease. While hormonal interventions have clearly been shown to decrease tumor burden and to prolong life, the majority of patients eventually succumb to their disease due to disease recurrence.
epigenetic priming for immune therapy
1.1.2 hormone-sensitive breast cancer

More than two-thirds of all advanced breast cancers are dependent on estrogen. Estrogen withdrawal by physical removal of the ovaries or inhibition of their function has been one of the earliest successful interventions against breast cancer; however, this intervention was limited to premenopausal women. The introduction of the SERMS (selective estrogen receptor modulators) has dramatically improved the survival of women with breast cancer, both pre- and post-menopausal. The most prominent SERM is tamoxifen, which until recently has been the gold standard for first-line therapy in women with advanced hormone-sensitive breast cancer. The production of estrogen in postmenopausal women requires an enzyme called aromatase, which can be successfully blocked chemically. The introduction of several aromatase inhibitors (AIs) has added greatly to the arsenal against breast cancer, and they have replaced tamoxifen as first-line therapy in the postmenopausal setting. The most commonly used aromatase inhibitors include letrozole, anastrozole, and exemestane. However, the AIs are reserved for women who have naturally entered menopause or have their ovarian function suppressed. For premenopausal women or women who are unable to tolerate AIs, tamoxifen remains the gold standard. The response rate to first-line hormonal therapy, determined from four pivotal randomized Phase III trials, ranges from 20-33%4-7. The response rate to second-line hormonal therapy is considerably less (10-20%)4, suggesting a clear need for more effective therapy8-10.
epigenetic priming for immune therapy
1.1.3 therapy resistance in breast

Breast cancer has now been subclassified into several major subgroups: hormone therapy sensitive, hormone therapy-resistant (HTR), HER2 positive (HER2+), and triple negative (TN). The treatment approaches to these clinical subtypes vastly differ. Hormone resistance has been attributed to a number of different mechanisms, namely loss of ER expression, alterations in apoptosis and cell signaling pathways, changes in ER coregulators, and the development of escape pathways11. Therefore, these women receive minimal benefit from endocrine therapy.  
The development of hormone therapy resistance is one of the most frequent causes of cancer death in women worldwide.
epigenetic priming for immune therapy
1.2 background on the investigational

Refer to the Investigator's Brochure (IB) for detailed background information on MK-3475. Pembrolizumab (previously known as SCH 900475) is a potent and highly selective humanized monoclonal antibody (mAb) of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2.
epigenetic priming for immune therapy
1.2.1.1 pharmaceutical and therapeutic background

The importance of intact immune surveillance in controlling outgrowth of neoplastic transformation has been known for decades12. Accumulating evidence shows a correlation between tumor-infiltrating lymphocytes (TILs) in cancer tissue and favorable prognosis in various malignancies13-17. In particular, the presence of CD8+ T-cells and the ratio of CD8+
effector T-cells / FoxP3+ regulatory T-cells correlates with improved prognosis and long-term survival in many solid tumors. The PD-1 receptor-ligand interaction is a major pathway hijacked by tumors to suppress immune control. The normal function of PD-1 expressed on the cell surface of activated T-cells under healthy conditions, is to down-modulate unwanted or excessive immune responses, including autoimmune reactions. PD-1 (encoded by the gene Pdcd1) is an Ig superfamily member related to CD28 and CTLA-4 which has been shown to negatively regulate antigen receptor signaling upon engagement of its ligands (PD-L1 and/or PD L2)1,2. The structure of murine PD-1 has been resolved3. PD-1 and family members are type I transmembrane glycoproteins containing an Ig Variable-type (V-type) domain responsible for ligand binding and a cytoplasmic tail which is responsible for the binding of signaling molecules. The cytoplasmic tail of PD-1 contains 2 tyrosine-based signaling motifs, an immunoreceptor tyrosine-based inhibition motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Following T-cell stimulation, PD 1 recruits the tyrosine phosphatases SHP-1 and SHP-2 to the ITSM motif within its cytoplasmic tail, leading to the dephosphorylation of effector molecules such as CD3ζ, PKCθ, and ZAP70 which are involved in the CD3 T-cell signaling cascade1,4-6.  
The mechanism by which PD-1 downmodulates T-cell responses is similar to, but distinct from that of CTLA-4 as both molecules regulate an overlapping set of signaling proteins7,8. PD-1 was shown to be expressed on activated lymphocytes including peripheral CD4+ and CD8+ T-cells, B-cells, T regs and Natural Killer cells9,10. Expression has also been shown during thymic development on CD4-CD8- (double negative) T-cells as well as subsets of macrophages and dendritic cells11. The ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed or can be induced in a variety of cell types, including non-hematopoietic tissues as well as in various tumors7,18-20. Both ligands are type I transmembrane receptors containing both IgV- and IgC-like domains in the extracellular region and contain short cytoplasmic regions with no known signaling motifs. Binding of either PD-1 ligand to PD-1 inhibits T-cell activation triggered through the T-cell receptor. PD-L1 is expressed at low levels on various non-hematopoietic tissues, most notably on vascular endothelium, whereas PD-L2 protein is only detectably expressed on antigen-presenting cells found in lymphoid tissue or chronic inflammatory environments. PD-L2 is thought to control immune T-cell activation in lymphoid organs, whereas PD-L1 serves to dampen unwarranted T-cell function in peripheral tissues7. Although healthy organs express little (if any) PD-L1, a variety of cancers were demonstrated to express abundant levels of this T-cell inhibitor. PD-1 has been suggested to regulate tumor-specific T-cell expansion in subjects with melanoma (MEL)21. This suggests that the PD-1/PD-L1 pathway plays a critical role in tumor immune evasion and should be considered as an attractive target for therapeutic intervention.
epigenetic priming for immune therapy
1.2.1.2 pre-clinical and clinical trials

Therapeutic studies in mouse models have shown that administration of antibodies blocking PD1/PD-L1 interaction enhances infiltration of tumor-specific CD8+ T-cells and leads ultimately to tumor rejection, either as a monotherapy or in combination with other treatment modalities. Antimouse PD-1 or anti-mouse PD-L1 antibodies have demonstrated anti-tumor responses as a mono-therapy in models of squamous cell carcinoma, pancreatic carcinoma, MEL, and colorectal carcinoma. Blockade of the PD-1 pathway effectively promoted CD8+ T-cell infiltration into the tumor and the presence of IFN-ɣ, granzyme B, and perforin, indicating that the mechanism of action involved local infiltration and activation of effector T-cell function in vivo22-27. Experiments have confirmed the in vivo efficacy of PD-1 blockade as a monotherapy as well as in combination with chemotherapy in syngeneic mouse tumor models (see the Investigator's Brochure [IB]). For trial details, please refer to the Investigator's Brochure. Pembrolizumab has been approved in several indications, see FDA label and IB v 16.
epigenetic priming for immune therapy
1.2.2 suberoylanilide hydroxamic acid (vorinostat

Vorinostat (Suberoylanilide hydroxamic acid, SAHA; NSC 701852) is a small molecule inhibitor of histone deacetylase (HDAC) that binds directly in the enzyme's active site in the presence of a zinc ion28. Because aberrant HDAC activity has been implicated in a variety of cancers, development of HDAC inhibitors is a rational approach to the design of targeted anticancer therapeutics. Several HDAC inhibitors from multiple chemical classes have been developed and are currently in clinical trials. Trichostatin A and butyric acid were among the first HDAC inhibitors to be administered to patients, but these were found to be clinically unsuitable due to potency and formulation issues29,30. Depsipeptide was originally selected for clinical study based on its antiproliferative effects; subsequently, it was discovered to antagonize HDACs and was the first HDAC inhibitor to demonstrate clinical efficacy31. Of the three classes of HDACs, vorinostat targets most human Class 1 (related to the yeast transcriptional regulator Rpd3) and Class 2 (similar to the yeast Hda1) enzymes32,33. The third class of HDACs (homologs of yeast sir2) requires NAD+ for activity and is not inhibited by vorinostat. Among those currently in clinical trials, vorinostat is the most potent HDAC inhibitor that can be administered orally with excellent bioavailability. Vorinostat was the first HDAC approved for CTCL in 2006.  
Vorinostat was identified originally by its ability to induce differentiation of murine erythroleukemia cells at micromolar concentrations34. Subsequently, it was found to induce differentiation or arrest growth of a wide variety of human carcinoma cells. To date, vorinostat activity has been reported in transformed hematopoietic cells, including multiple myeloma (MM), acute promyelocytic leukemia (APL), acute lymphocytic leukemia, chronic myelogenous leukemia, Waldenstrom's macroglobulinemia, and cutaneous T-cell lymphoma (CTCL)35-39.  
Activity has also been reported in cell lines representing other tumor types, including bladder transitional cell carcinoma, breast cancer, prostate cancer, head and neck squamous carcinoma, and colon carcinoma40-45.  
The antitumor activity of vorinostat was demonstrated in several in vivo models of cancer, including a xenograft model of human CWR22 prostate cancer cells41, a mouse model of APL
containing the promyelocytic leukemia zinc-finger-retinoic acid receptor α fusion gene (PLZFRARα)46, and an N-methylnitrosourea-induced mammary tumor model in rodents47. Vorinostat has also shown activity when administered daily by intraperitoneal (IP) injections in the CWR22 and PLZF-RARα models and by oral (PO) administration in the carcinogen-induced mammary tumor model.
epigenetic priming for immune therapy
1.2.3 tamoxifen

Tamoxifen is the most commonly used selective estrogen receptor modulator, SERM. It is currently being used for the prevention of breast cancer, for ductal carcinoma in situ, in women with early stage breast cancer and for those with metastatic cancer. It is generally well tolerated, and due to its extensive use, its toxicities and long-term sequelae are well characterized. Women being treated with tamoxifen may experience flushing (similar to the flushing women experience during menopause), vaginal dryness, and vaginal discharge. The most serious side effect is the slightly increased risk of thromboembolic events. In a trial involving 900 women treated with either tamoxifen or letrozole, 9 out of 455 patients experienced a thromboembolic event (2%), compared to 3 patients out of 455 in the group treated with letrozole (<1%). Other side effects included hot flashes (25%), headaches (5%), fatigue (5%), and nausea (8%)7. The observation of endometrial cancer (<0.5%) has been predominantly observed when used in the preventive or adjuvant setting48. Hence, the cumulative administration of tamoxifen is rarely hampered by toxicity, but rather by the emergence of resistance suggested by clinical progression while on the drug.
epigenetic priming for immune therapy
1.2.4 histone deacetylases (hdac) and

A promising new class of drugs showing activity in cancer is the histone deacetylase inhibitors
(HDACi)32,49. While these agents may have single-agent activity in cancer, they may also sensitize certain cancers to other types of therapeutic agents or overcome resistance. Histone deacetylases (HDAC) function to modulate gene expression by the removal of acetyl groups from histones50,51. The addition of acetyl groups to lysine residues on the N-terminal tail of histones by histone acetyltransferases (HAT) results in the weakening of the bond between histones and DNA51.  
In contrast, removal of acetyl groups by HDACs results in the condensation of the nucleosomes and has been correlated with gene silencing51. HDACs can be divided into at least three different classes, I-III. Each class contains several members of structurally and functionally variable HDACs52,53. Alterations in the regulatory function of HATs and HDACs have been associated with the development of certain cancers54. For example, mutations in the HAT gene CBP have been correlated with the development of certain colorectal and gastric cancers55, whereas altered HDAC function has been associated with the onset of acute promyelocytic leukemia (APL)54. There are several inhibitors of HDACs that show anti-cancer activity in vitro and in vivo56. These include the short-chain fatty acids (sodium butyrate, valproic acid (VPA)),
the hydroxamic acids (suberoylanilide hydroxamic acid (SAHA), trichostatin A), the cyclic tetrapeptides (depsipeptide (FK-228)) and the benzamides (MS-275). Vorinostat, FK-228, MS275, and others are currently undergoing early clinical testing.
epigenetic priming for immune therapy
1.3 rationale for the proposed

