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Version Date: 06/17/2020 Abbreviated Title: ONC201 in advanced cancers CC **Protocol \#:** 18-C-0034 Amendment: E
Version Date: 06/17/2020 NCT Number: NCT03394027 Title: A Phase 2 Study of ONC201 in recurrent/refractory metastatic breast cancer and advanced endometrial carcinoma  
| NCI Principal Investigator:   | Alexandra Zimmer, MD Women's Malignancies Branch (WMB), CCR, NCI  National Cancer Institute  Building 10, Room 2B50A 9000 Rockville Pike  Bethesda, MD 20892  Phone: 240-760-6132 Email: alexandra.zimmer@nih.gov   |
|-------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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investigational agents

| Drug Name:    | ONC201                     |
|---------------|----------------------------|
| IND Number:   | 136939                     |
| Sponsor:      | Center for Cancer Research |
| Manufacturer: | Oncoceutics, Inc           |  
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precis

Background:
- Advanced breast cancer and endometrial cancer have limited treatment options. Current treatments provide a modest improvement in progression free survival but no treatments improve survival.  
- ONC201 is the founding member of a novel class of anticancer drugs called imipridones.  
The exact mechanism of ONC201 is unknown at this time, but preclinical data suggests that it causes global downregulation of mitochondrial genes leading to mitochondrial damage and ultimately non-apoptotic cell death.  
- Preclinical studies have demonstrated that ONC201 selectively kills various cancer cells, including breast cancer cells (hormone-receptor positive cell lines, HER2+ cell line as well as triple negative breast cancer cell lines) and endometrial cancer cells, while having little effect on normal cells.  
- An on-going phase I study of ONC201 has demonstrated clinical benefit in some solid tumors, including endometrial cancer.  
Objectives:
- Cohort 1: To determine the progression free survival (PFS) at 8 months of ONC201 in metastatic hormone receptor positive breast cancer (HR+BC)
- Cohort 2: To determine the overall response rate (ORR) of ONC201 in metastatic triple negative breast cancer (TNBC)
- Cohort 3: To determine the ORR of ONC201 in advanced endometrial cancer (EC)
Eligibility: Selected Inclusion Criteria
- Histologically confirmed metastatic breast cancer or endometrial cancer with appropriate IHC testing and confirmation of HER2 non-amplification required for the breast cancer cohorts (cohorts 1 and 2)
- Age 18 years or older
- Female and male breast cancer patients are eligible for the breast cancer cohorts - ECOG 0 or 1
- Measurable metastatic disease with >1 biopsiable lesion with willingness to undergo a biopsy
- Cohort 1 (HR+BC) requires prior treatment with >2 lines of hormonal treatment. No prior treatment required for cohorts 2/3 (TNBC and EC).  
- Adequate hematopoietic, hepatic and renal function Selected Exclusion Criteria
- Patients who have received chemotherapy in the previous 3 weeks (6 weeks for nitrosoureas or mitomycin); other investigational agents within 3 weeks or a PD1/PDL1 agent within 4 weeks prior to first dose of study treatment.  
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design

- Radiotherapy < 4 weeks before first dose of study treatment
- Symptomatic CNS metastases. Asymptomatic or brain metastases treated > 4 weeks from first dose of study treatment are allowed.  
- History of invasive malignancy < 3 years - Known history of cardiac arrhythmias including uncontrolled atrial fibrillation, tachyarrhythmias or bradycardia.  
- History of CHF, or MI or stroke in the previous 3 months will be excluded.  
- Started denosumab or bisphosphonate therapy within 28 days prior to Cycle 1 Day 1
- HIV, Hepatitis B, or Hepatitis C infection
- This is a phase II single arm study of ONC201 divided in three cohorts, each cohort with a different type of metastatic, advanced disease:
o Cohort 1: HR+ breast cancer (male and female) o Cohort 2: Triple negative breast cancer (male and female) o Cohort 3: Endometrial cancer (female only)
- All patients will receive ONC201 at the recommended phase 2 dose (RP2D) of 625mg by mouth every 7 days with each cycle being 28 days long. Patients will receive ONC201 as long as they derive clinical benefit or toxicity becomes impeditive
- Patients will be evaluated for toxicity every 4 weeks by CTCAE v5.0 and for response every two cycles (8 weeks) by RECIST 1.1.
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study schema

Study Schema Figure  
  
*See **Table 1** for dose reductions Table 1: ONC201 Dose Reduction Schedule Dose Level
(DL)
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onc201

(by mouth)
1 (Starting Dose) 625mg every 7 days
-1 500mg every 7 days -2 375mg every 7 days -3 250mg every 7 days
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version date 06/17/2020

| TABLE OF CONTENTS                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| PRÉCIS 2 TABLE OF CONTENTS 5 1 INTRODUCTION 10 1.1 Study Objectives 10 1.1.1 Primary Objective 10 1.1.2 Secondary Objectives 10 1.1.3 Exporatory Studies: 10 1.2 Background and Rationale 10 1.2.1 Current Standards of Care for Recurrent/Refractory Invasive Breast Cancer 10 1.2.2 Current Standards of Care for Endometrial Cancer 11 1.2.3 Development of ONC201 12 1.2.4 Pharmaceutical Manufacturer Updates 16 2 ELIGIBILITY ASSESSMENT AND ENROLLMENT 17 2.1 Eligibility Criteria for Cohort 1: Hormone Receptor Positive Breast Cancer 17 2.1.1 Inclusion Criteria 17 2.1.2 Exclusion Criteria 18 2.2 Eligibility Criteria for Cohort 2: Triple Negative Breast Cancer 19 2.2.1 Inclusion Criteria 19 2.2.2 Exclusion Criteria 21 2.3 Eligibility Criteria for Cohort 3: Endometrial Cancer 22 2.3.1 Inclusion Criteria 22 2.3.2 Exclusion Criteria 23 2.4 Recruitment Strategies 23 2.5 Screening Evaluation 24 2.5.1 Screening activities performed prior to obtaining informed consent 24 2.5.2 Screening activities performed after a consent for screening has been signed 24 2.6 Participant Registration and Status Update Procedures 26 2.7 Screen Failures 26 2.8 Treatment Assignment and Randomization/Stratification Procedures 26 2.9 Baseline Evaluation 27 3 STUDY IMPLEMENTATION 28 |
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abbreviated title onc201 in advanced