Immunotherapy responses have remained limited in ER+ breast cancer. Our preliminary and clinical data suggest that epigenetic priming may be a successful strategy to enhance immune response. However, epigenetic priming is predominantly successful in patients with tumors that have exhausted CD8 subpopulation. This proposal is focused on the potential role of epigenetic immune priming to reverse therapy resistance in pre- and postmenopausal breast cancer. Initially focused on all hormone therapy positive patients, data from our pilot study with epigenetic priming suggests that benefits are vastly different if the population is limited to those patients with a 20% or higher expression of CD8 cells with dual expression of PD-L1 and CTLA in tumors (or 10% in blood). The pilot study, therefore, has two objectives, a) to show that vorinostat can increase the efficacy of pembrolizumab in a select ER+ breast cancer patient population and b) a biomarker select patient population has a higher than expected response rate to pembrolizumab The goal of immune priming is to increase the number and composition of tumor-infiltrating lymphocytes, which renders this a relevant approach to therapy resistant ER+ breast cancer. The goal of the biomarker selection is to enrich for a population who is immune responsive
epigenetic priming for immune therapy
1.3.1.1 programmed cell death 1

Inhibition of the PD-L1/PD-1 axis has been a major milestone in many cancer types. Clinically, epigenetic immune regulation has been implicated in priming lung cancer cells to PD-1 inhibitors, which resulted in prolonged responses14,15. Increased PD-L1 expression has been observed in hormone therapy resistance, frequently reported with loss of progesterone receptor (PR)
expression, a measure of hormone therapy resistance16. Hence, PD-1 inhibitors may have a role in hormone therapy-resistant breast cancer, and PD-1 inhibitors may be more effective after epigenetic immune priming. Several investigators have shown that HDAC inhibitors have immune modulatory functions, including modulation of Tregs, FoxP3 expression, changes in tumor-infiltrating lymphocytes (TILs), induction of PD-L1 expression, and blockage of PD-1 signaling. Hence, the combination of HDAC inhibitors may potentiate the efficacy of PD-1 inhibitors17.
epigenetic priming for immune therapy
1.3.1.2 pilot study - exhausted

Our initial pilot study of 34 patients with ER+ positive patients suggests that vorinostat decreases T-reg with Foxp3 expression. However, these effects were only associated with clinical benefit in patients with high expression of CD8 (CTLA/PD-L1) in their blood or tumor samples. The pilot included 34 patients (median age 56, 88% ECOG 1) who had been treated with a median of 5 (2-12) prior regimens. A minority experienced grade 3-4 toxicity (autoimmune hepatitis, fatigue, thrombocytopenia), with each toxicity occurring in 2 out of 34 patients (6%). Grade 2 toxicities included fatigue (27%), thrombocytopenia (12%), colitis (3%), and pneumonitis (3%). Six patients did not receive Pembrolizumab due to rapid disease progression or toxicity. 18% (5/28) of patients experienced clinical benefit (defined as CR, PR, or stable disease >6 months). Tumor lymphocyte infiltration and PD-L1 expression were low. High expression of PD-1/CTLA-4 dual staining in CD8 cells (>15% in tumor or blood) was seen in 5 patients, with 80% (4/5) of those patients experiencing benefit, and in one other patient who withdrew from the study in week 3 because of immune hepatitis. 95% (21/22) of patients with less than 20% dual expression had no response. Both tumor and peripheral blood CD8 PD1/CTLA-4 dual expression strongly correlated with time to progression. Furthermore, reduction in Foxp3+/CTLA-4high Treg population in was seen in most tumors treated with vorinostat, but a more significant reduction Foxp3+/CTLA-4high Treg was seen in responding patients. All patients in the prior study received vorinostat and pembrolizumab, and it is not clear whether the high response rate in the biomarker-enriched group is due to the biomarker expression alone or the constellation of the presence of the biomarker in the setting of HDAC inhibitorinduced downregulation of Tregs.  
Table 1.1 Duration of Response by percentage of dual positivity of PD-1/CTLA-4 (20%)  
  
Table 1.2 Duration of Response by percentage of dual positivity of PD-1/CTLA-4 (10%)
epigenetic priming for immune therapy
1.3.1.3 prior studies with vorinostat

In a Phase II clinical trial, we evaluated the role of vorinostat (VOR) in reversing hormone therapy resistance in advanced breast cancer13. In a single arm study, 43 patients (median age 56 years (31–71)) were treated. A total of 25 patients (58%) received prior adjuvant tamoxifen (TAM), 29 (67%) progressed on at least one prior chemotherapy regimen, 42 (98%) progressed after one, and 23 (54%) after two aromatase inhibitors. The objective response rate by RECIST criteria was 19%, and the clinical benefit rate (response or stable disease >24 weeks) was 40%. The median response duration was 10.3 months (confidence interval: 8.1–12.4). Histone hyperacetylation and higher baseline HDAC2 levels correlated with response and responses were mainly seen in the 56% of patients who had a meaningful change in acetylation with vorinostat. Preclinical studies have further suggested that hormonal therapy must be continued even in the presence of an HDAC inhibitor.
epigenetic priming for immune therapy
1.3.1.4 prior studies with other

A second phase II (ENCORE 301) trial evaluated entinostat and exemestane and treated patients with advanced estrogen receptor (ER)-positive breast cancer57. This study recently received Breakthrough Therapy designation from the FDA for its potential to reverse resistance to hormonal therapies. The median PFS was 4.3 months versus 2.3 months, for entinostat and placebo, respectively (HR = 0.73; P = 0.06). The median overall survival in the combination arm was 28.1 m compared to 19.8 m in the exemestane arm (HR = 0.59; P = 0.036). The objective response rate to the combination was 6.3%, while to exemestane alone was 4.6% (P = 0.58).
epigenetic priming for immune therapy
1.4 rationale for the dose

The rationale and justification for pembrolizumab, vorinostat, and tamoxifen dosing, route of administration, planned maximum dosage, and regimen are as defined in the previously conducted pilot study (NCT02395627)58.
epigenetic priming for immune therapy
1.5 correlative studies

Immune checkpoint inhibitors have revolutionized cancer therapy, yet have limited efficacy in estrogen receptor (ER+) breast cancer. Implicated factors include scarcity of tumor-infiltrating lymphocytes (TILs), low PD-L1 expression, female gender, and liver involvement. In vitro and in vivo studies suggest that epigenetic modulation with HDAC inhibitors modulate regulatory T cells (Treg) and change TIL composition, which is further associated with the presence of a specific immune signature58.  
Patients with ER+ metastatic breast cancer, who progressed on multiple prior therapies, were treated with tamoxifen in combination with vorinostat and pembrolizumab either immediately or after 3 weeks of epigenetic priming in a phase II trial. Comprehensive flow-cytometric immunophenotyping, PD-L1 staining, and histone acetylation were evaluated on tumor and blood cells58.  
34 patients (median age 56 years (32-81), heavily pretreated with a median of 5 (2-13) prior regimens received at least one dose of vorinostat and evaluable for response. Grade ¾ toxicities in 2 pts each (6%) included immune hepatitis, fatigue, and thrombocytopenia. Grade 2 toxicities were pneumonitis and colitis in 3%, fatigue in 27% and thrombocytopenia in 12% pts. Six patients did not receive pembrolizumab due to rapid progression or toxicity. Clinical benefits rate defined as CR, PR, and stable disease > 6m was seen in 5/28 (18%) of patients. Tumor lymphocyte infiltration and PD-L1 expression were low. High expression of PD-1/CTLA-4 dual staining in CD8 cells of >20% in tumor or blood was seen in 5 patients overall, 4/5 (80%) patients with benefit and in one other patient, who was withdrawn due to immune hepatitis in week 3. Both tumor and peripheral blood CD8 PD-1/CTLA-4 dual expression strongly correlated with time to progression and reduction in Foxp3+/CTLA-4high Treg population in tumors by vorinostat58.  
Our data highlight the potential for patient selection based on exhausted CD8+ cells in blood or tumor to predict response to immune checkpoint inhibitors in a small subset of ER+ breast cancer patients (NCT02395627)58-69.
epigenetic priming for immune therapy
2 study objectives 2.1 hypothesis

Given the above data, we hypothesize that HDAC induced epigenetic priming in a biomarker enriched study will increase the efficacy of pembrolizumab with patient selection based on exhausted CD8+ cells in blood or tumor in a small subset of (ER)+ breast cancer patients. Biomarker threshold for exhausted CD8+ will be set at 10% to avoid under-enrollment of patients with potential response. Response further correlated with exhausted CD4 cells in blood (but not not tumors), thus we will allow enrollment of patients who have dual expression of PD-1 and CTLA4 on CD4 cells in blood only: biomarker threshold will be set at 5%. Based on the below described earlier study results, we hypothesize that the pembrolizumab and TAM arm even in the setting of biomarker selection will not pass the first stage and will be stopped early after 11 patients and not expanded to 30 patients, whereas the immune priming by vorinostat arm will proceed to the second stage enrolling a total of 35. Under this hypothesis, we will most likely enroll 46 evaluable patients (~65% chance), whereas the possible enrollment ranges from 24 to 65. For the comparison of the treatment arms, the proposed design will have 72.53% power for the Z-test for two proportions conditioning on the study design, when the true response rates are 0.4 and 0.1 for the immune priming and the no immune priming treatment arm.
epigenetic priming for immune therapy
2.2 primary objective and endpoints

| Primary Objective                                                                                                                             | Endpoint(s)                                                            | Time Frame                               |
|-----------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------|------------------------------------------|
| To define the role of epigenetic immune  priming in a biomarker enriched ER+ breast  cancer population on the basis of overall  response rate | ORR:                                                                   | From time of initial response to time of |
| CR+PR+SD                                                                                                                                      | progression or death, whichever  occurs first, assessed up to 24 weeks |                                          |
epigenetic priming for immune therapy
2.3 secondary objective(s) and endpoint(s)

| Secondary Objective   | Endpoint(s)                                                                                 | Time Frame                                                                                                  |                                                                                                                       |
|-----------------------|---------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------|
| 1.                    | To assess duration of response  (DOR) 24-week landmark  progression-free survival  (PFS:24) | DOR (CR+PR)                                                                                                 | From initiation of study treatment to time of  progression or death, whichever occurs first,  assessed up to 24 weeks |
| 2.                    | Median PFS and overall                                                                      |                                                                                                            | Median PFS                                                                                                            |
|                      | overall                                                                                     |                                                                                                             |                                                                                                                       |
| survival (OS)         | survival (OS)                                                                               | From initiation of study treatment to the time  of death from any cause on study, assessed  up to 48 months |                                                                                                                       |
| 3.                    | Tumor responses will also be  calculated by Immune Related  Response-Criteria (irRC)        | Tumor responses                                                                                             | Through study completion, assessed up to                                                                              |
| (irRC)                | 24 months                                                                                   |                                                                                                             |                                                                                                                       |
epigenetic priming for immune therapy
2.4 exploratory (correlative) objectives

| Exploratory Objective         | Endpoint(s)                                                                                                      |                                                                                                                              |
|-------------------------------|------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------|
| 1.                            | Evaluation of biomarker target threshold on  response rate (retrospective cut off of 20% vs  10%)                | Biomarker target threshold on response rate                                                                                  |
| 2.                            | To assess the ratio of effector T cells: regulatory  T cells in blood and tumor biopsies pre- and  post-therapy. | Ratio of effector T cells: regulatory T cells in  blood and tumor biopsies pre- and post-therapy                             |
| 3.                            | To evaluate inflammatory T cell signature  changes in blood and tumor biopsies pre- and  post-therapy.           | inflammatory T cell signature changes in blood  and tumor biopsies pre- and post-therapy                                     |
| 4.                            | To evaluate changes in number of myeloidderived suppressor cells (MDSCs) in peripheral  blood and tumor biopsies pre- and post-therapy.                                                                                                                  | changes in number of myeloid-derived  suppressor cells (MDSCs) in peripheral blood  and tumor biopsies pre- and post-therapy |
| 5.                            | To evaluate changes in histone acetylation in  peripheral blood cells and tumor biopsies preand post-therapy                                                                                                                  | changes in histone acetylation in peripheral  blood cells and tumor biopsies pre- and posttherapy                                                                                                                              |
| 6.                            | Initial comparison to vorinostat-induced PD-1 in                                                                 | Comparison to vorinostat-induced PD-1 in                                                                                     |
| lymphocytes, PD-L1 modulation | lymphocytes, PD-L1 modulation                                                                                    |                                                                                                                              |
| 7.                            | Nanostring and 10x sequencing and single cell  immune phenotyping (on stored tissue for  successful arms only)   | Completion of Nanostring and 10x sequencing  and single cell immune phenotyping (on stored  tissue for successful arms only) |
| 8.                            | Impact of HDAC inhibition of response to  pembrolizumab vs. pembrolizumab in biomarker  enriched population      | Impact of HDAC inhibition                                                                                                    |
epigenetic priming for immune therapy
3 study design 3.1 characteristics

We propose a single site Phase II, two-arm, open-label unblinded, 65-patient trial in patients with hormone therapy-resistant breast cancer (HTR) to test the role of epigenetic priming in a biomarker enriched patient population in HTR breast cancer. The two arms include Arm A: pembrolizumab, vorinostat, and tamoxifen and Arm B pembrolizumab and tamoxifen. Patients will be evaluated for response at 9 weeks (±1 week) by RECIST1.1 (using IR-RECIST) and will continue on therapy if they have stable disease or response at the time of disease evaluations. Patients with progression on imaging but are clinically stable, may stay on study but will require follow-up staging after 4-6 weeks. Patients will be assessed continually for safety and tolerability as well as for any adverse events. If progression on therapy with pembrolizumab, patient may continue if clinically indicated and be re-evaluated after 4-8 weeks. We will follow AEs of clinical interest as defined by Merck and will dose modify pembrolizumab as per Merck guidelines. We have significant prior experience with vorinostat, tamoxifen, and pembrolizumab combination and are facile with managing drug toxicities. Tissue biopsies of all patients will be collected prior to enrollment to assess for pre-treatment of our immune signature and PD-L-1 expression. Follow up biopsies and peripheral blood collection will be taken from all patients post-treatment at 8-10 weeks to characterize changes in local tissue effects and antigen-specific cellular and humoral immune responses in peripheral blood. Patients will be stratified by prior number of hormonal and chemotherapies.
epigenetic priming for immune therapy
3.2 sample size

We adopt a randomized two-arm trial with futility early stopping built-in within each treatment arm using a Simon's two-stage design (Simon, 1989). The effect of vorinostat on pembrolizumab and the effect of pembrolizumab and tamoxifen in this biomarker-selected population of ER+ breast cancer patients will be first tested against the respectively chosen null rate in the first stage within the treatment arms and then compared between the treatment arms. 65 patients to be treated under the study/enrolled in the investigational portion of the study (sample size for primary objective).  
For Arm A (vorinostat + TAM + pembro): The null hypothesis that the true response rate for pembrolizumab and vorinostat in ER+ pretreated patients alone without biomarker enrichment is 0.15 which will be tested against the alternative that response rate of pembrolizumab in a biomarker select population is 0.40. In the first stage, 13 patients will be accrued. If there are 2 or fewer responses in these 13 patients, the study will be stopped. Otherwise, 22 additional patients will be accrued for a total of 35. The null hypothesis will be rejected if 10 or more responses are observed in 35 patients. This design yields a type I error rate of 0.025 and power of 0.9 in this treatment arm when the true response rate is 0.4 in this patient subpopulation.  
For Arm B (pembro + TAM): The null hypothesis that the true response rate for pembrolizumab in ER+ pretreated patients alone is 0.10 which will be tested against the alternative that response rate of pembrolizumab in a biomarker select population is 0.35. In the first stage, 11 patients will be accrued. If there are 1 or fewer responses in these 11 patients, the study will be stopped. Otherwise, 19 additional patients will be accrued for a total of 30. The null hypothesis will be rejected if 7 or more responses are observed in 30 patients. This design yields a type I error rate of 0.025 and power of 0.9 in this treatment arm when the true response rate of pembro + TAM treatment is 0.35 in this patient subpopulation.
epigenetic priming for immune therapy
3.3 eligibility criteria

Patients must have baseline evaluations performed prior to the first dose of study drug and must meet all inclusion and exclusion criteria. In addition, the patient must be thoroughly informed about all aspects of the study, including the study visit schedule and required evaluations and all regulatory requirements for informed consent. The written informed consent must be obtained from the patient prior to enrollment. The following criteria apply to all patients enrolled in the study unless otherwise specified.
epigenetic priming for immune therapy
3.3.1 inclusion criteria

1. Pre and postmenopausal women or men with stage IV ER+ breast cancer histological or cytological confirmation, 18 years of age or older.
Because no dosing or adverse event data are currently available on the use of pembrolizumab, vorinostat, or tamoxifen in patients <18 years of age, children are excluded from this study but will be eligible for future pediatric trials.  
2. >10% expression of PD-1/CTLA-4 dual staining in CD8 cells in tumor or blood or >5%
expression of PD-1/CTLA-4 dual staining in CD4 in blood (only).  
3. Eastern Cooperative Oncology Group (ECOG) Performance Status of ≤ 1. 4. Understand and voluntarily sign an informed consent prior to any study-related assessments or procedures are conducted and are able to adhere to the study visit schedule and other protocol requirements.  
5. Consent to paired tumor biopsy 6. Measurable tumor by RECIST criteria 7. Per Good Clinical Practice, any toxicity related to prior therapies that, in the opinion of the investigator, would potentially be worsened with anti-PD1 therapy should be resolved to less than Grade 1 8. Adequate organ function:
o Absolute neutrophil count (ANC) ≥ 1.5 X 109/L
o Hemoglobin (Hgb) ≥9g/dL (may transfuse if clinically indicated)
o Platelets (plt) ≥ 100 x 109/L
o Potassium within normal range, or correctable with supplements; o AST and ALT ≤ 2.5 x Upper Limit Normal (ULN) or ≤ 5.0 x ULN if liver tumor is present; o Serum total bilirubin ≤ 1.5 x ULN o Serum creatinine ≤ 1.5 x ULN, or 24-hr clearance ≥ 60ml/min; and 9. Females of childbearing potential (defined as sexually mature women who):
o Has not undergone a hysterectomy (the surgical removal of the uterus) or bilateral oophorectomy (the surgical removal of both ovaries) or, o Has not been naturally postmenopausal for at least 24 consecutive months (i.e., has had menses at any time during the preceding 24 consecutive months) must have Negative serum pregnancy test within 14days before starting study treatment in females of childbearing potential (FCBP) and willingness to adhere to acceptable forms or birth control (a physician-approved contraceptive method (oral, injectable, or implantable hormonal contraceptive; tubal ligation; intra-uterine device; barrier contraceptive with spermicide; or vasectomized partner)
10. All female and male participants must agree to use approved contraception during the treatment period and for at least 18 weeks after the last dose of study treatment and refrain from donating sperm during this period.
epigenetic priming for immune therapy
3.3.2 exclusion criteria

1. Prior treatment with pembrolizumab or other PD-(L)1 2. Any significant medical condition, laboratory abnormalities, which places the subject at unacceptable risk if he/she were to participate in the study.  
3. Has a history of (non-infectious) pneumonitis/interstitial lung disease that required steroids or current pneumonitis/ interstitial lung disease.  
4. Has known active Hepatitis B (e.g., HBsAg reactive) or Hepatitis C (e.g., HCV RNA
[qualitative] is detected)"
5. Has a history of HBV 6. Has a history or current evidence of any condition, therapy, or laboratory abnormality that might confound the results of the study, interfere with the subject's participation for the full duration of the study, or is not in the best interest of the subject to participate, in the opinion of the treating investigator 7. Symptomatic central nervous system metastases. Subjects with brain metastases that have been previously treated and are stable for 6 weeks are allowed.  
8. Persistent diarrhea or malabsorption ≥ NCI CTCAE grade 2, despite medical management.  
9. Unstable angina, significant cardiac arrhythmia, or New York Heart Association (NYHA)
class 3 or 4 congestive heart failure.  
10. Prior systemic cancer-directed treatments or investigational modalities ≤ 5 half-lives or 4 weeks, whichever is shorter, prior to starting study drug or who have not recovered from side effects of such therapy (except alopecia).  
11. Active autoimmune disease except for vitiligo or hypothyroidism 12. Active and ongoing steroid use, except for non-systemically absorbed treatments (such as inhaled or topical steroid therapy for asthma, COPD, allergic rhinitis).  
13. Major surgery ≤ 2 weeks prior to starting a study drug or who have not recovered from side effects of such therapy.  
14. Pregnant or breastfeeding. 15. Known Human Immunodeficiency Virus (HIV) infection 16. Known history of tuberculosis 17. Known allergic reaction or intolerability to tamoxifen 18. Patients with prior history of DVTs must be on therapeutic or preventive anticoagulation
epigenetic priming for immune therapy
3.4 inclusion and recruitment of

Individuals of any sex/gender, race, or ethnicity may participate. The study recruitment strategy aims to achieve representation of minority groups that reflects the demographics of the affected population in the catchment area.
epigenetic priming for immune therapy
3.5 duration of treatment

Eligible patients will receive treatment as described above, in consecutive 3-week cycles, until progression of disease or unacceptable toxicity. Patients are expected to receive treatment for at least 3 cycles, at which time the first evaluation for efficacy will occur. If there is continued safety and tolerability, treatment may continue. All patients will be restaged during at 9 weeks (±1 week) and 18 weeks (±1 week) via imaging scans; staging will then be continued every 3 cycles until 6 months. After 6 months, staging may be spaced out to 6 cycles if clinically indicated. Patients will be followed for evaluation of safety for at least 30 days after the last dose of study drug. Any study drug-related serious adverse events will be followed until resolution, return to baseline grade, or deemed irreversible by the Investigator. Subjects who experience a recurrence of the same severe or life-threatening event at the same grade or greater with re-challenge of pembrolizumab should be discontinued from trial treatment.
epigenetic priming for immune therapy
3.6 duration of follow up

Patients will be followed for 30 days after end of study participation or removal from study. Patients removed from study for unacceptable treatment-related adverse event(s) will be followed until resolution or stabilization of all treatment-related adverse events to Grade 2 or lower. Subjects who discontinue for reasons other than progressive disease will have post-treatment follow-up for disease status until disease progression, initiating a non-study cancer treatment, withdrawing consent, or becoming lost to follow-up. After documented disease progression, each subject will be followed by telephone for overall survival until death, withdrawal of consent, or the end of the study, whichever occurs first. Subjects may withdraw consent at any time for any reason or be dropped from the trial at the discretion of the investigator should any untoward effect occur. In addition, a subject may be withdrawn by the investigator or the Sponsor if enrollment into the trial is inappropriate, the trial plan is violated, or for administrative and/or other safety reasons. Specific details regarding discontinuation or withdrawal are provided in Section 5.4.
epigenetic priming for immune therapy
3.7 randomization procedures

This is an open-label randomized trial; therefore, the sponsor, investigator, and subject will know the treatment administered. Two arms will be tested, with each independent assessment. Patients will be randomized to either arm using a computerized randomization program by Dr. Scott Thomas. Patients will be further stratified by prior number of chemo- and hormonal therapies.
epigenetic priming for immune therapy
3.8 primary completion

The estimated primary completion is 36 months after the study opens to accrual.
epigenetic priming for immune therapy
3.9 study completion

The expected study completion date is 48 months after the study opens to accrual. The study will conclude when all enrolled patients have been followed for more than 30 days following completion of the study. The estimated duration of study, from start of enrollment to final study report is 4 years.
epigenetic priming for immune therapy
mechanism of action

Pembrolizumab (previously known as SCH 900475) is a potent and highly selective humanized monoclonal antibody (mAb) of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances functional activity of the target lymphocytes to facilitate tumor regression and ultimately immune rejection.
epigenetic priming for immune therapy
metabolism

Pembrolizumab is catalyzed into small peptides and single amino acids via general protein degradation, but it does not rely on metabolism for clearance.
epigenetic priming for immune therapy
contraindications none

Formulation, Appearance, Packaging, and Labeling Pembrolizumab is supplied as 100 mg/4 mL solution in a single-use vial for intravenous administration. Pembrolizumab is manufactured by Merck. Supplies will be labeled in accordance with regulatory requirements. This trial is open-label; therefore, the participant, the trial site personnel, the Sponsor, and/or designee are not blinded to treatment. Drug identity (name, strength) is included in the label text; random code/disclosure envelopes or lists are not provided. Availability Pembrolizumab is being obtained as commercial supply provided by Merck.
epigenetic priming for immune therapy
storage and handling