| Version Date: 06/17/2020                                                                                  |                                                                                                               |
|-----------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------|
| 3.1                                                                                                       | Study Design .                                                                                                |
| 3.2                                                                                                       | Drug Administration                                                                                           |
| 3.2.1                                                                                                     | Self-Administered Study Drugs: .                                                                              |
| 3.3                                                                                                       | Dose Modifications/Delays and Management of Toxicities ................................................... 29 |
| 3.3.1                                                                                                     | Modifications and Toxicity Management                                                                         |
| Medication Delay                                                                                          |                                                                                                               |
| 3.3.2                                                                                                     |                                                                                                               |
| 3.3.3                                                                                                     | Instructions on Missed Doses .                                                                                |
| 3.4                                                                                                       | Study Calendar .                                                                                              |
| Criteria for Removal from Protocol Therapy and Off Study Criteria ................................. 35    |                                                                                                               |
| 3.5                                                                                                       |                                                                                                               |
| 3.5.1                                                                                                     | Criteria for removal from protocol therapy .                                                                  |
| 3.5.2                                                                                                     | OFF-Study Criteria .                                                                                          |
| 4                                                                                                         | CONCOMITANT MEDICATIONS/MEASURES .                                                                            |
| Restrictions                                                                                              |                                                                                                               |
| 4.1                                                                                                       |                                                                                                               |
| 4.2                                                                                                       | Contraception                                                                                                 |
| s                                                                                                         | BIOSPECIMEN COLLECTION .                                                                                      |
| Correlative Studies for Research/Pharmacokinetic Studies .                                                |                                                                                                               |
| 5.1                                                                                                       |                                                                                                               |
| 5.2                                                                                                       | Genomic Alterations in Breast and Endometrial Cancers                                                         |
| 5.3                                                                                                       | T Tumor biopsies .                                                                                            |
| Timing* .                                                                                                 |                                                                                                               |
| 5.3.1                                                                                                     | Tissue Sampling and Handling .                                                                                |
| 5.3.2                                                                                                     |                                                                                                               |
| 5.4                                                                                                       | Blood samples                                                                                                 |
| 5.4.1                                                                                                     | Timing                                                                                                        |
| 5.4.2                                                                                                     | Sampling and Handling.                                                                                        |
| 5.5                                                                                                       | Table 5: Specimen Collection and Storage .                                                                    |
| 5.6                                                                                                       | Sample Storage, Tracking and Disposition .                                                                    |
| 5.6.1                                                                                                     | Patient sample protections .                                                                                  |
| Sample Storage, Tracking and Disposition (CPC) ....................................................... 49 |                                                                                                               |
| 5.6.2                                                                                                     |                                                                                                               |
| 5.6.3                                                                                                     | Laboratory of Jane Trepel                                                                                     |
| 5.6.4                                                                                                     | Procedures for storage of tissue specimens in the Laboratory of Pathology ......... 51                        |
| End of Study .                                                                                            |                                                                                                               |
| 5.6.5                                                                                                     |                                                                                                               |
| 5.7                                                                                                       | Samples for Genetic/Genomic Analysis .                                                                        |
| 5.7.1                                                                                                     | Certificate of Confidentiality .                                                                              |  
6  
| Version Date: 06/17/2020            |                                                                                                                                       |                    |
|-------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------|--------------------|
| 6                                   | DATA COLLECTION AND EVALUATION                                                                                                        |                    |
| DATA COLLECTION                     |                                                                                                                                       |                    |
| 6.1                                 | 6.1.1                                                                                                                                 | SOURCE DOCUMENTS . |
| 6.1.2                               | Case Report Forms                                                                                                                     |                    |
| 6.2                                 | DATA Sharing Plans .                                                                                                                  |                    |
| Human Data Sharing Plan.            |                                                                                                                                       |                    |
| 6.2.1                               |                                                                                                                                       |                    |
| 6.2.2                               | Genomic Data Sharing Plan                                                                                                             |                    |
| 6.3                                 | Response Criteria                                                                                                                     |                    |
| Disease Parameters .                |                                                                                                                                       |                    |
| 6.3.1                               | Methods for Evaluation of Measurable Disease .                                                                                        |                    |
| 6.3.2                               |                                                                                                                                       |                    |
| 6.3.3                               | Response Criteria                                                                                                                     |                    |
| 6.3.4                               | D Duration of Response                                                                                                                |                    |
| Progression-Free Survival           |                                                                                                                                       |                    |
| 6.3.5                               |                                                                                                                                       |                    |
| 6.4                                 | Toxicity Criteria                                                                                                                     |                    |
| 7                                   | NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN .... 60                                                                    |                    |
| Definitions                         |                                                                                                                                       |                    |
| 7.1                                 |                                                                                                                                       |                    |
| 7.2                                 | OHSRP Office of Compliance and Training / IRB Reporting ............................................. 60                              |                    |
| 7.2.1                               | Expedited Reporting                                                                                                                   |                    |
| 7.2.2                               | IRB Requirements for PI Reporting at Continuing Review ........................................ 60                                    |                    |
| 7.3                                 | NCI CLINICAL DIRECTOR REPORTING                                                                                                       |                    |
| 7.4                                 | NIH Required Data and Safety Monitoring Plan ............................................................................ 60          |                    |
| 7.4.1                               | Principal Investigator/Research Team ............................................................................................. 60 |                    |
| 8                                   | SPONSOR SAFETY REPORTING .                                                                                                            |                    |
| 8.1                                 | DEFINITIONS                                                                                                                           |                    |
| 8.1.1                               | A Adverse Event                                                                                                                       |                    |
| 8.1.2                               | Serious Adverse Event (SAE)                                                                                                           |                    |
| Life-threatening                    |                                                                                                                                       |                    |
| 8.1.3                               |                                                                                                                                       |                    |
| 8.1.4                               | SEVERITY .                                                                                                                            |                    |
| 8.1.5                               | Relationship to Study Product                                                                                                         |                    |
| 8.2                                 | Assessment of Safety Events                                                                                                           |                    |
| Reporting of Serious Adverse Events |                                                                                                                                       |                    |
| 8.3                                 |                                                                                                                                       |                    |
| 8.4                                 | Safety Reporting Criteria To The Pharmaceutical Collaborators ........................................ 63                             |                    |  
| Version Date: 06/17/2020            |                                                                                                                              |                            |
|-------------------------------------|------------------------------------------------------------------------------------------------------------------------------|----------------------------|
| 8.5                                 | Reporting Pregnancy                                                                                                          |                            |
| Maternal exposure                   |                                                                                                                              |                            |
| 8.5.1                               |                                                                                                                              |                            |
| 8.5.2                               | Paternal exposure                                                                                                            |                            |
| 8.6                                 | Regulatory Reporting for Studies Conducted Under CCR-Sponsored IND .................. 64                                     |                            |
| 9                                   | Clinical Monitoring.                                                                                                         |                            |
| STATISTICAL CONSIDERATIONS          |                                                                                                                              |                            |
| 10                                  | 10.1                                                                                                                         | Statistical Hypothesis .   |
| 10.2                                | Sample Size Determination                                                                                                    |                            |
| Populations for Analyses .          |                                                                                                                              |                            |
| 10.3                                | Statistical Analyses                                                                                                         |                            |
| 10.4                                |                                                                                                                              |                            |
| 10.4.1                              | General Approach .                                                                                                           |                            |
| 10.4.2                              | Analysis of the Primary Endpoints                                                                                            |                            |
| Analysis of the Secondary Endpoints |                                                                                                                              |                            |
| 10.4.3                              |                                                                                                                              |                            |
| 10.4.4                              | Se                                                                                                                           |                            |
| 10.4.5                              | Baseline Descriptive Statistics                                                                                              |                            |
| Planned Interim Analyses .          |                                                                                                                              |                            |
| 10.4.6                              | Sub-Group Analyses .                                                                                                         |                            |
| 10.4.7                              |                                                                                                                              |                            |
| 10.4.8                              | Tabulation of individual Participant Data                                                                                    |                            |
| Exploratory Analyses                |                                                                                                                              |                            |
| 10.4.9                              |                                                                                                                              |                            |
| COLLABORATIVE AGREEMENTS .          |                                                                                                                              |                            |
| 11                                  | 1.1                                                                                                                          | Clinical Trial Agreement . |
| 12                                  | HUMAN SUBJECTS PROTECTIONS .                                                                                                 |                            |
| 12.1                                | Rationale For Subject Selection                                                                                              |                            |
| Participation of Children           |                                                                                                                              |                            |
| 12.2                                |                                                                                                                              |                            |
| 12.3                                | Participation of Subjects Unable to Give Consent ................................................................ 69         |                            |
| 12.4                                | Evaluation of Benefits and Risks/Discomforts ............................................................................ 69 |                            |
| Risks/Benefits Analysis             |                                                                                                                              |                            |
| 12.5                                |                                                                                                                              |                            |
| 12.5.1                              | Benefits .                                                                                                                   |                            |
| 12.5.2                              | Risks                                                                                                                        |                            |
| Risks/Benefits Analysis             |                                                                                                                              |                            |
| 12.5.3                              | Consent Process and Documentation                                                                                            |                            |
| 12.6                                |                                                                                                                              |                            |
| 12.6.1                              | Request for Waiver of Consent for Screening Activities.............................................. 71                      |                            |  
| Version Date: 06/17/2020                   |                                                                                                                                                       |
|--------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------|
| 13                                         | PHARMACEUTICAL INFORMATION                                                                                                                            |
| Drug ONC201 (IND # 136939)                 |                                                                                                                                                       |
| 13.1                                       | SOURCE .                                                                                                                                              |
| 13.1.1                                     |                                                                                                                                                       |
| 13.1.2                                     | T Toxicity .                                                                                                                                          |
| 13.1.3                                     | Formulation and preparation                                                                                                                           |
| Stability and Storage                      |                                                                                                                                                       |
| 13.1.4                                     |                                                                                                                                                       |
| 13.1.5                                     | Incompatibilities                                                                                                                                     |
| 14                                         | REFERENCES .                                                                                                                                          |
| APPENDICES .                               |                                                                                                                                                       |
| 15                                         | Appendix A-Performance Status Criteria                                                                                                                |
| 15.1                                       |                                                                                                                                                       |
| 15.2                                       | Appendix B- Method for Preparing PBMC Samples from Blood For                                                                                          |
| Pharmacodynamic (PD) Studies .             |                                                                                                                                                       |
| Appendix C - SOP for Cytokine Collection . |                                                                                                                                                       |
| 15.3                                       |                                                                                                                                                       |
| 15.4                                       | Appendix D- Standard Operating Procedure for Plasma and Buffy coat collection for                                                                     |
| circulating tumor DNA                      |                                                                                                                                                       |
| Appendix E- Oral Medication Diary          |                                                                                                                                                       |
| 15.5                                       | Appendix F- Oncoceutics Specimen Collection, Processing, Storage and Shipping . 89                                                                    |
| 15.6                                       | Serum Sample Supplies ............................................................................................................................ 90 |
| 15.6.1                                     |                                                                                                                                                       |
| 15.6.2                                     | Plasma Processing                                                                                                                                     |
| 15.6.3                                     | Serum Processing                                                                                                                                      |
| Arranging for Shipments.                   |                                                                                                                                                       |
| 15.6.4                                     |                                                                                                                                                       |
| 15.6.5                                     | Ococeutics Specimen Shipping Form ................................................................................................ 99                 |  
Version Date: 06/17/2020
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1.1.1 primary objective

- Cohort 1 HR+BC - To determine the progression free survival at 8 months (PFS by RECIST) of ONC201 in patients with refractory, metastatic hormone receptor positive breast cancer
- Cohort 2 TNBC - To determine the overall response rate (ORR; CR + PR by RECIST) of ONC201 in patients with metastatic triple negative breast cancer
- Cohort 3 Endometrial Cancer - To determine the ORR of ONC201 in patients with advanced or metastatic endometrial cancer
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1.1.2 secondary objectives

- Cohort 1 HR+BC - Safety, ORR, clinical benefit rate (CBR; CR + PR + SD by RECIST) - Cohort 2 TNBC - Safety, CBR, PFS
- Cohort 3 Endometrial Cancer - Safety, CBR, PFS
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1.1.3 exporatory studies

The primary goal of the exploratory studies is to identify potential predictive biomarkers of response as well as additional evidence of the mechanism of action of and resistance to ONC201. These will be performed in an exploratory fashion on select patients if there are adequate samples for analysis. 1.2 BACKGROUND AND RATIONALE This is a phase II study of ONC201 for the treatment of metastatic hormone receptor positive breast cancer, metastatic triple negative breast cancer, and endometrial cancer. At this time, there is no clear benefit to the combination of ONC201 plus anti-HER2 therapies. However, we plan to develop preclinical data for ONC201 in combination with HER2 targeted drugs (*e.g.*, trastuzumab, trastuzumab emtansine, and lapatinib) and we anticipate initiating a Phase I run-in study at a later date.
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1.2.1 current standards of care

Invasive breast cancer remains the most common malignancy in women in the United States with an annual incidence of 123.1 cases per 100,000 women (5). Treatment of invasive breast cancer involves local control with surgery and/or radiation where appropriate, as well as systemic control with endocrine therapy, HER2 targeted therapy, and chemotherapy. Despite advances in early detection and effective treatments, breast cancer is the second most common cause of US cancer deaths in women with an annual rate of 21.9 deaths per 100,000 women (5). It is estimated that in 2017, there will be 252,710 new cases of breast cancer with 40,610 deaths (6).
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1.2.1.1 hormone receptor positive breast

Hormone receptor positive breast cancer (HR+BC) accounts for approximately 75% of all breast cancers and is defined by immunohistochemical (IHC) staining of at least  1% of tumor cells with estrogen receptors and/or  1% with progesterone receptors. Despite optimal treatment, up to 30% of women with node-negative breast cancer recur after their initial diagnosis. While the risk is greatest within the first two years of diagnosis, women with HR+BC have an increased risk for recurrence up to 25 years after diagnosis (7, 8). Recurrence can be loco-regional or metastatic. Once metastatic, there are limited effective treatments and overall prognosis is poor. Generally, patients with metastatic or recurred disease are first treated with endocrine therapy alone (e.g. tamoxifen, aromatase inhibitors, or fulvestrant) or, more recently, endocrine therapy in combination with CDK 4/6 inhibitors or mTOR inhibitors (9). However, once the cancer is determined to be hormone refractory, chemotherapy is the next treatment option. Combination chemotherapy regimens have higher response rates and longer progression free survival (PFS), but with higher toxicity profiles and no added survival benefit when compared to single agent chemotherapy regimens (10). Therefore, the use of sequential single agents is the favored regimen once the cancer is refractory to endocrine therapy. Preferred single agents, like paclitaxel or capecitabine, carry a PFS of 5.7 to 8.0 months (11, 12). Both endocrine therapy and chemotherapy in this setting have been shown to have palliative benefit but no curative therapies exist.
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1.2.1.2 triple negative breast cancer

Triple-negative breast cancer (TNBC) accounts for 10-20% of all breast cancers, and is defined by the absence of estrogen and progesterone receptors (ER, PR) and the absence of HER2 amplification or over-expression (13). Thus, patients with TNBC do not benefit from known targeted therapies to ER or HER2 (14). Standard therapy for metastatic TNBC is single agent chemotherapy and, in metastatic patients, ORRs are around 30-35%, with median PFS 4.5-6 months, and median OS around 12 months (15). Clearly, new approaches to the treatment of TNBC are needed to improve outcomes.
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1.2.2 current standards of care

Endometrial adenocarcinoma is the most common malignancy of the female genital tract with an estimated 61,380 new cases and 10,920 deaths in 2017 (6). While endometrial cancer is often found in early stages due to irregular vaginal bleeding in a predominantly postmenopausal population, there has been a recent increase in cancer-related mortality (16). This is suspected to be due to an increase in advanced-stage cancers, high risk histologies (e.g., serous carcinomas or clear cell carcinomas) and patients being diagnosed at an older age (16). In addition, there is an increased risk of endometrial cancer in patients with Lynch Syndrome (17). Multi-agent chemotherapy regimens are preferred in the relapsed or advanced/metastatic setting. Current first line chemotherapy regimens have response rates ranging from 21% to 81%. These responses are of short duration with median progression free survival of approximately one year (18, 19). Due to a relatively low toxicity profile and multiple studies demonstrating improved survival, carboplatin and paclitaxel is the preferred regimen for advanced/metastatic or recurrent endometrial cancer with response rates of 40 to 62% and overall survival from 13 to 29 months (20). If a multi-drug regimen cannot be tolerated, single agent treatment with paclitaxel (most active), cisplatin, carboplatin, doxorubicin, liposomal doxorubicin, topotecan, or docetaxel can be used. However, response rates with single agents are lower (first-line treatment response rate 21% to 36%; second line treatment response rates 4% to 27%) (21). None of these therapies are curative and second line therapies have not been shown to improve survival (21). New therapies are needed to improve outcomes in patients with advanced stage endometrial cancers.
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1.2.3 development of onc201 1.2.3.1

ONC201 (also known as TIC10 and NSC350625) is the founding member of a novel class of anti-cancer compounds called imipridones (22). It is an orally active small molecule that was initially identified by a screen for small molecules that could induce tumor necrosis factor (TNF) –related apoptosis-inducing ligand (TRAIL) on colon cancer cells, and activate the TRAIL death receptors on those cells inducing an autocrine apoptotic death. TRAIL is a type II transmembrane  
 protein that is expressed in various tissues and cells including the surface of Natural Killer (NK) and T cells, macrophages and dendritic cells (23, 24). On immune cells, the expression of TRAIL plays a role in immune surveillance in the prevention of tumors and metastasis (25). Humans have five distinct TRAIL receptors (26, 27). Death receptor 4 (DR4) and DR5 contain intracellular death domains and are capable of signaling apoptosis. There are two membranebound decoy receptors called DcR1 and DcR2 which lack a functional death domain and while unable to activate apoptosis, are able to inhibit TRAIL signaling. Osteoprotogerin is the fifth TRAIL-binding receptor which also functions as a decoy/inhibitor by sequestering TRAIL extracellularly. TRAIL is attractive as an anti-tumor agent because of its capability to induce apoptosis in cancer cells by activating DR4 and DR5 with little toxicity against normal cells (23, 28). In our preclinical work, we have shown that TRAIL receptor agonists are most active against triple negative breast cancer and in particular those with mesenchymal features (29, 30). Despite their robust cell killing of tumor cells *in vitro* and in animal models *in vivo,* efficacy of TRAIL receptor agonists has been limited in clinical trials. The limited activity is thought to be due to lack of reliable predictive biomarkers of sensitivity and drug properties that limit efficacy such as short serum half-life, stability, and bio-distribution (25, 28, 31). There is a need to find alternative approaches to activating the DR pathways. ONC201 induces sustained up-regulation of TRAIL in tumor cell lines, which activates DR through autocrine and paracrine mechanisms.  
Figure 1: Structure and mechanism of action of ONC201. In the TRAIL-dependent pathway, ONC201 inhibits both AKT and MEK, resulting in the dephosphorylation of the transcription factor FOXO3a. This enables its translocation from the cytoplasm into the nucleus where it binds to the TRAIL promoter and upregulates gene transcription of TRAIL. Apoptosis occurs through activation of TRAIL-DR receptors. In the TRAIL-independent pathway, ONC201 induces a stress response through an unknown mechanism which differs between solid tumors and hematologic malignancies. The mechanism of ONC201 appears to be different in breast cancer cell lines, where ONC201 demonstrates caspase-independent, DR4/5 independent mechanisms distinct from TRAIL-induced apoptosis. Instead it induces mitochondrial dysfunction and ATP depletion. Image adapted from Greer and Lipkowitz, 2016: (3).  
Several alternative mechanisms have also been proposed for the anticancer activity of ONC201, including inhibition of the dopamine receptor D2 (DRD2), and activation of an atypical integrated stress response in tumor cells (32, 33).  
  
ONC201 has been extensively investigated in the Lipkowitz lab as a promising means to kill tumor cells. Rather than a simple induction of TRAIL
and induction or DR receptordependent apoptosis, both the Lipkowitz lab and independent labs have shown that ONC201 has both TRAIL-dependent and TRAIL-independent pathways of inducing death
(**Figure 1**– two right sided pathways). We have investigated the mechanism by which ONC201 kills breast cancer cells. Unexpectedly, ONC201 did not induce caspase 3 or PARP cleavage, and its toxicity was not inhibited by Z-VADFMK, nor by siRNA
knockdown of DR4 or DR5. By contrast GST-TRAIL induced caspase 3 and PARP cleavage and GST-TRAIL-induced cell death was inhibited by Z-VAD- FMK and by siRNA knockdown of DR5. Live cell imaging revealed ONC201 induces cell membrane ballooning followed by rupture consistent with necrosis, whereas GST-TRAIL induced classic apoptosis morphology. Together these results suggest that ONC201 kills breast cancer cells via a caspase-independent, DR4/5-independent mechanism distinct from TRAIL-induced apoptosis. ONC201 does induce ATF4 and CHOP, consistent with the recently published observations of another group (32, 33).  
Figure 2: IC50 for breast cancer cell lines treated with ONC201. Cells were treated with a dose curve of ONC201 for 5 days and viability measured by MTS assay. The data represent the mean IC50 for each cell line (Greer and Lipkowitz, unpublished observations).  
Further mechanistic studies in the Lipkowitz lab showed that ONC201 induced transcriptional down regulation of multiple nuclear encoded mitochondrial genes, resulting in decreased mitochondrial oxidative phosphorylation and ATP production (**Figure 1**– right pathway). Our data suggest that in addition to the published mechanisms, ONC201 can kill breast cancer cells by a novel mechanism involving inhibition of mitochondrial respiration. In breast cancer cells this appears to be the dominant mechanism. One of the reasons for conducting this trial in the Intramural program is to validate and expand our understanding of ONC201's mechanism of action in the human. Such data will translate back to the bench, enabling the development of combination therapies and plans for overcoming resistance. We have tested ONC201 across a range of breast cancer cell lines and have found that all subtypes are inhibited by ONC201, and the IC50s for the cell lines range from 0.8-5µM (Figure 2). This is within the achievable concentrations that were seen in pharmacokinetic studies from phase I studies (2).  
ONC201 has been evaluated as a therapeutic agent *in vitro* as well as *in vivo* in various cell lines.  
Single-dose experiments in HCT116 wild-type (human colon cancer) xenograft–bearing mice corroborated the antitumor activity of ONC201 (TIC10)
(**Figure 3**). *In vivo* experiments have shown promising anti-tumor effects in 12 subcutaneous xenographs (including triple negative breast cancer, non-small cell lung cancer, hepatocellular carcinoma, glioblastoma, colon cancer and head and neck squamous cell carcinoma), one orthotopic xenograph (glioblastoma) and one transgenic (lymphoma) model. Dr. El-Deiry's group has demonstrated that ONC201 inhibits xenografts of the triple negative breast cancer cell line MDA-MB-231 (1).
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1.2.3.2 clinical development of onc201

Due to preclinical results, ONC201 is currently being investigated in phase I and II trials in patients with refractory solid tumors including glioblastoma as well as in patients with relapsed/refractory non-Hodgkin's lymphoma, relapsed/refractory acute leukemias and high-risk myelodysplastic syndromes. Many of these trials are ongoing, but results from the first-in-human phase I trial of ONC201 in patients with refractory solid tumors are promising (2, 4). The trial enrolled 28 patients with 15 different types of tumors including prostate cancer, endometrial cancer, ovarian cancer, colon cancer, gallbladder cancer and sarcoma. In this heavily treated population (average of 5-6 prior treatments), researchers found prolonged stable disease in many patients, as well as disease reduction in others patients, most notably in those with endometrial or prostate cancer. This trial also evaluated the safety and tolerability of ONC201. The recommended phase 2 dose (RP2D) was determined to be 625mg by mouth q3 weeks and dosing was subsequently adjusted to weekly dosing due to pharmacokinetic studies. There have been no deaths attributed to study drug. There have been no SAEs assessed by the investigators as "probably" or "definitely" related to study drug, and no discontinuation of ONC201 due to toxicity has been reported.The most common AEs reported as possibly related to ONC201 were mild/moderate nausea, vomiting, anorexia, pyrexia and fatigue. More than 70 patients with advanced solid tumors and hematological malignancies were evaluated in phase I clinical trials and no serious side effects were found attributable to ONC201. Grade I adverse events that have been observed and reported include fever, nausea, vomiting, fatigue, and elevated serum amylase that were attributed as possiblyrelated to ONC201. One Grade II allergic reaction occurred that was managed with standard medications. One Grade III event neutropenia was transiently observed. No other Grade III or IV events were reported so far.  
The lesion-bylesion results of this phase 1 study were recently reported
(**Figure 4**) (2, 4).  
In this phase I study, 3 of 5 patients with endometrial cancer had stable disease by RECIST criteria (> 4 months in 2 and >10 months in one). The Lipkowitz lab has also confirmed that ONC201 had a similar effect on the killing of various endometrial cancer cell lines (data not shown) as it did on breast cancer cell lines. Based on these result we propose to include a cohort of patients with endometrial cancer in our trial.  
  