Pembrolizumab is stored at the UCSF investigational pharmacy. Clinical supplies must be stored in a secure, limited-access location under the storage conditions specified on the label. Receipt and dispensing of trial medication must be recorded by an authorized person at the trial site. Clinical supplies may not be used for any purpose other than that stated in the protocol. Store the diluted solution from the KEYTRUDA 100 mg/4 mL vial either:  
At room temperature for no more than 6 hours from the time of dilution. This includes room temperature storage of the diluted solution and the duration of infusion.  
Under refrigeration at 2°C to 8°C (36°F to 46°F) for no more than 24 hours from the time of dilution. If refrigerated, allow the diluted solution to come to room temperature prior to administration.
Discard after 6 hours at room temperature or after 24 hours under refrigeration. Do not freeze.
epigenetic priming for immune therapy
side effects

Complete and updated adverse event information is available in the Investigational Drug Brochure (IB) and/or product package insert.
epigenetic priming for immune therapy
returns and reconciliation

The investigator is responsible for keeping accurate records of the clinical supplies received from Merck or designee, the amount dispensed to and returned by the participants and the amount remaining at the conclusion of the trial. Upon completion or termination of the study, all unused and/or partially used investigational product will be destroyed at the site per institutional policy. It is the Investigator's responsibility to arrange for disposal of all empty containers, provided that procedures for proper disposal have been established according to applicable federal, state, local and institutional guidelines and procedures, and provided that appropriate records of disposal are kept.
epigenetic priming for immune therapy
4.1.2 investigational product vorinostat

Classification Antineoplastic Mechanism of Action Vorinostat, a histone deacetylase (HDAC) inhibitor. Histone deacetylases (HDACs) are a family of enzymes that regulate chromatin remodeling and gene transcription via the dynamic process of acetylation and deacetylation of core histones. Vorinostat, a potent inhibitor of HDAC activity, binds directly to the catalytic pocket of HDAC
enzymes. It causes G1 or G2 phase cell-cycle arrest, apoptosis, or differentiation in cultured transformed cells.
epigenetic priming for immune therapy
metabolism

The major pathways of vorinostat metabolism involve glucuronidation and hydrolysis, followed by β-oxidation. Human serum levels of two metabolites, O-glucuronide of vorinostat and 4anilino4-oxobutanoic acid were measured. Both metabolites are pharmacologically inactive.  
Compared to vorinostat, the mean steady-state serum exposures in humans of the Oglucuronide of vorinostat and 4-anilino-4-oxobutanoic acid were 4-fold and 13-fold higher, respectively. In vitro studies using human liver microsomes indicate negligible biotransformation by cytochromes P450 (CYP).
epigenetic priming for immune therapy
contraindications none

Formulation, Appearance, Packaging, and Labeling Vorinostat is supplied as size 3 100 mg capsules for oral administration. Vorinostat will be supplied in a 100-120 capsule bottle. Vorinostat will be given at 400 mg per day (4x 100 mg tablets), 4 of 7 days. Vorinostat is manufactured by Merck & Co., Inc. Availability Vorinostat is being obtained as commercial supply provided by Merck & Co., Inc.
epigenetic priming for immune therapy
storage and handling

Vorinostat is stored at the UCSF investigational pharmacy. Store vorinostat capsules at room temperature, 15 to 30 .C (59 to 86 .F). Do not store above 30.C. Avoid exposure to excessive moisture. Vorinostat is an anticancer drug. If clean powder is spilled from broken or damaged vorinostat capsules care should be taken to minimize inhalation or direct contact with skin. Wash spill area at least 3 times with ethyl alcohol, followed by water.
epigenetic priming for immune therapy
side effects

Complete and updated adverse event information is available in the current IB and/or product package insert.
epigenetic priming for immune therapy
mechanism of action

Following a single oral dose of 20 mg tamoxifen, an average peak plasma concentration of 40 ng/mL (range 35 to 45 ng/mL) occurred approximately 5 hours after dosing. The decline in plasma concentrations of tamoxifen is biphasic with a terminal elimination half-life of about 5 to 7 days. The average peak plasma concentration of N-desmethyl tamoxifen is 15 ng/mL (range 10 to 20 ng/mL). Chronic administration of 10 mg tamoxifen given twice daily for 3 months to patients results in average steady-state plasma concentrations of 120 ng/mL (range 67-183 ng/mL) for tamoxifen and 336 ng/mL (range 148-654 ng/mL) for N-desmethyl tamoxifen. The average steady-state plasma concentrations of tamoxifen and N-desmethyl tamoxifen after administration of 20 mg tamoxifen once daily for 3 months are 122 ng/mL (range 71-183 ng/mL)
and 353ng/mL (range 152-706 ng/mL), respectively.  
After initiation of therapy, steady-state concentrations for tamoxifen are achieved in about 4 weeks, and steady-state concentrations for N-desmethyl tamoxifen are achieved in about 8 weeks, suggesting a half-life of approximately 14 days for this metabolite. In a steady state, crossover study of 10 mg NOLVADEX tablets given twice a day vs. a 20 mg NOLVADEX tablet given once daily, the 20 mg NOLVADEX tablet was bioequivalent to the 10 mg NOLVADEX tablets.
epigenetic priming for immune therapy
metabolism

Tamoxifen is extensively metabolized after oral administration. N-desmethyl tamoxifen is the major metabolite found in patients' plasma. The biological activity of N-desmethyl tamoxifen appears to be similar to that of tamoxifen. 4-Hydroxytamoxifen and a side chain primary alcohol derivative of tamoxifen have been identified as minor metabolites in plasma. Tamoxifen is a substrate of cytochrome P-450 3A, 2C9 and 2D6, and an inhibitor of P-glycoprotein.
epigenetic priming for immune therapy
excretion

Studies in women receiving 20 mg of 14C tamoxifen have shown that approximately 65% of the administered dose was excreted from the body over a period of 2 weeks with fecal excretion as the primary route of elimination. The drug is excreted mainly as polar conjugates, with unchanged drug and unconjugated metabolites accounting for less than 30% of the total fecal radioactivity.
epigenetic priming for immune therapy
contraindications

Tamoxifen is contraindicated in patients with known hypersensitivity to the drug or any of its ingredients.
epigenetic priming for immune therapy
drug-drug interactions

In vitro studies showed that erythromycin, cyclosporin, nifedipine, and diltiazem competitively inhibited formation of N-desmethyl tamoxifen with apparent K1 of 20, 1, 45 and 30 μM, respectively. The clinical significance of these in vitro studies is unknown. Formulation, Appearance, Packaging, and Labeling Tamoxifen is available in the following doses for oral administration:  
10 mg Tablets. Each tablet contains 15.2 mg of tamoxifen citrate, which is equivalent to 10 mg of tamoxifen.
20 mg Tablets. Each tablet contains 30.4 mg of tamoxifen citrate, which is equivalent to 20 mg of tamoxifen.  
The administered dose will be 20 mg daily. Availability Tamoxifen is commercially available. It is being obtained as commercial supply. Storage and handling Tamoxifen is commercially available. Patients will obtain tamoxifen from their local pharmacy. Side Effects Complete and updated adverse event information is available in the current IB and/or product package insert.
epigenetic priming for immune therapy
4.2 accountability records for investigational

UCSF Investigational Drug Services (IDS) will manage drug accountability records for UCSF.
epigenetic priming for immune therapy
4.3 ordering investigational product(s)

UCSF will obtain pembrolizumab and vorinostat directly from Merck & Co., Inc. Tamoxifen is commercially available.
epigenetic priming for immune therapy
5 treatment plan 5.1 dosage

All trial treatments will be administered on an outpatient basis. Pembrolizumab 200 mg will be administered as a 30-minute IV infusion every 3 weeks. Treatment cycle intervals may be increased due to toxicity, as described in Section 5.2. Every effort should be made to target infusion timing to be as close to 30 minutes as possible.  
However, given the variability of infusion pumps, a window of -5 minutes and +10 minutes is permitted (i.e., infusion time is 30 minutes: -5 min/+10 min). The Pharmacy Manual contains specific instructions for the preparation of the pembrolizumab infusion fluid and administration of infusion solution. Selection of 200 mg as the appropriate dose for a switch to fixed dosing is based on simulation results indicating that 200 mg will provide exposures that are reasonably consistent with those obtained with 2 mg/kg dose and importantly will maintain individual patient exposures within the exposure range established in melanoma as associated with maximal clinical response. A population PK model, which characterized the influence of body weight and other patient covariates on exposure, has been developed using available data from 476 subjects from PN001. The distribution of exposures from the 200 mg fixed dose is predicted to considerably overlap those obtained with the 2 mg/kg dose, with some tendency for individual values to range slightly higher with the 200 mg fixed dose. The slight increase in PK variability predicted for the fixed-dose relative to weight-based dosing is not expected to be clinically important given that the range of individual exposures is well contained within the range of exposures shown in the melanoma studies of 2 and 10 mg/kg to provide similar efficacy and safety. The population PK evaluation revealed that there was no significant impact of tumor burden on exposure. In addition, exposure was similar between the NSCLC and melanoma indications. Therefore, there are no anticipated changes in exposure between different tumor types and indication settings. As vorinostat and tamoxifen are oral, self-administered drugs, drug diaries will be provided for assessing compliance with treatment. The diary will be provided each time vorinostat and tamoxifen are provided to the study participant to take home. Missed doses will be noted and will not be replaced. The participant will proceed with the next scheduled dose at the next scheduled time.  
| Dose/           | Dose    | Route of   | Regimen/ Treatment   |            |              |
|-----------------|---------|------------|----------------------|------------|--------------|
| Drug            | Potency | Frequency  | Administration       | Period     | Use          |
| Day 1 of each 3 |         |            |                      |            |              |
| Pembrolizumab   | 200 mg  | Q3W        | IV infusion          | week cycle | Experimental |  
Trial treatment should begin on the day of randomization or as close as possible to the date on which treatment is allocated/assigned. Trial treatment should be administered on Day 1 of each cycle after all procedures/assessments have been completed as detailed on the Trial Flow Chart (Section 6.0). Trial treatment may be administered up to 3 days before or after the scheduled Day 1 of each cycle due to administrative reasons.
epigenetic priming for immune therapy
table 5.2 arm a regimen

| Investigational   | Premedication;        | Cycle            |                       |                    |        |
|-------------------|-----------------------|------------------|-----------------------|--------------------|--------|
| Product           | precautions           | Dose             | Route                 | Schedule           | Length |
| Day 1, Week       |                       |                  |                       |                    |        |
| Pembrolizumab     | No premedications     | 1                |                       |                    |        |
| No precautions    | 200 mg                | Intravenous      | Cycles 1+ Days 4 of 7 |                    |        |
| Vorinostat        | Administer whole with | days             |                       |                    |        |
| food, if possible | 400 mg                | Oral             | All cycles            | 3 weeks  (21 days) |        |
| Daily             |                       |                  |                       |                    |        |
| Tamoxifen         | No premedications     | 20 mg  (tablets) | Oral                  | continuous         |        |
| No precautions    | All cycles            |                  |                       |                    |        |
epigenetic priming for immune therapy
table 5.3 arm b regimen

| Investigational                                          | Premedication;                   | Cycle   |             |          |        |
|----------------------------------------------------------|----------------------------------|---------|-------------|----------|--------|
| Product                                                  | precautions                      | Dose    | Route       | Schedule | Length |
| Day 1, Week                                              |                                  |         |             |          |        |
| Pembrolizumab                                            | No premedications No precautions | 200 mg  | Intravenous | 1        |        |
| Cycles 1+                                                | 3 weeks  (21 days)               |         |             |          |        |
| Daily                                                    |                                  |         |             |          |        |
| Tamoxifen                                                | No premedications                | 20 mg   | continuous  |          |        |
| (tablets)                                                | Oral                             |         |             |          |        |
| No precautions                                           | All cycles                       |         |             |          |        |
| Tamoxifen may be omitted in Arm B at the PI's discretion |                                  |         |             |          |        |
epigenetic priming for immune therapy
5.2 dose modifications and dosing

The following dose modification rules will be used with respect to potential toxicity. Toxicity will be assessed according to the NCI CTCAE version 5.0. The following dose modifications should be made for febrile neutropenia, and blood counts obtained on day one of each cycle. If more than one of these conditions applies, use the most stringent (i.e., the greatest dose reduction). **The AE may be due to pembrolizumab or**
vorinostat alone in which case the respective agent should be reduced. If overlapping toxicity is suspected, both drugs may be reduced with the potential to re-escalate the non-offending agent. Given the very different nature of pembrolizumab and vorinostat/tamoxifen toxicities, separate tables are shown for individual agents. No dose modification will be pursued for tamoxifen.
epigenetic priming for immune therapy
hematologic toxicity

| other days and counts have recovered upon retreatment on days 1, 8, 15.  Neutropenia Febrile neutropenia Management/Next Dose for Vorinostat ANC >1000 100% ANC: 500-1000 Hold for until <grade 2 # ,  Restart at 75%: 300 mg qd for all subsequent cycles Second occurrence resume at 200 mg qd for all subsequent cycles Third occurrence: remove from study ANC: 0-500 Hold for until <grade 2 or less# Restart at 75%: 300 mg qd for all subsequent cycles Second occurrence resume at 200 mg qd for all subsequent cycles Third occurrence: discuss with sponsor and principal investigator, consider  omitting alternate weeks  #Patient with a dose delay for more than 3 weeks in the first 2 cycles will need to be replaced for toxicity  and efficacy assessments.   |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
epigenetic priming for immune therapy
adverse event anemia

| Adverse Event: Anemia Hemoglobin                                                                  | Management/Next Dose for Vorinostat   |
|---------------------------------------------------------------------------------------------------|---------------------------------------|
| >8 mg/dl                                                                                          | 100 %: 400 mg qd                      |
| 6.5-8 mg/dl                                                                                       | 75 %: 300 mg qd                       |
| <6.5 mg/dl                                                                                        | 50 %: 200 mg qd                       |
| An alternative to dose modification in cases of anemia only would be the use of PRBC transfusion. |                                       |  
| persists for day 1, 8, and 15 of each cycle. Dose modifications are not required if labs are drawn on other days and  counts have recovered upon retreatment on days 1, 8, 15. Platelets Management/Next Dose for Vorinostat >75'000 100 %: 400 mg qd <75'000 100 %: 400 mg qd ≤50'000/mm3 Hold until <grade 2 or less Restart at 75%: 300 mg qd for all subsequent cycles Second occurrence resume at 200 mg qd for all subsequent cycles Third occurrence: discuss with sponsor and principal investigator, consider  omitting alternate weeks  ≤25'000/mm3 Hold until <grade 2 or less Restart at 75%: 300 mg qd for all subsequent cycles Second occurrence resume at 200 mg qd for all subsequent cycles Third occurrence: remove from study  An alternative to dose modification in cases of anemia only would be the use of PRBC transfusion.   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| modifications, in case of uncertainty doses may be re-escalated if toxicities recover. Bilirubin Management/Next Dose for Vorinostat ≤1.5 mg/dl 100 %: 400 mg qd 1.6 - 3.0 mg/dl 75 %: 300 mg qd > 3.0 mg/dl First occurrence: hold until <grade 2, 75 %: 300 mg qd  Second occurrence: Hold until grade 2 or less, 50 %: 200 mg qd AST/ALT Management/Next Dose for Vorinostat ≤2.5 x ULN 100 %: 400 mg qd 2.5-5 x ULN First occurrence: 75 %: 300 mg qd Second occurrence: 50 %: 200 mg qd > 5 x ULN First occurrence: hold until <grade 2, resume at 75 %: 300 mg qd  Second occurrence: Hold until grade 2 or less, 50 %: 200 mg qd Third occurrence: remove from study   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Hepatic Dysfunction: Any new increase in LFTs should raise the concern to rule out progressive metastatic disease. If the rise is not due to progressive metastatic disease, give the following dose based on lab values obtained on day 1 of each cycle:
Effects on the liver: Tamoxifen has been associated with changes in liver enzyme levels, and on rare occasions, a spectrum of more severe liver abnormalities including fatty liver, cholestasis, hepatitis, and hepatic necrosis. A few of these serious cases included fatalities. In most reported cases, the relationship to tamoxifen was uncertain. However, some positive rechallenges and dechallenges have been reported. If grade 3 or 4 liver toxicity is maintained after a temporary stopping of vorinostat, both drugs should be stopped. Any rechallenge should be discussed with the study chair. If treatment is delayed for more than 3 weeks, contact the Principal Investigator.
epigenetic priming for immune therapy
gastrointestinal toxicity

Nausea/Vomiting/Dehydration: In this protocol, the routine use of any and all anti-emetics is allowed as clinically appropriate; these include the use of dexamethasone and 5-HT3 receptor antagonists. Patients should maintain adequate fluid and food intake because vorinostat's dose-limiting toxicities are anorexia, dehydration, diarrhea, and fatigue. In the setting of dysgeusia, popsicles and Gatorade have been found to be useful by some investigators to maintain adequate hydration. Patients should also be encouraged to seek a nutritional consult.  
Diarrhea: Treat diarrhea promptly with appropriate supportive care, including loperamide.  
Instruct patients to begin taking loperamide at the first signs of 1) poorly formed or loose stool, 2) occurrence of more bowel movements than usual in one day, or 3) unusually high volume of stool. Loperamide should be taken in the following manner: 4 mg at first onset of diarrhea then 2 mg after each unformed stool. The daily dose should not exceed 16 mg. Loperamide should not be taken prophylactically. Advise patients to drink plenty of clear fluids to help prevent dehydration caused by diarrhea. Avoid loperamide if there is the presence of blood or mucus in the stool or if diarrhea is accompanied by fever. If diarrhea is suspected to be linked to pembrolizumab and is ≥ grade 3, hold dose until toxicity resolves to ≤ grade 1, then resume at dose given in prior cycle.  
Page 31 of 82
epigenetic priming for immune therapy
other non-hematological toxicities

Laboratory abnormalities must be clinically relevant, and dose and drug-related to prompt dose modifications, in case of uncertainty doses may be re-escalated if toxicities recover Give the following dose based on the worst toxicity encountered during the previous cycle:  
| Fatigue    | Management/Next Dose for Vorinostat                                                                                                                                                    |
|------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Grade 2    | Hold until toxicity is ≤grade 1, then resume at dose given in prior cycle.                                                                                                             |
| Grade 3, 4 | Hold until toxicity is ≤ grade 1, then resume at 75% (or less) of dose given in the prior cycle.  If grade 3 toxicity recurs despite dose reduction, decrease to 50 % of initial dose. |
| Other      | Management/Next Dose for Vorinostat                                                                                                                                                    |
| Grade 2    | Hold until toxicity is ≤grade 1, then resume at dose given in prior cycle.                                                                                                             |
| Grade 3    | Hold until toxicity is ≤grade 1, then resume at 75% (or less) of dose given in the prior cycle.  If grade 3 toxicity recurs despite dose reduction, decrease to 50 % of initial dose.  |
| Grade 4    | Discontinue protocol therapy.                                                                                                                                                          |
epigenetic priming for immune therapy
vorinostat may be reduced for

Effects on the Eye: Ocular disturbances, including corneal changes, decrement in color vision perception, retinal vein thrombosis, and retinopathy has been reported in patients receiving tamoxifen. An increased incidence of cataracts and the need for cataract surgery have been reported in patients receiving tamoxifen. If grade 3 or 4 ocular toxicities occur, both drugs should be stopped. Any rechallenge should be discussed with the study chair.
epigenetic priming for immune therapy
5.2.1 dose modifications and dosing

Subjects should receive appropriate supportive care measures as deemed necessary by the treating investigator, including but not limited to the items outlined below:  
Diarrhea: Subjects should be carefully monitored for signs and symptoms of enterocolitis
(such as diarrhea, abdominal pain, blood or mucus in stool, with or without fever) and of bowel perforation (such as peritoneal signs and ileus). In symptomatic subjects, infectious etiologies should be ruled out, and if symptoms are persistent and/or severe, endoscopic evaluation should be considered.  
o In subjects with severe enterocolitis (Grade 3), pembrolizumab will be permanently discontinued, and treatment with systemic corticosteroids should be initiated at a dose of 1 to 2 mg/kg/day of prednisone or equivalent. When symptoms improve to Grade 1 or less, corticosteroid taper should be started and continued over at least 1 month.  
o In subjects with moderate enterocolitis (Grade 2), pembrolizumab should be withheld, and anti-diarrheal treatment should be started. If symptoms are persistent for more than one week, systemic corticosteroids should be initiated (e.g., 0.5 mg/kg/day of prednisone or equivalent). When symptoms improve to Grade 1 or less, corticosteroid taper should be started and continued over at
least 1 month. Regarding guidelines for continuing treatment with pembrolizumab, see Section 5.2.  
o All subjects who experience diarrhea should be advised to drink liberal quantities of clear fluids. If sufficient oral fluid intake is not feasible, fluid and electrolytes should be substituted via IV infusion.  
Nausea/vomiting: Nausea and vomiting should be treated aggressively, and consideration should be given in subsequent cycles to the administration of prophylactic antiemetic therapy according to standard institutional practice. Subjects should be strongly encouraged to maintain liberal oral fluid intake.  
Anti-infectives: Subjects with a documented infectious complication should receive oral or IV antibiotics or other anti-infective agents as considered appropriate by the treating investigator for a given infectious condition, according to standard institutional practice.  
Immune-related adverse events: Please see Section 5.2.2 below and the separate guidance document in the administrative binder regarding diagnosis and management of adverse experiences of a potential immunologic etiology. Specific immune-related adverse events (irAEs) will be collected and designated as immune-related events of clinical interest (ECIs) as described in Section 5.2.2
epigenetic priming for immune therapy
5.2.2 dose modification and toxicity

AEs associated with pembrolizumab exposure, including coadministration with additional compounds, may represent an immunologic etiology. These immune-related AEs (irAEs) may occur shortly after the first dose or several months after the last dose of pembrolizumab/combination treatment and may affect more than one body system simultaneously. Therefore, early recognition and initiation of treatment are critical to reduce complications. Based on existing clinical study data, most irAEs were reversible and could be managed with interruptions of pembrolizumab//combination treatment, administration of corticosteroids and/or other supportive care. For suspected irAEs, ensure adequate evaluation to confirm etiology or exclude other causes. Additional procedures or tests such as bronchoscopy, endoscopy, skin biopsy may be included as part of the evaluation. Based on the severity of irAEs, withhold or permanently discontinue pembrolizumab and administer corticosteroids. Dose modification and toxicity management guidelines for irAEs associated with pembrolizumab/combination treatment are provided in Table 5.4.
epigenetic priming for immune therapy
attribution of toxicity

When study interventions are administered in combination, attribution of an adverse event to a single component is likely to be difficult. Therefore, while the investigator may attribute a toxicity event to the combination, to Vorinostat or Tamoxifen alone, or to pembrolizumab alone, for adverse events listed in Table 5.4, both interventions must be held according to the criteria in Table 5.4 Dose Modification and Toxicity Management Guidelines for Immune-Related Adverse Events Associated with Pembrolizumab.
epigenetic priming for immune therapy
holding study interventions

When study interventions are administered in combination, if the AE is considered immunerelated, both interventions should be held according to recommended dose modifications.
epigenetic priming for immune therapy
restarting study interventions