Only one triple negative breast cancer patient was recruited for the phase 1 study, and unfortunately had disease progression while on ONC201. Nevertheless, the preclinical data on breast cancer cell lines performed by the drug developer and confirmed by the Lipkowitz lab is encouraging that women with breast cancer may derive clinical benefit from ONC201.
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1.2.4 pharmaceutical manufacturer updates 1.2.4.1

As of October 31, 2019, the majority of adverse events (AEs) experienced by participants on clinical trials who received ONC201 were mild or moderate in severity. The most frequently reported AEs of any grade (in greater than 25% of patients) were fatigue, nausea, headache and vomiting. These AEs were generally Grade 1/2, with fatigue being the only Grade 3/4 AE that occurred in more than one patient on the ONC013 study (17.4%). Eleven patients experienced serious adverse events (SAEs) that were considered possibly related to ONC201. The list of SAEs reported by investigators that were attributed as possibly-related to ONC201 by the investigator are:  
- Grade 1: Fatigue, vomiting
- Grade 3: Encephalopathy, cognitive impairment, neutropenia, upper respiratory infection, respiratory distress, pneumonia, stroke, tremor, fatigue, bone pain, anorexia, weakness
- Grade 4 Dysphagia, dyspnea
Two suspected unexpected serious adverse (SUSAR) events occurred at a dose of 625mg daily. Both events were encephalitis that occurred on the investigator-initiated trial NCT02392572 in patients with MDS or AML and are considered dose-limiting toxicities(DLT). No other DLTs occurred in other indications or at lower doses both as monotherapy or in combination with other agents. Only one dose reduction (625mg to 500mg) occurred in one patient with a Grade 3 neutropenia assessed by the PI as possibly related to ONC201. However, upon re-challenge neutropenia did not recur. Based on the clinical safety data summarized above, ONC201 administered at the doses and schedules below 625mg daily appears to be well tolerated in adults and children.
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2 eligibility assessment and enrollment

2.1 ELIGIBILITY CRITERIA FOR COHORT 1: HORMONE RECEPTOR POSITIVE BREAST CANCER
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2.1.1 inclusion criteria

2.1.1.1 Patients must have histologically confirmed persistent or recurrent invasive metastatic hormone receptor positive, HER2 normal breast cancer for which standard curative measures do not exist or are no longer effective. Hormone receptor positive is defined as estrogen receptor (ER) positive  10% by immunohistochemistry (IHC) and/or progesterone receptor (PR) positive  10% by IHC.HER2 will be considered negative per ASCO-CAP guidelines (HER2 test result as negative if a single test (or both tests) performed show: 1) IHC 1+ as defined by incomplete membrane staining that is faint/barely perceptible and within >10% of the invasive tumor cells; 2) IHC 0 as defined by no staining observed or membrane staining that is incomplete and is faint/barely perceptible and within ≤10% of the invasive tumor cells; or 3) ISH negative based on: a) Single-probe average HER2 copy number <4.0 signals/cell or b) Dualprobe HER2/CEP17 ratio <2.0 with an average HER2 copy number <4.0 signals/cell)and HER2 testing must have been performed in a laboratory accredited by the College of American Pathologists (CAP) or another accrediting entity.  
2.1.1.2 Patients must have measurable disease, per RECIST 1.1. See Section **6.3.1** for the evaluation of measurable disease.  
2.1.1.3 Patients must have at least one lesion deemed safe to biopsy and be willing to undergo mandatory biopsies.  
2.1.1.4 HR+BC patients must have received prior treatment with at least 2 lines of hormonal treatment (SERM, AI, or fulvestrant) and deemed ineligible for further hormonal therapy . Patients may have received prior chemotherapy and there is no limit to the number of prior chemotherapy.  
2.1.1.5 Age >18 years.  
2.1.1.6 ECOG performance status 0 or 1 2.1.1.7 Adequate renal function, defined as serum creatinine ≤ 1.5 X upper limit of normal
(ULN), or measured creatinine clearance ≥ 60 mL/min/1.  
2.1.1.8 Adequate hepatic function, defined as AST and ALT levels ≤ 3 X ULN and total bilirubin < 1.5 X ULN, unless known diagnosis of Gilbert's syndrome, where bilirubin ≤ 5 mg/dL will be permitted. Gilbert's syndrome will be defined as elevated unconjugated bilirubin, with conjugated (direct) bilirubin within the normal range and less than 20% of the total. Total bilirubin will be permitted up to 5 mg/dL, if patients have historical readings consistent with the definition of Gilbert's syndrome prior to entering study.  
2.1.1.9 Adequate bone marrow function, defined as absolute neutrophil (ANC) ≥ 1,500/mm3
(≥1.5 X106/L), platelet count ≥ 75,000/mm3 (≥ 75 X106/L), and hemoglobin ≥ 9 mg/dL
(transfusion to obtain hemoglobin ≥ 9 mg/dL within 24 hours prior to dosing is allowed).  
2.1.1.10 Patients must be able to swallow oral medications (capsules) without chewing, breaking, crushing, opening or otherwise altering the product formulation.  
2.1.1.11 The effects of *ONC201* on the developing human fetus are unknown. For this reason and because *imipridone* agents are known to be teratogenic, female patients must either be of non-reproductive potential (i.e., post-menopausal by history: ≥60 years old and no menses for ≥1 year without an alternative medical cause; OR history of hysterectomy, OR history of bilateral tubal ligation, OR history of bilateral oophorectomy) or must have a negative serum pregnancy test upon study entry and agree to use contraception or abstinence during the study and for and for at least 4 weeks after the final dose of any study-related medications. Male patients must use at least two forms of contraception during the study and for at least 4 weeks after the final dose of any study-related medications or have a partner who is not of reproductive potential. (See Section 4.2)
2.1.1.12 Ability of subject to understand and the willingness to sign a written informed consent document.  
2.1.2 **Exclusion Criteria** 2.1.2.1 Patients who have received chemotherapy in the previous 3 weeks (6 weeks for nitrosoureas or mitomycin); other investigational agents within 3 weeks or a PD1/PDL1 agent within 4 weeks prior to first dose of study treatment.  
2.1.2.2 Patients who have undergone radiotherapy within 4 weeks of first dose of study treatment.  
2.1.2.3 Patients with a history of another invasive malignancy within the last 3 years. 2.1.2.4 Patients with symptomatic brain metastases or leptomeningeal involvement. Patients with asymptomatic or brain metastases that have been treated with radiation at least 4 weeks prior to first dose of study treatment are allowed.  
2.1.2.5 History of allergic reactions attributed to compounds of similar chemical or biologic composition to *imipridones* or other agents used in study.  
2.1.2.6 Patients with a mean QTcF interval of > 500 msec or receiving therapeutic agents known to prolong the QT interval 2.1.2.7 Known history of cardiac arrhythmias including uncontrolled atrial fibrillation, tachyarrhythmias or bradycardia, history of congestive heart failure, or myocardial infarction or stroke in the previous 3 months will be excluded.  
2.1.2.8 Known history of gastrointestinal illnesses that would preclude the absorption of ONC201, which is an oral agent 2.1.2.9 Patients with bone metastases who have initiated denosumab or bisphosphonate therapy within 28 days prior to Cycle 1 Day 1.  
2.1.2.10 Pregnant women are excluded from this study because ONC201 has the potential for teratogenic or abortifacient effects. Because there is an unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with ONC201, breastfeeding should be discontinued if the mother is treated with ONC201. These potential risks may also apply to other agents used in this study.  
2.1.2.11 HIV-positive patients on combination antiretroviral therapy are ineligible because of the potential for pharmacokinetic interactions with ONC201.  
2.1.2.12 Patients who have known active Hepatitis B, or Hepatitis C infections. 2.2 ELIGIBILITY CRITERIA FOR COHORT 2: TRIPLE NEGATIVE BREAST CANCER
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2.2.1 inclusion criteria