Participants may not have any dose modifications (no change in dose or schedule) of pembrolizumab in this study, as described in Table 5.4.  
If the toxicity does not resolve or the criteria for resuming treatment are not met, the participant must be discontinued from all study interventions.  
If the toxicities do resolve and conditions are aligned with what is defined in Table 5.4, the combination of Vorinostat, Tamoxifen and pembrolizumab may be restarted at the discretion of the investigator. In these cases where the toxicity is attributed to the combination or to Vorinostat or Tamoxifen alone, re-initiation of pembrolizumab as a monotherapy may be considered at the PI's discretion.  
| monotherapy and IO Combinations Toxicity  Grade  Action With                                                                                                                             | Corticosteroid and/or Other                                                                                |                                                  |                                                  |                                                  |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------|--------------------------------------------------|--------------------------------------------------|--------------------------------------------------|
| (CTCAE v5.0)                                                                                                                                                                             | Pembrolizumab                                                                                              | Therapies                                        | Monitoring and Follow-up                         |                                                  |
| irAEs                                                                                                                                                                                    | Grade 2                                                                                                    | Withhold                                         |  Administer corticosteroids (initial dose of    |  Monitor participants for signs and symptoms of |
| 1 to 2 mg/kg prednisone or equivalent)                                                                                                                                                   | pneumonitis                                                                                                |                                                  |                                                  |                                                  |
| followed by taper                                                                                                                                                                        |                                                                                                            |                                                  |                                                  |                                                  |
| Recurrent  Grade 2,  Grade 3 or 4                                                                                                                                                        | Permanently  discontinue                                                                                   |  Evaluate participants with suspected           |                                                  |                                                  |
| Pneumonitis                                                                                                                                                                              |  Add prophylactic antibiotics for                                                                         | pneumonitis with radiographic imaging and        |                                                  |                                                  |
| opportunistic infections                                                                                                                                                                 | initiate corticosteroid treatment                                                                          |                                                  |                                                  |                                                  |
| Grade 2 or 3                                                                                                                                                                             | Withhold                                                                                                   |  Administer corticosteroids (initial dose of    |  Monitor participants for signs and symptoms of |                                                  |
| 1 to 2 mg/kg prednisone or equivalent)                                                                                                                                                   | enterocolitis (ie, diarrhea, abdominal pain,                                                               |                                                  |                                                  |                                                  |
| followed by taper                                                                                                                                                                        | blood or mucus in stool with or without fever)  and of bowel perforation (ie, peritoneal signs  and ileus) |                                                  |                                                  |                                                  |
| Recurrent  Grade 3 or  Grade 4                                                                                                                                                           | Permanently  discontinue                                                                                   |  Participants with ≥Grade 2 diarrhea suspecting |                                                  |                                                  |
| Diarrhea/Colitis                                                                                                                                                                         | colitis should consider GI consultation and  performing endoscopy to rule out colitis                      |                                                  |                                                  |                                                  |
|  Participants with diarrhea/colitis should be  advised to drink liberal quantities of clear  fluids. If sufficient oral fluid intake is not  feasible, fluid and electrolytes should be |                                                                                                            |                                                  |                                                  |                                                  |  
| Toxicity  Grade                                                                               | Action With                                                        | Corticosteroid and/or Other                   |                                                   |                |
|-----------------------------------------------------------------------------------------------|--------------------------------------------------------------------|-----------------------------------------------|---------------------------------------------------|----------------|
| (CTCAE v5.0)                                                                                  | Pembrolizumab                                                      | Therapies                                     | Monitoring and Follow-up                          |                |
| irAEs                                                                                         | substituted via IV infusion                                        |                                               |                                                   |                |
| Grade 2 a                                                                                     | Withhold                                                           |  Administer corticosteroids (initial dose of |  Monitor with liver function tests (consider     |                |
| 0.5 to 1 mg/kg prednisone or equivalent)                                                      | weekly or more frequently until liver enzyme                       |                                               |                                                   |                |
| AST or ALT                                                                                    | followed by taper                                                  |                                               |                                                   |                |
| Elevation or  Increased                                                                       | Grade 3 b or 4 c                                                   | Permanently                                   |  Administer corticosteroids (initial dose of     |                |
| Bilirubin                                                                                     | discontinue                                                        | value returned to baseline or is stable)      |                                                   |                |
| 1 to 2 mg/kg prednisone or equivalent)  followed by taper                                     |                                                                    |                                               |                                                   |                |
| New onset  T1DM or  Grade 3 or 4  hyperglycemia  associated with  evidence of  -cell failure | Withhold d                                                         |  Initiate insulin replacement therapy for    |  Monitor participants for hyperglycemia or other |                |
| participants with T1DM                                                                        | signs and symptoms of diabetes                                     |                                               |                                                   |                |
| T1DM or  Hyperglycemia                                                                        |  Administer antihyperglycemic in  participants with hyperglycemia |                                               |                                                   |                |
| Grade 2                                                                                       | Withhold                                                           |  Administer corticosteroids and initiate     |  Monitor for signs and symptoms of hypophysitis  |                |
| hormonal replacements as clinically                                                           | (including hypopituitarism and adrenal                             |                                               |                                                   |                |
| Hypophysitis                                                                                  | Grade 3 or 4                                                       | Withhold or  permanently  discontinue d       | indicated                                         | insufficiency) |  
| Toxicity  Grade                                                       | Action With                                | Corticosteroid and/or Other             |                                                  |                                                  |
|-----------------------------------------------------------------------|--------------------------------------------|-----------------------------------------|--------------------------------------------------|--------------------------------------------------|
| (CTCAE v5.0)                                                          | Pembrolizumab                              | Therapies                               | Monitoring and Follow-up                         |                                                  |
| irAEs                                                                 | Grade 2                                    | Continue                                |  Treat with nonselective beta-blockers          |  Monitor for signs and symptoms of thyroid      |
| (eg, propranolol) or thionamides as                                   | disorders                                  |                                         |                                                  |                                                  |
| Hyperthyroidism                                                       | Grade 3 or 4                               | Withhold or  permanently  discontinue d | appropriate                                      |                                                  |
| Grade 2, 3 or 4                                                       | Continue                                   |  Initiate thyroid replacement hormones |  Monitor for signs and symptoms of thyroid      |                                                  |
| Hypothyroidism                                                        | (eg, levothyroxine or liothyronine) per    | disorders                               |                                                  |                                                  |
| standard of care                                                      |                                            |                                         |                                                  |                                                  |
| Nephritis:                                                            | Grade 2                                    | Withhold                                |                                                  |                                                  |
| grading  according to  increased  creatinine or  acute kidney  injury |  Administer corticosteroids (prednisone 1 |  Monitor changes of renal function     |                                                  |                                                  |
| to 2 mg/kg or equivalent) followed by                                 |                                            |                                         |                                                  |                                                  |
| Grade 3 or 4                                                          | Permanently  discontinue                   | taper                                   |                                                  |                                                  |
| Grade 2                                                               | Withhold                                   |                                         |                                                  |                                                  |
| Neurological  Toxicities                                              | Grade 3 or 4                               | Permanently  discontinue                |  Based on severity of AE administer             |  Ensure adequate evaluation to confirm etiology |
| corticosteroids                                                       | and/or exclude other causes                |                                         |                                                  |                                                  |
| Grade 1                                                               | Withhold                                   |  Based on severity of AE administer    |  Ensure adequate evaluation to confirm etiology |                                                  |
| corticosteroids                                                       | and/or exclude other causes                |                                         |                                                  |                                                  |
| Myocarditis                                                           | Grade 2, 3 or 4                            | Permanently  discontinue                |                                                  |                                                  |
| Exfoliative  Dermatologic  Conditions                                 | Suspected SJS,  TEN, or  DRESS             | Withhold                                |  Based on severity of AE administer             |  Ensure adequate evaluation to confirm etiology |  
| Toxicity  Grade                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | Action With                                  | Corticosteroid and/or Other                      |                          |                         |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------|--------------------------------------------------|--------------------------|-------------------------|
| (CTCAE v5.0)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | Pembrolizumab                                | Therapies                                        | Monitoring and Follow-up |                         |
| irAEs                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Confirmed SJS,  TEN, or  DRESS               | Permanently                                      | corticosteroids          | or exclude other causes |
| discontinue                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |                                              |                                                  |                          |                         |
| Persistent Grade 2 Withhold                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           |  Based on severity of AE administer         |  Ensure adequate evaluation to confirm etiology |                          |                         |
| corticosteroids                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | or exclude other causes                      |                                                  |                          |                         |
| Grade 3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | Withhold or  discontinue based  on the event |                                                  |                          |                         |
| All Other irAEs                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | e                                            |                                                  |                          |                         |
| Recurrent  Grade 3 or  Grade 4                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | Permanently  discontinue                     |                                                  |                          |                         |
| AE(s)=adverse event(s); ALT= alanine aminotransferase; AST=aspartate aminotransferase; CTCAE=Common Terminology Criteria for Adverse Events; DRESS=Drug  Rash with Eosinophilia and Systemic Symptom; GI=gastrointestinal; IO=immuno-oncology; ir=immune related; IV=intravenous; SJS=Stevens-Johnson Syndrome;  T1DM=type 1 diabetes mellitus; TEN=Toxic Epidermal Necrolysis; ULN=upper limit of normal. Note: Non-irAE will be managed as appropriate, following clinical practice recommendations. a AST/ALT: >3.0 to5.0 x ULN if baseline normal; >3.0 to 5.0 x baseline, if baseline abnormal;  bilirubin:>1.5 to 3.0 x ULN if baseline normal; >1.5 to 3.0 x baseline if baseline abnormal b AST/ALT: >5.0 to 20.0 x ULN, if baseline normal; >5.0 to 20.0 x baseline, if baseline abnormal; bilirubin:>3.0 to 10.0 x ULN if baseline normal; >3.0 to 10.0 x baseline  if baseline abnormal c AST/ALT: >20.0 x ULN, if baseline normal; >20.0 x baseline, if baseline abnormal;  bilirubin: >10.0 x ULN if baseline normal; >10.0 x baseline if baseline abnormal d The decision to withhold or permanently discontinue pembrolizumab is at the discretion of the investigator or treating physician. If control achieved or ≤ Grade 2,  pembrolizumab may be resumed. e Events that require discontinuation include, but are not limited to: encephalitis and other clinically important irAEs (e.g., vasculitis and sclerosing cholangitis). |                                              |                                                  |                          |                         |
epigenetic priming for immune therapy
5.2.3 dose modification and toxicity

Pembrolizumab may cause severe or life-threatening infusion-reactions, including severe hypersensitivity or anaphylaxis. Signs and symptoms usually develop during or shortly after drug infusion and generally resolve completely within 24 hours of completion of infusion. Dose modification and toxicity management guidelines on pembrolizumab associated infusion reaction are provided in Table 5.5.  
| Table 5.5 Pembrolizumab Infusion Reaction Dose modification and Treatment Guidelines NCI CTCAE Grade Treatment Premedication at Subsequent Dosing Grade 1 Mild reaction; infusion  interruption not indicated;  intervention not indicated Increase monitoring of vital signs as medically indicated until the participant  None is deemed medically stable in the opinion of the investigator. Grade 2 Requires therapy or infusion  interruption but responds  promptly to symptomatic  treatment (e.g., antihistamines,  NSAIDs, narcotics, IV fluids);  prophylactic medications  indicated for ≤24 hrs Stop Infusion. Additional appropriate medical therapy may include but is not limited to: IV fluids Antihistamines NSAIDs Acetaminophen Narcotics Increase monitoring of vital signs as medically indicated until the participant  is deemed medically stable in the opinion of the investigator. If symptoms resolve within 1 hour of stopping drug infusion, the infusion  may be restarted at 50% of the original infusion rate (e.g., from 100 mL/hr  to 50 mL/hr). Otherwise dosing will be held until symptoms resolve, and the  participant should be premedicated for the next scheduled dose. Participants who develop Grade 2 toxicity despite adequate  premedication should be permanently discontinued from further  study drug treatment Participant may be premedicated 1.5h  (± 30 minutes) prior to infusion of  pembrolizumab with: Diphenhydramine 50 mg po (or  equivalent dose of antihistamine). Acetaminophen 500-1000 mg po (or  equivalent dose of analgesic). Grades 3 or 4 Grade 3: Prolonged (i.e., not rapidly  responsive to symptomatic  medication and/or brief  interruption of infusion);  recurrence of symptoms  following initial improvement;  hospitalization indicated for  other clinical sequelae (e.g.,  renal impairment, pulmonary  infiltrates) Grade 4: Life-threatening; pressor or  ventilatory support indicated Stop Infusion. Additional appropriate medical therapy may include but is not limited to: Epinephrine** IV fluids Antihistamines NSAIDs Acetaminophen Narcotics Oxygen Pressors Corticosteroids Increase monitoring of vital signs as medically indicated until the participant  is deemed medically stable in the opinion of the investigator. Hospitalization may be indicated. **In cases of anaphylaxis, epinephrine should be used immediately. Participant is permanently discontinued from further study drug  treatment. No subsequent dosing Appropriate resuscitation equipment should be available at the bedside and a physician readily available during the period of drug administration. For further information, please refer to the Common Terminology Criteria for Adverse Events v5.0 (CTCAE) at http://ctep.cancer.gov   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Other allowed dose interruption for pembrolizumab Pembrolizumab may be interrupted for situations other than treatment-related AEs such as medical/surgical events or logistical reasons not related to study therapy. Participants should be placed back on study therapy within 3 weeks of the scheduled interruption unless otherwise discussed with the Sponsor. The reason for interruption should be documented in the patient's study record.
epigenetic priming for immune therapy
5.2.4 second course

All participants who stop study treatment with SD or better may be eligible for up to an additional 17 cycles (approximately 1 year) of pembrolizumab treatment if they progress after stopping study treatment from the initial treatment phase. This retreatment is termed the Second Course Phase of this study and is only available if the study remains open, and the participant meets the following conditions:
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either

Stopped initial treatment with study treatment after attaining an investigator-determined confirmed CR based on RECIST 1.1, and  
o Was treated with at least 8 cycles of study treatment before discontinuing treatment, and o Received at least 2 treatments with pembrolizumab beyond the date when the initial CR was declared
OR  
Had SD, PR, or CR and stopped study treatment after completion of 35 administrations
(approximately 2 years) of study treatment for reasons other than disease progression or intolerability
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and

Experienced an investigator-determined radiographic disease progression by RECIST 1.1 after stopping initial treatment, and o Upon unblinding at the time of centrally verified disease progression were found to have received pembrolizumab, and o No new anticancer treatment was administered after the last dose of study treatment, and o The participant meets all of the safety parameters listed in the inclusion criteria and none of the safety parameters listed in the exclusion criteria, and o The study is ongoing
An objective response or disease progression that occurs during the Second Course Phase for a participant will not be counted as an event for the primary analysis of either endpoint in this study. Note: patients must have measurable disease at the start of protocol treatment to be eligible for this provision.
epigenetic priming for immune therapy
5.2.5 monitoring and toxicity management

Each patient receiving pembrolizumab in combination with Tamoxifen and Vorinostat will be evaluable for safety. The safety parameters include all laboratory tests and hematological abnormalities, physical findings, and spontaneous reports of adverse events reported to the investigator by patients. Each patient will be assessed periodically for the development of any toxicity as outlined in Section 6 Study Procedures and Observations. Toxicity will be assessed according to the NCI CTCAE v5.0. Dose adjustments will be made according to the system showing the greatest degree of toxicity.
epigenetic priming for immune therapy
5.2.5.1 events of clinical interest

Selected non-serious and serious adverse events are also known as Events of Clinical Interest (ECI) and must be reported to the Sponsor. For the time period beginning when the consent form is signed until treatment allocation/randomization, any ECI, or follow up to an ECI, that occurs to any subject must be reported within 24 hours to the Sponsor if it causes the subject to be excluded from the trial, or is the result of a protocol-specified intervention, including but not limited to washout or discontinuation of usual therapy, diet, placebo treatment or a procedure. For the time period beginning at treatment allocation/randomization through 30 days following cessation of treatment if the subject initiates new anticancer therapy, whichever is earlier, any ECI, or follow up to an ECI, whether or not related to the Sponsor's product, must be reported within 24 hours to the Sponsor, either by electronic media or paper. Electronic reporting procedures can be found in the EDC data entry guidelines. Paper reporting procedures can be found in the Investigator Trial File Binder (or equivalent). Events of Clinical Interest for this trial include:  
1. an overdose of Sponsor's product, as defined in Section 7.3.7 - Definition of an Overdose for This Protocol and Reporting of Overdose to the Sponsor, that is not associated with clinical symptoms or abnormal laboratory results.  
2. an elevated AST or ALT lab value that is greater than or equal to 3X the upper limit of normal and an elevated total bilirubin lab value that is greater than or equal to 2X the upper limit of normal and, at the same time, an alkaline phosphatase lab value that is less than 2X the upper limit of normal, as determined by way of protocol-specified laboratory testing or unscheduled laboratory testing.*
*Note: These criteria are based upon available regulatory guidance documents. The purpose of the criteria is to specify a threshold of abnormal hepatic tests that may require an additional evaluation for an underlying etiology. The trial site guidance for assessment and follow up of these criteria can be found in the Investigator Trial File Binder (or equivalent).
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5.3 stopping rules

There is a futility early stopping built-in within each treatment arm. For Arm A (vorinostat + TAM + pembro): In the first stage, 13 patients will be accrued. If there are 2 or fewer responses in these 13 patients, the study will be stopped. For Arm B (pembro and TAM): In the first stage, 11 patients will be accrued. If there are 1 or fewer responses in these 11 patients, the study will be stopped. Clinical Criteria for Early Trial Termination 1. Early trial termination will be the result of the criteria specified below:  
2. Quality or quantity of data recording is inaccurate or incomplete 3. Poor adherence to protocol and regulatory requirements 4. Incidence or severity of adverse drug reaction in this or other studies indicates a potential health hazard to subjects 5. Plans to modify or discontinue the development of the study drug
In the event of Merck decision to no longer supply study drug, ample notification will be provided so appropriate adjustments to subject treatment can be made.
epigenetic priming for immune therapy
5.4 discontinuation and withdrawal

Participants may discontinue study treatment at any time for any reason or be dropped from the study treatment at the discretion of the investigator should any untoward effect occur. In addition, a participant may be discontinued from study treatment by the investigator or the Sponsor if study treatment is inappropriate, the trial plan is violated, or for administrative and/or other safety reasons. Specific details regarding procedures to be performed at study treatment discontinuation are provided in Section 6.3.2.6 - Study Procedures Restaging or Off-Study Visit. A participant must be discontinued from study treatment but continue to be monitored in the study for any of the following reasons:  
The participant or participant's legally acceptable representative requests to discontinue study treatment Confirmed radiographic disease progression outlined in Section 3.5. Any progression or recurrence of any malignancy, or any occurrence of another malignancy that requires active treatment Unacceptable adverse experiences, as described in Section 7.3.5.  
The participant has a medical condition or personal circumstance which, in the opinion of the investigator and/or sponsor, placed the participant at unnecessary risk from continued administration of study treatment.  
The participant has a confirmed positive serum pregnancy test Noncompliance with study treatment or procedure requirements Recurrent Grade 2 pneumonitis Discontinuation of treatment may be considered for participants who have attained a confirmed complete response (CR) and have been treated for at least 8 cycles (at least 24 weeks), receiving [for monotherapy: at least 2 doses of pembrolizumab; for combination treatment: 2 cycles of the combination including 2 doses of pembrolizumab and at least 80% of the planned doses of vorinostat beyond the date when the initial CR was declared. These participants may be eligible for second-course treatment described in Section 5.3.  
The participant is lost to follow-up Completion of 35 treatments (approximately 2 years) with pembrolizumab. (Patients may continue vorinostat and tamoxifen.)
Note: The number of treatments is calculated, starting with the first dose. Participants who stop the combination or pembrolizumab after receiving 35 doses may be eligible for retreatment if they progress after stopping study treatment provided they meet the requirements detailed in Section 5.3. Participants may be retreated in the Second Course Phase (Retreatment) for up to an additional 17 cycles (approximately 1 year).
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note to author

- *Guidance on duration of pembrolizumab for solid tumors:*  
o *When there is disease present on imaging (i.e., metastatic, locally advanced,*
bulky disease NOS, etc.) - 24 months of pembrolizumab (35 cycles)
o When there is no disease present on imaging (i.e., adjuvant, following resection, maintenance following induction chemo, etc.) - 12 months of pembrolizumab)
- *Guidance on duration of pembrolizumab for hematologic malignancies:*
o For widespread, recurrent disease - 24 months of pembrolizumab
When using pembrolizumab as consolidative therapy/ limited disease - 12 months. A subject must be discontinued from the trial for any of the following reasons:
· The subject or legal representative (such as a parent or legal guardian)
withdraws consent.  
· Confirmed radiographic disease progression Note: For unconfirmed radiographic disease progression, but clear clinical progression in the opinion of the treating physician or investigator, the patient should be withdrawn from study for progression. Note: A subject may be granted an exception to continue on treatment with confirmed radiographic progression if clinically stable or clinically improving by symptoms or tumor markers.  
Unacceptable adverse experiences as described in Section 7.3.5
· Intercurrent illness that prevents further administration of treatment  
· Investigator's decision to withdraw the subject · The subject has a confirmed positive serum pregnancy test · Noncompliance with trial treatment or procedure requirements · The subject is lost to follow-up
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5.4.1 supportive care guidelines for

Subjects with symptomatic pneumonitis should immediately stop receiving pembrolizumab and have an evaluation. The evaluation may include bronchoscopy and pulmonary function tests to rule out other causes such as infection. If the subject is determined to have study drug associated pneumonitis, the suggested treatment plan is detailed in Table 5.6.  
| Study drug  associated  pneumonitis   | Withhold/Discontinue                                                                                                                                                         | Supportive Care                                              |
|---------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------|
| pembrolizumab                         |                                                                                                                                                                              |                                                              |
| Grade 1                               | No action                                                                                                                                                                    | Intervention not indicated                                   |
| (asymptomatic) Grade 2                | Withhold pembrolizumab, may  return to treatment if improves to  Grade 1 or resolves within 12  weeks                                                                        | Systemic corticosteroids are indicated. Taper if  necessary. |
| Grade 3 and                           | Discontinue pembrolizumab                                                                                                                                                    | Systemic corticosteroids are indicated. The use              |
| Grade 4                               | of infliximab may be indicated as appropriate.  Refer to the Event of Clinical Interest and  Immune-related Adverse Event Guidance  Document for additional recommendations. |                                                              |  
For Grade 2 pneumonitis that improves to ≤ Grade 1 within 12 weeks, the following rules should apply:
First episode of pneumonitis  
o May increase dosing interval by one week in subsequent cycles Second episode of pneumonitis - permanently discontinue pembrolizumab if upon rechallenge subject develops pneumonitis  Grade 2
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6 study procedures and schedule

The study-specific procedures and assessments are detailed in this section and outlined in the Study Calendar - Section 6.1. Screening assessments must be performed within 28 days prior to the first dose of investigational product unless otherwise noted. Any results falling outside of the reference ranges may be repeated at the discretion of the investigator.  
All on-study visit procedures are allowed **a window of ± 5 days** unless otherwise noted.  
Treatment or visit delays for public holidays or weather conditions or patient's personal reasons that do not compromise safety do not constitute a protocol violation. Treatment delays may not exceed 3 weeks unless discussed with the study team for patients with documented benefit. Patients with proven systemic benefit, who have localized progression in the sanctuary site, may receive localized treatment to that site, including but not limited to radiation therapy, gamma knife or surgery.
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6.1 study calendar