Patients must have histologically or cytologically confirmed persistent or recurrent invasive, metastatic triple negative breast cancer (TNBC) for which standard curative measures do not exist or are no longer effective. TNBC, defined as ER negative (ER < 10%), PR negative (PR <10%). HER2 will be considered negative per ASCO-CAP guidelines (HER2 test result as negative if a single test (or both tests) performed show: 1) IHC 1+ as defined by incomplete membrane staining that is faint/barely perceptible and within >10% of the invasive tumor cells; 2) IHC 0 as defined by no staining observed or membrane staining that is incomplete and is faint/barely perceptible and within ≤10% of the invasive tumor cells; or 3) ISH negative based on: a) Single-probe average HER2 copy number <4.0 signals/cell or b) Dual-probe HER2/CEP17 ratio <2.0 with an average HER2 copy number <4.0 signals/cell) and HER2 testing must have been performed in a laboratory accredited by the College of American Pathologists (CAP) or another accrediting entity. Patients must have received at least one line of prior chemotherapy in the metastatic setting.  
2.2.1.1 Patients must have measurable disease, per RECIST 1.1. See Section **6.3.1** for the evaluation of measurable disease.  
2.2.1.2 Patients must have at least one lesion deemed safe to biopsy and be willing to undergo mandatory biopsies.  
2.2.1.3 Eligible patients may or may not have received prior chemotherapy and there is no limit to the number of prior chemotherapy. Patients are also eligible if they have received treatment with immunotherapy, such PD-1 inhibitors, PD-L1 inhibitors or CTLA4 inhibitors.  
2.2.1.4 Age >18 years. 2.2.1.5 ECOG performance status 0 or 1 2.2.1.6 Adequate renal function, defined as serum creatinine ≤ 1.5 X upper limit of normal
(ULN), or measured creatinine clearance ≥ 60 mL/min/1.  
2.2.1.7 Adequate hepatic function, defined as AST and ALT levels ≤ 3 X ULN and total bilirubin < 1.5 X ULN, unless known diagnosis of Gilbert's syndrome, where bilirubin ≤ 5 mg/dL will be permitted. Gilbert's syndrome will be defined as elevated unconjugated bilirubin, with conjugated (direct) bilirubin within the normal range and less than 20% of the total. Total bilirubin will be permitted up to 5 mg/dL, if patients have historical readings consistent with the definition of Gilbert's syndrome prior to entering study.  
2.2.1.8 Adequate bone marrow function, defined as absolute neutrophil (ANC) ≥ 1,500/mm3
(≥1.5 X106/L), platelet count ≥ 75,000/mm3 (≥ 75 X106/L), and hemoglobin ≥ 9 mg/dL
(transfusion to obtain hemoglobin ≥ 9 mg/dL within 24 hours prior to dosing is allowed).  
2.2.1.9 Patients must be able to swallow oral medications (capsules) without chewing, breaking, crushing, opening or otherwise altering the product formulation.  
2.2.1.10 The effects of *ONC201* on the developing human fetus are unknown. For this reason and because *imipridone* agents as well as other therapeutic agents used in this trial are known to be teratogenic. Female patients must either be of non-reproductive potential (i.e., post-menopausal by history: ≥60 years old and no menses for ≥1 year without an alternative medical cause; OR history of hysterectomy, OR history of bilateral tubal ligation, OR history of bilateral oophorectomy) or must have a negative serum pregnancy test upon study entry and agree to use contraception or abstinence during the study and for and for at least 4 weeks after the final dose of any study-related medications. Male patients must use at least two forms of contraception during the study and for and for at least 4 weeks after the final dose of any study-related medications or have a partner who is not of reproductive potential. (See Section 4.2).  
2.2.1.11 Ability of subject to understand and the willingness to sign a written informed consent document.  
2.2.2 **Exclusion Criteria** 2.2.2.1 Patients who have received chemotherapy in the previous 3 weeks (6 weeks for nitrosoureas or mitomycin); other investigational agents within 3 weeks or a PD1/PDL1 agent within 4 weeks prior to study enrollment.  
2.2.2.2 Patients who have undergone radiotherapy within 4 weeks of first dose of study treatment.  
2.2.2.3 Patients with a history of another invasive malignancy within the last 3 years. 2.2.2.4 Patients with symptomatic brain metastases or leptomeningeal involvement. Patients with asymptomatic or brain metastases that have been treated with radiation at least 4 weeks prior to first dose of study treatment are allowed.  
2.2.2.5 History of allergic reactions attributed to compounds of similar chemical or biologic composition to *imipridones* or other agents used in study.  
2.2.2.6 Patients with a mean QTcF interval of > 500 msec or receiving therapeutic agents known to prolong the QT interval 2.2.2.7 Known history of cardiac arrhythmias including uncontrolled atrial fibrillation, tachyarrhythmias or bradycardia, history of congestive heart failure, or myocardial infarction or stroke in the previous 3 months will be excluded.  
2.2.2.8 Known history of gastrointestinal illnesses that would preclude the absorption of ONC201, which is an oral agent 2.2.2.9 Patients with bone metastases who have initiated denosumab or bisphosphonate therapy within 28 days prior to Cycle 1 Day 1.  
2.2.2.10 Pregnant women are excluded from this study because ONC201 has the potential for teratogenic or abortifacient effects. Because there is an unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with ONC201, breastfeeding should be discontinued if the mother is treated with ONC201. These potential risks may also apply to other agents used in this study.  
2.2.2.11 HIV-positive patients on combination antiretroviral therapy are ineligible because of the potential for pharmacokinetic interactions with ONC201.  
2.2.2.12 Patients who have known active Hepatitis B, or Hepatitis C infections. 2.3 ELIGIBILITY CRITERIA FOR COHORT 3: ENDOMETRIAL CANCER 2.3.1 **Inclusion Criteria** 2.3.1.1 Patients must have histologically or cytologically confirmed persistent or recurrent advanced or metastatic invasive endometrial cancer (EC) for which standard curative measures do not exist or are no longer effective.  
2.3.1.2 Patients must have measurable disease, per RECIST 1.1.. See Section **6.3.1** for the evaluation of measurable disease.  
2.3.1.3 Patients must have at least one lesion deemed safe to biopsy and be willing to undergo mandatory biopsies.  
2.3.1.4 Women with endometrial cancer must have had at least one prior line of therapy in the metastatic/recurrent setting but there is no limit to the number of prior chemotherapy lines. Patients are eligible if they have received treatment with immunotherapy, such PD-1 inhibitors, PD-L1 inhibitors or CTLA4 inhibitors.  
2.3.1.5 Age >18 years. 2.3.1.6 ECOG performance status 0 or 1 2.3.1.7 Adequate renal function, defined as serum creatinine ≤ 1.5 X upper limit of normal
(ULN), or measured creatinine clearance ≥ 60 mL/min/1.  
2.3.1.8 Adequate hepatic function, defined as AST and ALT levels ≤ 3 X ULN and total bilirubin < 1.5 X ULN, unless known diagnosis of Gilbert's syndrome, where bilirubin ≤ 5 mg/dl will be permitted. Gilbert's syndrome will be defined as elevated unconjugated bilirubin, with conjugated (direct) bilirubin within the normal range and less than 20% of the total. Total bilirubin will be permitted up to 5 mg/dL, if patients have historical readings consistent with the definition of Gilbert's syndrome prior to entering study.  
2.3.1.9 Adequate bone marrow function, defined as absolute neutrophil (ANC) ≥ 1,500/mm3
(≥1.5 X106/L), platelet count ≥ 75,000/mm3(≥ 75 X106/L), and hemoglobin ≥ 9 mg/dL
(transfusion to obtain hemoglobin ≥ 9 mg/dL within 24 hours prior to dosing is allowed).  
2.3.1.10 Patients must be able to swallow oral medications (capsules) without chewing, breaking, crushing, opening or otherwise altering the product formulation.  
2.3.1.11 The effects of *ONC201* on the developing human fetus are unknown. For this reason and because *imipridone* agents as well as other therapeutic agents used in this trial are known to be teratogenic. Female patients must either be of non-reproductive potential (i.e., post-menopausal by history: ≥60 years old and no menses for ≥1 year without an alternative medical cause; OR history of hysterectomy, OR history of bilateral tubal ligation, OR history of bilateral oophorectomy) or must have a negative serum pregnancy test upon study entry and agree to use contraception or abstinence during the study and for and for at least 4 weeks after the final dose of any study-related medications. Male patients must use at least two forms of contraception during the study and for and for at least 4 weeks after the final dose of any study-related medications or have a partner who is not of reproductive potential. (See Section 4.2).  
2.3.1.12 Ability of subject to understand and the willingness to sign a written informed consent document.  
2.3.2 **Exclusion Criteria** 2.3.2.1 Patients who have received chemotherapy in the previous 3 weeks (6 weeks for nitrosoureas or mitomycin); other investigational agents within 3 weeks or a PD1/PDL1 agent within 4 weeks prior to study enrollment.  
2.3.2.2 Patients who have undergone radiotherapy within 4 weeks of first dose of study treatment.  
2.3.2.3 Patients with a history of another invasive malignancy within the last 3 years. 2.3.2.4 Patients with symptomatic brain metastases or leptomeningeal involvement. Patients with asymptomatic or brain metastases that have been treated with radiation at least 4 weeks prior to first dose of study treatment are allowed.  
2.3.2.5 History of allergic reactions attributed to compounds of similar chemical or biologic composition to *imipridones* or other agents used in study.  
2.3.2.6 Patients with a mean QTc interval of > 500 msec or receiving therapeutic agents known to prolong the QT interval 2.3.2.7 Known history of cardiac arrhythmias including uncontrolled atrial fibrillation, tachyarrhythmias or bradycardia, history of congestive heart failure, or myocardial infarction or stroke in the previous 3 months will be excluded.  
2.3.2.8 Known history of gastrointestinal illnesses that would preclude the absorption of ONC201, which is an oral agent 2.3.2.9 Patients with bone metastases who have initiated denosumab or bisphosphonate therapy within 28 days prior to Cycle 1 Day 1.  
2.3.2.10 Pregnant women are excluded from this study because ONC201 has the potential for teratogenic or abortifacient effects. Because there is an unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with ONC201, breastfeeding should be discontinued if the mother is treated with ONC201. These potential risks may also apply to other agents used in this study.  
2.3.2.11 HIV-positive patients on combination antiretroviral therapy are ineligible because of the potential for pharmacokinetic interactions with ONC201.  
2.3.2.12 Patients who have known active Hepatitis B, or Hepatitis C infections.
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2.4 recruitment strategies

This protocol may be abstracted into a plain language announcement posted on NIH websites (e.g. clinicaltrials.gov) and on NIH social media platforms. Participants may also be identified through referrals from physicians, or from populations in NIH Clinics.
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2.5 screening evaluation 2.5.1 screening