| Period/                                           | Pre                                                              |          |         |            |                              |        |        |                               |
|---------------------------------------------------|--------------------------------------------------------------|----------|---------|------------|------------------------------|--------|--------|-------------------------------|
| Screening                                         | Screening1                                                   | Cycle 1  | Cycle 2 | Future     | Restaging or Off-Study Visit |        |        |                               |
| Cycles2,3                                         | Survival Follow-up                                           |          |         |            |                              |        |        |                               |
| Procedure                                         |                                                              |          |         |            |                              |        |        |                               |
| Study Day/Visit Day*                              | -365 to -28                                                  | -28 (±3) | 1 (±3)  | 10-12 (±3) | 15 (±3)                      | 1 (±3) | 1 (±3) | 9 weeks Post Start of Tx (±7) |
| Study Treatment/Drug Administration Pembrolizumab | 200 mg q every 3 weeks; start on C1D1                        |          |         |            |                              |        |        |                               |
| Vorinostat                                        | 400 mg once a day, 4 days on, 3 days off, weekly every cycle |          |         |            |                              |        |        |                               |
| Tamoxifen                                         | 20 mg once a day continuously                                |          |         |            |                              |        |        |                               |
| Administrative Procedures Prescreening ICF        | X                                                            |          |         |            |                              |        |        |                               |
| Informed consent                                  | X                                                            |          |         |            |                              |        |        |                               |
| Clinical Assessments Physical exam                | X                                                            | X        | X       | X          | X                            |        |        |                               |
| Vital signs                                       | X                                                            | X        | X       | X          | X                            |        |        |                               |
| Performance status                                | X                                                            | X        | X       | X          | X                            |        |        |                               |
| Medical history                                   | X                                                            |          |         |            |                              |        |        |                               |
| Concomitant medications                           | X                                                            | X        | X       | X          | X                            |        |        |                               |
| Toxicity Assessment4                              | X                                                            | X        | X       | X          | X                            | X 4    |        |                               |
| Drug Diaries5                                     | X                                                            | X        | X       |            |                              |        |        |                               |
| Documentation of Response                         | X                                                            | X        |         |            |                              |        |        |                               |  
1 All screening procedures must be performed within 28 days unless otherwise indicated. Pre-study tests, History and Physical may be used for Day 1 tests if within 2 weeks 2 Cycle delays up to 14 days for non-medical reason (e.g. holidays) are acceptable without notification of regulatory agencies 3 Therapy delay for palliative radiation should be based on discussion with the study sponsor and the treating physician, every effort should be made not to hold therapy, patients, where vorinostat is being held for more than 4 weeks, may stay on study if there is felt to be a benefit 4 Toxicity assessment must be performed until all treatment-related toxicities have been resolved or decreased to grade 1. Adverse events must be recorded up to 30 days after termination on study 5 Drug diaries will be administered for vorinostat and tamoxifen (oral drugs) only on the first day of every cycle  
| Period/                                      | Pre                    |               |                  |            |                              |        |        |                               |
|----------------------------------------------|--------------------|---------------|------------------|------------|------------------------------|--------|--------|-------------------------------|
| Screening                                    | Screening1         | Cycle 1       | Cycle 2          | Future     | Restaging or Off-Study Visit |        |        |                               |
| Cycles2,3                                    | Survival Follow-up |               |                  |            |                              |        |        |                               |
| Procedure                                    |                    |               |                  |            |                              |        |        |                               |
| Study Day/Visit Day*                         | -365 to -28        | -28 (±3)      | 1 (±3)           | 10-12 (±3) | 15 (±3)                      | 1 (±3) | 1 (±3) | 9 weeks Post Start of Tx (±7) |
| Laboratory Assessments Hematology6           | X                  | X             | X                | X          | X                            |        |        |                               |
| Comprehensive Metabolic Panel7               | X                  | X             | X                | X          | X                            |        |        |                               |
| Thyroid Function8                            | X                  | X             | X (odd cycles)   |            |                              |        |        |                               |
| Tumor Markers9                               | X                  | X             | X                | X          |                              |        |        |                               |
| Pregnancy test10                             | X                  | X             |                  |            |                              |        |        |                               |
| Lymphocyte Subset                            | X or C1D111        | X12           | X 11 (end of C3) |            |                              |        |        |                               |
| PBMC11                                       | X or C1D111        | X 12          | X 11 (end of C3) |            |                              |        |        |                               |
| PK testing                                   | X or C1D1          | X             | X (end of C3)    |            |                              |        |        |                               |
| Biomarker testing (prescreening)             | X                  |               |                  |            |                              |        |        |                               |
| Imaging Procedures CT/MRI13                  | X                  | X             | X                |            |                              |        |        |                               |
| Tissue Collection/ Biopsy Tumor FNA/Biopsy11 | X or C1D1          | X (end of C3) |                  |            |                              |        |        |                               |  
6 Including CBC with differential and platelet count 7 Sodium, Potassium, Chloride, blood urea nitrogen, Creatinine, Non-fasting glucose, Calcium, ALT, AST, Alkaline Phosphatase, Albumin, Total Bilirubin, Total Protein 8 Including FT4 and TSH, TSH only for patients on thyroid replacement therapy 9 CEA, Ca15-3, Ca 125 (if clinically indicated) 10 For women of childbearing potential, performed within 14 days of starting treatment. Pregnancy test will be repeated if clinically indicated 11 Correlative studies: Pre-study within 14 days, and end of cycle 3 (preferably day 17-19 while on vorinostat) prior to cycle 4 day 1 pembrolizumab infusion 12 On day 10-12 pre-dose 13 Staging studies within 30 days prior to day of start, restaging after cycle 3 (week 9 +1w), 6 (week 18+1w), then 9, 12, then every 6 cycles if clinically indicated. Partial and complete responses must be confirmed at least one month apart from the first documentation of objective response. Scans may be performed more frequently if clinically indicated *Note: Window for all study procedures is ±3 days except for scans which is ±7 days
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6.2 schedule of procedures and

Prescreening ICF Laboratory Assessments o Biomarkers testing (1 tbsp. blood draw)
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6.3 participant registration

A written, signed, informed consent form (ICF) and a Health Insurance Portability and Accountability Act (HIPAA) authorization must be obtained before any study-specific assessments are initiated. A copy of the signed ICF will be given to the subject, and a copy will be filed in the medical record. The original will be kept on file with the study records.  
All participants consented to the study will be registered in OnCore®, the UCSF Helen Diller Family Comprehensive Cancer Center Clinical Trial Management System (CTMS). The system is password protected and meets HIPAA requirements.
epigenetic priming for immune therapy
6.3.1 pretreatment period 6.3.1.1 screening

The Screening procedures and assessments must be completed within 28 days of initiating study treatment.  
Clinical Assessments  
o Documentation of disease assessment (CT scan) o Physical examination o Vital signs o Performance status o Complete medical history o Concomitant medication review o Toxicity assessment o Documentation of response Laboratory Assessments o Hematology labs - CBC with differential and platelet count o Comprehensive Metabolic Panel o Thyroid function o PK testing (or at C1D1) o Tumor markers o Pregnancy test o Lymphocyte Subset (or at C1D1) o PBMC (or at C1D1)
Imaging Procedures
o CT / MRI of breast Tumor Tissue Collection o Tumor FNA/biopsy (or at C1D1)
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6.3.2 treatment period 6.3.2.1 study

Study Treatment/Drug Administration o Pembrolizumab (200 mg q every 3 weeks) o Vorinostat (400 mg once a day, 4 days on, 3 days off, weekly every cycle) o Tamoxifen (20 mg once a day continuously)
Clinical Assessments o Physical examination o Vital signs o Performance status o Concomitant medication review o Toxicity assessment o Drug diaries Laboratory Assessments o Hematology labs - CBC with differential and platelet count o Comprehensive Metabolic Panel o PK testing (if not conducted at screening) o Tumor markers o Lymphocyte Subset (if not conducted at screening) o PBMC (if not conducted at screening)
Tumor Tissue Collection o Tumor FNA/biopsy (if not conducted at screening)
epigenetic priming for immune therapy
6.3.2.2 study procedures cycle 1

Study Treatment/Drug Administration o Pembrolizumab (200 mg q every 3 weeks) o Vorinostat (400 mg once a day, 4 days on, 3 days off, weekly every cycle) o Tamoxifen (20 mg once a day continuously)
Laboratory Assessments o PK testing o Lymphocyte Subset o PBMC
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6.3.2.3 study procedures cycle 1

Study Treatment/Drug Administration o Pembrolizumab (200 mg q every 3 weeks)
o Vorinostat (400 mg once a day, 4 days on, 3 days off, weekly every cycle) o Tamoxifen (20 mg once a day continuously)
Clinical Assessments o Physical examination o Vital signs o Performance status o Concomitant medication review o Toxicity assessment Laboratory Assessments o Hematology labs - CBC with differential and platelet count o Comprehensive Metabolic Panel
epigenetic priming for immune therapy
6.3.2.4 study procedures cycle 2

Study Treatment/Drug Administration o Pembrolizumab (200 mg q every 3 weeks) o Vorinostat (400 mg once a day, 4 days on, 3 days off, weekly every cycle) o Tamoxifen (20 mg once a day continuously)
Clinical Assessments o Physical examination o Vital signs o Performance status o Concomitant medication review o Toxicity assessment o Drug diaries Laboratory Assessments o Hematology labs - CBC with differential and platelet count o Comprehensive Metabolic Panel o Thyroid function o Tumor markers
epigenetic priming for immune therapy
6.3.2.5 study procedures future cycles

Study Treatment/Drug Administration o Pembrolizumab (200 mg q every 3 weeks) o Vorinostat (400 mg once a day, 4 days on, 3 days off, weekly every cycle) o Tamoxifen (20 mg once a day continuously)
Clinical Assessments  
o Physical examination o Vital signs o Performance status o Concomitant medication review o Toxicity assessment o Drug diaries o Documentation of response Laboratory Assessments o Hematology labs - CBC with differential and platelet count o Comprehensive Metabolic Panel o Thyroid function (odd cycles) o PK testing (end of C3) o Tumor markers o Pregnancy test o Lymphocyte Subset (end of C3) o PBMC (end of C3)
Imaging Procedures o CT / MRI of breast Tumor Tissue Collection o Tumor FNA/biopsy (end of C3)
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6.3.2.6 study procedures restaging or

Study Treatment/Drug Administration o Pembrolizumab (200 mg q every 3 weeks)  
o Vorinostat (400 mg once a day, 4 days on, 3 days off, weekly every cycle)
o Tamoxifen (20 mg once a day continuously)
Clinical Assessments o Toxicity assessment Imaging Procedures o CT / MRI of breast
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6.3.3 survival follow-up

After completing the follow-up period, participants will be contacted by telephone every 12 weeks to assess for survival/anti-cancer therapy status until death, withdrawal of consent, or the end of the study, whichever occurs first.
epigenetic priming for immune therapy
6.4 correlative studies 6.4.1 histone

Exposure to HDAC inhibitors results in inhibition of histone deacetylation. Histone acetylation (mainly histone H3) has been used as a biological marker of HDAC inhibitor activity. H3 acetylation has been measured successfully in PBMCs in clinical trials using vorinostat and other HDAC inhibitors. We will, therefore, measure H3 and H4 acetylation in peripheral blood mononuclear cells and tumor samples of patients before and after treatment with vorinostat. Mononuclear cells will be obtained prior to treatment initiation and repeated on day 10 (+2) and at the end of cycle 3 (day 17-19). Cells will be isolated from patient blood by centrifugation at 400 x g (Sorvall, HS4 rotor) for 30 min using a Ficoll-Paque gradient (Sigma, St. Louis, MO) as described by Sandor et al. Histone acetylation will be quantified by WB analysis of PBMCs using an acid extraction method to collect nuclear histone proteins.
epigenetic priming for immune therapy
6.4.2 evaluation of vorinostat effects

Post-treatment tumor samples and PBMCs (as well as the vorinostat PK samples) will be obtained on cycle 3 (days 17-19) after the vorinostat morning dose. Fine needle aspirations (FNA) and core biopsies will be obtained either by an interventional radiologist (visceral disease, lymph node) or a cytopathologist (subcutaneous and skin lesions). Diff-Quick air-dry method (FNA) or touch prep analysis (core biopsies) at time of biopsy will be used to confirm the presence of tumor. Tumor samples will be divided and processed for specific techniques as listed below. An attempt for three cores will be made using the appropriate needle. Tissues will be evaluated for the effects of vorinostat on histone acetylation and estrogen expression (by IHC). For IHC, tumors will be fixed in formalin (1 hour/mm thickness), dehydrated, blocked in paraffin. Five-micron sections will be stained with the appropriate antibodies and visualized by light microscopy. Staining will be evaluated using Adobe Photoshop. As the tumor biopsies may contain a mixture of tumor and surrounding cells, cells will be stained (in addition to ER and H3) with specific antibodies used to distinguish tumor versus non-tumor tissues (e.g., cytokeratin (Keratin (5D3)) for breast cancers. PBMCs histone acetylation and ER expression will be quantified by Western blot.
epigenetic priming for immune therapy
6.4.3 logistics on immune modulation

For immune assays we will collect 60cc of blood in green top tubes at baseline and day 10-12 in cycle 1 and on day 17-18 in cycle 3, at the time points specified above for tumor biopsies, FFPE for immune cell infiltrate and a frozen section will be obtained for TCR sequencing.
epigenetic priming for immune therapy
6.5 use of concurrent/concomitant medications

All treatments that the investigator considers necessary for a subject's welfare may be administered at the discretion of the investigator in keeping with the community standards of medical care. All concomitant medication will be recorded on the case report form (CRF) including all prescription, over-the-counter (OTC), herbal supplements, and IV medications and fluids. If changes occur during the trial period, documentation of drug dosage, frequency, route, and date may also be included on the CRF. All concomitant medications received within 28 days before the first dose of trial treatment and 30 days after the last dose of trial treatment should be recorded. Concomitant medications  
administered after 30 days after the last dose of trial treatment should be recorded for SAEs and ECIs as defined in Section 7.3.  
Anti-diarrheal and anti-emetic treatment may be given as required, either therapeutically or prophylactically, after such events have occurred in conjunction with a prior dose given without antidiarrheals or anti-emetics. If needed, efforts will be made to standardize such supportive regimens in agreement with participating Investigators. Other examples of supportive care medications include prophylactic or therapeutic use of erythropoietin for anemia and bisphosphonates (e.g., pamidronate).