Minimal risk activities that may be performed before the subject has signed a consent include the following:
- Email, written, in person or telephone communications with prospective subjects regarding their medical issues and possible eligibility to the clinical trial
- Review of existing medical records to include H&P, laboratory studies, etc. - Review of existing MRI, x-ray, or CT images - Review of existing photographs or videos - Review of existing pathology specimens/reports from a specimen obtained for diagnostic purposes A waiver of consent for these activities has been requested in section **12.6.1**.  
The following activities will be performed only after the subject has signed the study consent OR the consent for study 01-C-0129 (provided the procedure is permitted on that study) on which screening activities may also be performed. Assessments performed at outside facilities or on another NIH protocol within the timeframes below may also be used to determine eligibility once a patient has signed the consent. The screening evaluations or tests will be conducted as described below and in the Study Calendar (Section 3.4) after the participant has signed the consent document.  
For baseline evaluations, please see section 2.9.  
Confirmation of tumor histology in NCI Laboratory of Pathology (archival tumor samples will be requested; if unavailable, the subject may choose to undergo fresh biopsy). Pathological confirmation of diagnosis of breast cancer or endometrial cancer in the Laboratory of Pathology at NIH Clinical Center or Walter Reed National Military Medical Center at Bethesda. However, if no pathologic specimen is available, patients may enroll with a pathologist's report showing a histologic diagnosis of HR+BC, TNBC or Endometrial Cancer in a College of American Pathologists (CAP) accredited laboratory and a clinical course consistent with the disease. Medical history and physical examination, including vitals signs and concomitant medications, will be conducted at screening and at subsequent visits as indicated in the schedule of assessments. Results of the physical examination including any abnormalities will be documented and can be used for the baseline examination if conducted within 17 days prior to enrolling on trial. Abnormal findings will be reassessed at subsequent visits. On subsequent visits, any newly diagnosed or worsening conditions, signs and symptoms, whether related or unrelated to the trial, will be reported as adverse events.
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2.5.2.3 performance status

The ECOG performance status (Appendix A) will be assessed at screening and at subsequent visits as indicated in the schedule of assessments and documented in the clinical record. 2.5.2.4 Scans for disease evaluation at screening.  
- CT of chest/abdomen /pelvis (MRI may be substituted at investigator's discretion)
- Bone scan may be done at the investigator's discretion is there is concern for bone disease
- Brain MRI (preferred) or head CT with contrast may be done at the investigator's discretion if there is concern for CNS metastasis If the screening scan has been obtained within 17 days prior to first dose of study treatment , it may be used for baseline evaluation.
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2.5.2.5 laboratory studies

Screening laboratory studies to include:
- CBC with differential, PT/INR/PTT, Biochemical profile for Screening: 14 comprehensive metabolic panel (sodium, potassium, chloride, carbon dioxide, BUN, creatinine or measured creatinine clearance, glucose, AST (SGOT), ALT (SGPT), total bilirubin, serum creatinine. If the labs have been obtained within 10 days prior to initiation of therapy, they may be used for baseline evaluation.  
- Anti-HIV, anti-HCV, Hepatitis B surface Ag: viral titers will be obtained within 28 days prior to enrollment.
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2.5.2.6 pregnancy testing

For female subjects of childbearing potential, urine or serum HCG will be performed on initial screening.  
- A urine beta-HCG will be performed before each administration of ONC201 during the treatment phase, at the end-of-treatment visit, and at the post-treatment follow-up visit.  
- Patients who are postmenopausal (age-related amenorrhea for 12 or more consecutive months, or documented FSH > 40 mIU/mL) / Estradiol < 20mIU/mL), or who had undergone hysterectomy or bilateral oophorectomy are exempt from pregnancy testing.
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2.5.2.7 tumor block

A primary tumor paraffin block will be requested to perform comparative genomic analysis as part of correlative studies planned for this clinical trial. 2.5.2.8 Tumor Biopsy A biopsy of fresh frozen tissue will be planned after consent but prior to the first dose of ONC201. 2.5.2.9 Research Blood Samples Peripheral blood samples for the correlative studies will be collected after consent but prior to the first dose of ONC201, per Section 5 Biospecimen Collection.  
2.6 PARTICIPANT REGISTRATION AND STATUS UPDATE PROCEDURES Registration and status updates (e.g., when a participant is taken off protocol therapy and when a participant is taken off-study) will take place per CCR SOP ADCR-2, CCR Participant Registration & Status Updates found **here**.
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2.7 screen failures

Screen failures are defined as participants who consent to participate in the clinical trial but are not subsequently assigned to the study intervention or entered in the study. A minimal set of screen failure information is required to ensure transparent reporting of screen failure participants, to meet the Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to respond to queries from regulatory authorities. Minimal information includes demography, screen failure details, eligibility criteria, and any serious adverse event (SAE). 2.8 TREATMENT ASSIGNMENT AND RANDOMIZATION/STRATIFICATION PROCEDURES
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cohorts

| Number   | Name               | Description                                     |
|----------|--------------------|-------------------------------------------------|
| 1        | HR+ breast cancer  | HR+ breast cancer (male and female)             |
| 2        | TNBC               | Triple negative breast cancer (male and female) |
| 3        | Endometrial cancer | Endometrial cancer (female only)                |
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arms

| Number   | Name   | Description                                                                                                                                                     |
|----------|--------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 1        | ONC201 | All patients from all 3 cohorts will receive ONC201 at the  recommended phase 2 dose (RP2D) of 625mg by mouth every 7  days with each cycle being 28 days long. |
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randomization/stratification procedures

Randomization and stratification procedures are not applicable to this study.  
- All patients from Cohort 1 will be assigned to Arm 1.  
- All patients from Cohort 2 will be assigned to Arm 1. - All patients from Cohort 3 will be assigned to Arm 1.
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2.9 baseline evaluation

Scans and x-rays must be done within 17 days prior to the initiation of study therapy. All other studies should be performed within 10 days prior to initiation of study therapy, unless otherwise specified. If the tests were performed within these timeframes at screening, they do not need to be repeated at start of treatment.  
- Start the process for documentation of germline BRCA mutation status (Cohort 1 and 2).  
- Start the process for documentation of Lynch syndrome (germline evaluation) or MSI
status of tumor (somatic evaluation by genomics, IHC or demonstration of Microsatellite instability of the tumor). (Cohort 3 only)
- History and physical examination with ECOG evaluation (Appendix A), history of prior therapy, review of concurrent medications, and review of baseline symptoms.  
- Assessment of disease by imaging (may include CT chest, abdomen and pelvis or MRI)
- EKG obtained within 7 days of initiating therapy - Laboratory evaluations o CBC with differential and platelet count o Sodium, potassium, chloride, CO2, creatinine or measured creatinine clearance, glucose, BUN
o Mineral panel (magnesium, calcium, albumin, phosphorus), lactic acid and a lipid panel o Hepatic panel (alkaline phosphatase, AST/ALT/total bilirubin) with total protein o PT/INR/PTT o β-HCG in women of childbearing potential o Hormone panel: Prolactin, Estrogen o Lymphocyte phenotyping - TBNK
o Tumor Markers: Breast cancer (cohorts 1 and 2) - CEA, CA15-3, CA 27.29; Endometrial cancer (cohort 3) - CA-125 o Urinalysis o Germline mutation analysis of BRCA or Lynch Syndrome (germline evaluation)
or MSI status of tumor (somatic evaluation by genomics, IHC or demonstration of Microsatellite instability of the tumor) (if not previously documented)
- Correlative Tests (Refer to the tables in section 5.5 for additional information)
o Tumor biopsy o Blood sampling
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3 study implementation 3.1 study

This is an open label, phase II single arm study of ONC201 divided in three cohorts, each cohort with a different type of metastatic, advanced disease: ER+ BC, TNBC and endometrial cancer. All proposed cohorts require measurable disease based on RECIST criteria and disease that can be biopsied safely. The study will specify a required biopsy prior to treatment and a biopsy during the second cycle after drug treatment. There will also be an optional biopsy at the time of progression. Participants will receive ONC201 starting at the RP2D of 625mg q7 days. Each cycle is 28 days long. ONC201 can be given +/- 1 day due to holidays, inclement weather, conflicts, or similar reasons. During Cycle 1, patients will receive weekly phone calls by research staff to ask about changes to health and any potential side effects. While the drug has activity against HER2 amplified breast cancer cells in vitro, we would not initiate a single agent HER2+ cohort at this time because of the clear benefit to HER2 targeted therapies in this subset of breast cancer. We will develop preclinical data for ONC201 in combination with HER2 targeted drugs (*e.g.*, trastuzumab, trastuzumab emtansine, and lapatinib) and plan to initiate a phase I run-in trial at a later date.
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3.2 drug administration

At the start of each cycle, enough study drug for one complete cycle will be dispensed by the pharmacy. Patients will be given a medication diary (see **Appendix E**) to record dates of administration as well as side effects. Patients in all cohorts will self-administer five capsules (each capsule 125mg x 5 = 625mg) of ONC201 by mouth every 7 days with each cycle being 28 days in duration. The capsules may be taken one hour before or two hours after eating, according to patient preference. If patients are unable to tolerate this dose, they can have three dose reductions before being removed from the study (see **Table 2**). Patients will receive ONC201 as long as they derive clinical benefit or toxicity becomes impeditive.
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3.2.1 self-administered study drugs

The ONC201 used in this study is a self-administered investigational agent. Such agents are dispensed from the pharmacy to a participant or to a Patient Care Unit for self-medication and a record of the dispensed investigational agent is generated and kept by the dispensing pharmacy. As indicated above, patients will be asked to keep a medication diary and bring it with them on each study visit (See **Appendix E**). Patients will also bring any remaining pills to each study visit. The research nurse will review and validate the completeness and accuracy of the participant's diary with the participant. If a participant goes off study while at home, the research nurse will ensure and document the return of the unused oral investigational agents from the participant. Unused investigational agent will be destroyed per dispensing pharmacy procedure.  
3.3 DOSE MODIFICATIONS/DELAYS AND MANAGEMENT OF TOXICITIES
- In the case of toxicity, appropriate medical treatment should be used (including anti-emetics, anti-diarrheals, etc.).  
- Once a patient has a dose reduction for toxicity, the dose will not be increased.  
- Participants continuing to experience toxicity at the off treatment visit will be contacted for additional assessments until the toxicity has resolved or is deemed irreversible. Patients must remain on the study to have additional assessments completed.  
- For AEs that are unrelated to the study drugs, study drug may be held for up to 14 days at the discretion of the PI.
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3.3.1 modifications and toxicity management

| Dose Level        | ONC201             | Number of Capsules*   |
|-------------------|--------------------|-----------------------|
| (DL)              | (by mouth)         |                       |
| 1 (Starting Dose) | 625mg every 7 days | 5 capsules            |
| -1                | 500mg every 7 days | 4 capsules            |
| -2                | 375mg every 7 days | 3 capsules            |
| -3                | 250mg every 7 days | 2 capsules            |  
* Each ONC201 capsule is 125mg Summary of Dose Modifications or Discontinuation for ONC201-Related Adverse Events Worst Treatment-Related Non-Hematologic AE (except for controlled nausea and vomiting)
During the Previous Cycles  
| Grade   | Dose modification                                                  |
|---------|--------------------------------------------------------------------|
| 0-2     | No dose modifications for non-hematologic AEs.                     |
| 3       | Reduce by one dose level (except controlled nausea, and vomiting). |
| 4       | Stop (except controlled nausea and vomiting).                      |
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worst treatment-related hematologic ae during

Worst AE Platelets  
| ≥75 x 109 /L       | 50 - 75 x 109 /L   | < 50 x 109 /L   |                      |               |
|--------------------|--------------------|-----------------|----------------------|---------------|
| ≥ 1.5 x 109 /L     | Dose unchanged     | Dose unchanged  | Reduce 1 dose  level |               |
| ≥1 to 1.5 x 109 /L | Dose unchanged     | Dose unchanged  | Reduce 1 dose  level |               |
| ANC                | < 1 x 109 /L       | Reduce 1 dose   | Reduce 1 dose        | Reduce 1 dose |
| level              | level              | level           |                      |               |  
Dose reductions: If any treatment related non-hematologic AE observed was grade > 2 (except controlled nausea and vomiting) and/or if platelets < 50 x 109/L and/or ANC < 1 x 109/L, then the dose should be reduced by one dose level. Patients who require more than three dose reductions will have treatment stopped. If any treatment-related non-hematologic AE observed was grade 4 (except controlled nausea and vomiting) then ONC201 treatment should be stopped. Any dose reductions of ONC201 will be determined according to: (1) non-hematologic AE during the preceding treatment cycle, as well as (2) the lowest ANC and platelets observed. No dose escalation should be attempted. Important: If the dose was reduced or delayed for AEs, there will be no dose re-escalation in subsequent treatment cycles.
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3.3.2 medication delay

| Hematologic AE on Day 1 of Each Cycle  AE              | Delay Delay up to 4 weeks until all resolved. If not  resolved after 4 weeks, then remove from study  regimen.   |
|--------------------------------------------------------|------------------------------------------------------------------------------------------------------------------|
| ANC< 1.0 x 109 /L and/or  Platelet count < 75 x 109 /L |                                                                                                                  |  
Non-Hematologic AE (except for controlled nausea, and vomiting) on Day 1 of Each Cycle (within the prior 72 hours)  
| Grade   | Delay   |
|---------|---------|  
| Delay by 1 week up to 4 consecutive weeks until   |                                                                                              |
|---------------------------------------------------|----------------------------------------------------------------------------------------------|
| 2-4                                               | all resolved (to grade ≤ 1). If not resolved after 4  weeks, then remove from study regimen. |  
On day 1 of each cycle, patients must have adequate hematopoietic function (i.e., ANC ≥ 1.0 x 109/L, platelet count ≥ 75 x 109/L) and all grade 2, 3 or 4 non-hematologic AEs (except for controlled nausea, and vomiting) must have resolved to grade ≤ 1. If AEs persists, treatment should be delayed by 1 week for up to 4 consecutive weeks. If, after 4 weeks of delay, all treatment related AEs have still not resolved (to grade ≤ 1): then any further treatment with ONC201 should be stopped.
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3.3.3 instructions on missed doses

ONC201 can be given +/- **1 day** due to holidays, inclement weather, conflicts, or similar reasons.  
If the first dose of the 4-week cycle not taken within 1 day of day 1 of the cycle, the cycle will be delayed one week. If a mid-cycle dose (i.e., D8, D15, D22) is more than 1 day late, the weekly dose will be skipped and the next dose will be resumed in accordance with the planned administration day.  
| 3.4                                            | STUDY CALENDAR   |          |          |                                   |                                    |        |
|------------------------------------------------|------------------|----------|----------|-----------------------------------|------------------------------------|--------|
| Procedure                                      | Screening/       | Cycle 1  | Cycle 2  | Subsequent  Odd  numbered  cycles | Subsequent  Even  numbered  cycles | End of |
| treatment  visit (~ 30  days  from last  dose) |                  |          |          |                                   |                                    |        |
| Baseline                                       | Day 1            | Day 1    | Day 1    | Day 1                             |                                    |        |
| (±3days)                                       | (±3days)         | (±3days) | (±3days) |                                   |                                    |        |
| Informed consent                               | X                |          |          |                                   |                                    |        |
| History and PE2                                | X                | X        | X        | X                                 | X                                  | X      |
| Vital signs                                    | X                | X        | X        | X                                 | X                                  | X      |
| Performance Score                              | X                | X        | X        | X                                 | X                                  | X      |
| Labs                                           | X3               | X4 (< 10 | X4       | X4                                | X4                                 | X4     |
| days)                                          |                  |          |          |                                   |                                    |        |
| Pathology                                      | X                |          |          |                                   |                                    |        |
| confirmation5 Tumor block                      | X6               |          |          |                                   |                                    |        |
| Fresh frozen tissue                            | X                | X        | X        |                                   |                                    |        |
| (tumor biopsy)7                                | (optional)       |          |          |                                   |                                    |        |
| Correlative  Research Studies  (blood samples) | X                | X        | X        | X                                 | X                                  | X      |
| PK/PD                                          | X                |          |          |                                   |                                    |        |
| NIH Advance                                    | X                |          |          |                                   |                                    |        |
| Directives Form                                | (optional)       |          |          |                                   |                                    |        |
| Radiological                                   | X (< 17          | X        |          |                                   |                                    |        |
| Assessments8                                   | days)            |          |          |                                   |                                    |        |
| Response                                       | X                |          |          |                                   |                                    |        |
| Evaluation                                     |                  |          |          |                                   |                                    |        |  
| Procedure                                      | Screening/   | Cycle 1   | Cycle 2   | Subsequent  Odd  numbered  cycles   | Subsequent  Even  numbered  cycles   | End of   |
|------------------------------------------------|--------------|-----------|-----------|-------------------------------------|--------------------------------------|----------|
| treatment  visit (~ 30  days  from last  dose) |              |           |           |                                     |                                      |          |
| Baseline                                       | Day 1        | Day 1     | Day 1     | Day 1                               |                                      |          |
| (±3days)                                       | (±3days)     | (±3days)  | (±3days)  |                                     |                                      |          |
| Adverse Events                                 | X            | X         | X         | X                                   | X                                    |          |
| Concomitant                                    | X            | X         | X         | X                                   | X                                    | X        |
| Medications Electrocardiogram9                 | X1           | X         |           |                                     |                                      |          |  
1 Up to 7 days before enrolling the trial 2 History including prior treatments and review of baseline adverse events and concurrent medications. Physical examination includes neurological exam. Height (at screening only) and weight are also included.  
3 CBC with differential, PT/INR/PTT, AST (SGOT), ALT (SGPT), total protein, total bilirubin, serum creatinine.Urine or serum HCG on day of screening for women of childbearing potential. Anti-HIV, anti-HCV, Hepatitis B Surface Ag (within 28 days prior to first dose of study treatment).  
4 Labs must be obtained <10 days of initiating treatment: CBC with differential, PT/INR/PTT,
sodium, potassium, chloride, carbon dioxide, BUN, creatinine or measured creatinine clearance, glucose, AST, ALT, bilirubin, calcium, total protein, albumin, alkaline phosphatase, phosphorus, magnesium, urinalysis, prolactin, estrogen, lactic acid and lipid panel. Lymphocyte phenotyping (TBNK). Urine or serum HCG for women of childbearing potential. Tumor Markers (CEA, CA15-3, CA27.29 for breast cancer and CA125 for endometrial cancer). Evaluation of germline BRCA1/2 (cohorts 1 and 2) or Lynch syndrome (germline evaluation) or MSI status of tumor (somatic evaluation by genomics, IHC or demonstration of Microsatellite instability of the tumor) (cohort 3) if no documentation of mutational analysis provided.  
5 Pathological confirmation of diagnosis (plus ER, PR and HER2 expression for breast cancer patients) in the Laboratory of Pathology at NIH Clinical Center or Walter Reed National Military Medical Center at Bethesda. However, if no pathologic specimen is available, patients may enroll with a pathologist's report showing a histologic diagnosis of HR+BC, TNBC or Endometrial Cancer in a College of American Pathologists (CAP) accredited laboratory and a clinical course consistent with the disease.  
6 Primary tumor paraffin block will be requested at enrollment to perform comparative genomic analysis as part of correlative studies planned for this clinical trial.  
7 Planned biopsy after consent but prior to first dose of ONC201 as well as a planned biopsy on C2D2 after the 5th dose of ONC201. There is also an optional biopsy at the time of progression. 8Baseline images within 17 days prior to initiation of therapy: CT of chest/abdomen /pelvis (MRI
may be substituted at investigator's discretion), Bone scan only at baseline. Brain MRI may be ordered at the discretion of the investigator if there is concern for CNS metastasis. CT of chest/abdomen/pelvis to be repeated every 8 weeks +/- 4 days after start of treatment (odd numbered cycles). Additional imaging to be performed at the investigator's discretion.  
9 12-lead EKG within 7 days prior to initiation of therapy and on C2D1, with monthly repeats only for abnormal baseline EKGs or if patient is symptomatic. Note: Allowance for scheduling changes: Brief interruption and delay in the 28-day cycle may occasionally be required due to travel delays, airport closure, inclement weather, family responsibilities, security alerts and government holidays, etc., for scheduling convenience. This can also extend to complications of disease not attributable to disease progression or protocol therapy. These delays will not be considered protocol deviation. 3.5 CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA Prior to removal from study, effort must be made to have all subjects complete a safety visit approximately 30 days following the last dose of study therapy.
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3.5.1 criteria for removal from

- Progressive disease - Participant requests to be withdrawn from active therapy
- Unacceptable Toxicity as defined in section 3.3.  
- Investigator discretion
- Positive pregnancy test
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3.5.2 off-study criteria

- Screen Failure - Completed study follow-up period
- Study closure
- Participant requests to be withdrawn from study
- Death
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4 concomitant medications/measures

The investigator's brochure for ONC201 by Oncoceutics does not describe any known drug-drug interactions with ONC201. In vitro cytochrome P450 assays were conducted in human hepatocytes. In these studies, ONC201 is not an inducer of the CYP450 system (CYP1A2, 2B6, and 3A4). ONC201 was observed to be a mild inhibitor of the CYP450 enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4) at 35-429 uM, i.e., at least 3.5-fold above the Cmax observed in the first-in-human trial. Drugs that antagonize DRD2 are commonly used as antipsychotics. However, comparison between ONC201 and other typical antipsychotics (such as haloperidol and risperidone) reveal substantial differences in anti-tumor activity, therapeutic window, specificity and receptor pharmacology. Moreover, the current administration schedule of ONC201 dosing is atypical for antipsychotic drugs. Taken together, there is no clear expectation that side effects that are commonly observed with antipsychotic drugs will be observed with ONC201 in the current schedule. Nevertheless, it is possible that side effects associated with antipsychotics could be observed with ONC201including weight gain, diabetes, metabolic disorders, dystonia, akinesia, akathisia, tremors, muscle rigidity, insomnia, blurry vision, Parkinsonian symptoms, cognitive impairment, sexual dysfunction, osteoporosis, sedation, behavioral changes, and cardiac disorders.
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4.1 restrictions

As a general precaution, the use of metformin and similars are prohibited due to potential similarities in mechanisms and mitochondrial damage. Patients will also be prohibited from using herbs and magic potions.
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breast feeding

It is not known whether ONC201 is excreted in breast milk. It is recommended that women do not breastfeed during treatment with ONC201 and for at least 2 months after the last dose.
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blood donation

Patients should not donate blood while participating in this study or for 90 days following the dose of ONC201. 4.2 CONTRACEPTION The effects of ONC201 on the developing human fetus are unknown. For this reason, women of childbearing potential and men must agree to use two methods of adequate contraception (see Table 3) prior to study entry, for the duration of study participation and for at least 4 weeks after the final dose of any study-related medications. Cessation of birth control after this point should be discussed with a responsible physician. Periodic abstinence, the rhythm method and the withdrawal method are not acceptable methods of birth control. Table 3: Effective methods of contraception (two methods must be used)  
| Barrier Methods                                             | Intrauterine Device Methods                                   | Hormonal Methods             |
|-------------------------------------------------------------|---------------------------------------------------------------|------------------------------|
| Male condom plus spermicide                                 | Copper T                                                      | Implants                     |
| Cap plus spermicide                                         | Progesterone Ta                                               | Hormone shot or injection    |
| Diaphragm plus spermicide                                   | Levonorgestrel-releasing  intrauterine system  (e.g., Mirena) | Combined pill Minipill Patch |
| aThis is also considered a hormonal method of birth control |                                                               |                              |
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5 biospecimen collection

5.1 CORRELATIVE STUDIES FOR RESEARCH/PHARMACOKINETIC STUDIES
The primary goal of the correlative studies is to identify potential predictive biomarkers as well as additional evidence of the mechanism of action of and resistance to ONC201. Other correlative study aims include investigation of proof-of-concept biomarkers. These will be performed in an exploratory fashion on select patients if there are adequate samples for analysis.  
(Table 4; **Table 5**)
While the exact mechanism of ONC201 is unclear at this time, preclinical data as well as the phase I data have identified multiple potential mechanisms of action. As mentioned above, ONC201 is thought to have both TRAIL-dependent and TRAIL-independent mechanisms of killing. ONC201 has been identified as a selective antagonist of DRD2 that activates the integrated stress response in tumor cells leading to TRAIL activation. Serum prolactin will be evaluated as a surrogate marker for target engagement, which is induced by DRD2 antagonism in the pituitary gland (34). In the solid tumor phase I study, prolactin was found to increase in patients taking ONC201 and was loosely associated with response in the small trial. However, there is no evidence that DRD2 is present on tumors. In addition to serum prolactin levels, we will also evaluate select biopsy samples for the presence of DRD2. ONC201 has shown TRAIL-dependent apoptosis as well as caspase-independent, DR4/5independent, TRAIL-independent induction of the integrated stress response through inhibition of mitochondrial respiration. Due to these conflicting mechanisms, we will evaluate select tissue biopsy samples for markers of apoptosis including cleaved caspase-3, cleaved poly(ADP ribose) polymerase (PARP), cleaved terminal deoxynucleotidyl transferase dUPT nick end labeling (TUNEL), ATF4 expression, CHOP expression, phospho-AMPK levels, annexin-V, TRAIL receptor and TRAIL expression. We will also evaluate markers of apoptosis in the serum including TRAIL expression, M30 assays, and M65 assays. We also plan to evaluate serum cytokine levels including IL6, L24, IL32, IL1RAP, which were found to be increased after ONC201 administration on RNAseq. Like Metformin, ONC201 induces inhibition of mitochondrial respiration. Metformin induces mitochondrial stress which in turn initiates the integrated stress response through AFT4 which increases fibroblast growth factor 21 (FGF21). Due to similarities between these two drugs, we will evaluate the lipid profile and serum lactic levels due to known effects of Metformin on these labs (38). Recent data from the El-Deiry lab suggests that ONC201 increases NK cell numbers and that this may contribute to the anti-tumor activity.(39) If sufficient PBMCs are available, PBMCs will be processed and stored at Trepel Lab according to SOP (Table 5), and will be assessed using multiparameter flow cytometry for immune subsets including but not necessarily limited to Tregs, myeloid-derived suppressor cells, effector CD4+ and CD8+ T-cells, NK cells and monocyte subsets. Assessment will include functional markers, i.e. PD-1, Tim-3, CTLA-4, CD40, HLADR and PD-L1. This study will be done in collaboration with Jane Trepel of Developmental Therapeutics Branch/CCR/NCI. Peripheral blood will be collected at baseline and post-therapy to enumerate circulating tumor cell (CTC) levels at baseline and post-therapy, and analyze for correlation with clinical response or survival. CTCs will be assessed using ferrofluidic enrichment and multiparameter flow cytometric detection. CTCs will be identified as viable, nucleated cells, that positively express one or more epithelial or tumor markers and are negative for expression of hematopoietic markers. Cell free DNA (cfDNA) and mitochondrial DNA will also be evaluated. Finally, due to impressive preclinical data involving mitochondrial stress following by ballooning after exposure to ONC201, tumor biopsy samples will be evaluated by electron microscopy to evaluate the impact of ONC201 on mitochondrial and cellular morphology. All samples will be barcoded, with data entered and stored in Labmatrix, the patient sample data management system utilized by the NCI Clinical Pharmacology Core (CPC). This is a secure program, with access to the Labmatrix system limited to defined CPC personnel who are issued individual user accounts. The program creates a unique barcode ID for every sample and sample box, which cannot be traced back to patients without Labmatrix access. The data recorded for each sample includes the patient ID, name, trial name/protocol number, sample collection time, cycle time point, dose, material type, as well as box and freezer locations. Patient demographics associated with the clinical center patient number are provided in the system. Samples will be stored until requested by the PI. All requests are monitored and tracked in Labmatrix.  
| Table 4: Correlative Studies* PMBC Plasma/Whole Blood                                           | Archived Tissue       | Tumor and/or            |                         |
|-------------------------------------------------------------------------------------------------|-----------------------|-------------------------|-------------------------|
| Samples                                                                                         | Effusion Cells        |                         |                         |
| Immune phenotyping                                                                              | TRAIL levels          | Whole exome  sequencing | Whole exome  sequencing |
| Dendritic cell  maturation                                                                      | Serum M30 assay       | RNASeq                  | RNASeq                  |
| Serum M65 assay                                                                                 | Markers of  Apoptosis | Dopamine Receptor  D2   |                         |
| Cytokines                                                                                       |                       |                         |                         |
| Circulating Tumor  Cells                                                                        | TRAIL expression      | Electron Microscopy     |                         |
| TRAIL receptor  expression                                                                      |                       |                         |                         |
| Cell free DNA                                                                                   |                       |                         |                         |
| Cell free Mitochondrial  DNA                                                                    | Dopamine Receptor  D2 |                         |                         |
| * Correlative studies will include but are not limited to the studies listed above. Correlative |                       |                         |                         |  
* Correlative studies will include but are not limited to the studies listed above. Correlative studies will be performed if adequate samples are collected and on select patients.
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5.2 genomic alterations in breast

Patients with breast or endometrial cancer who are being evaluated for treatment may have tumor tissue obtained through the course of work-up, diagnosis or staging of their disease. This tissue may be submitted from outside (i.e. FFPE tissue) or obtained during tests at the Clinical Center. This tumor tissue will be submitted to Frederick for next-generation DNA sequencing using comprehensive cancer gene panel testing. This tumor sequencing is exploratory and will not be reported to the patient. Germline testing will be performed with the sole purpose to help identify the somatic mutations. As many as 20 patient tumor samples will be sent for molecular profiling per year of the protocol. No exploratory germline testing will be performed on this protocol. Patients with endometrial cancer and associated malignant peritoneal cytology that is obtained at the time of staging laparoscopy will have that peritoneal fluid submitted for clinical diagnostic cytopathologic analysis in the Laboratory of Pathology. In addition, any peritoneal or pleural fluid collected during enrollment on the study will be examined for malignant cells in the Laboratory of Pathology's Cytopathology section. 5.3 TUMOR BIOPSIES
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5.3.1 timing

Biopsies will be performed at the following times: - Mandatory - after consent, prior to treatment on cycle 1 day 1 - Mandatory - cycle 2 day 2 (< 24 hours of ONC201 dose)  
- Optional - at the time of progression
*Biopsies may be not performed on the specific dates and times due to the following reasons, including but not limited to, delayed recovery of hematologic toxicities, delayed clinic schedule, or national holidays.
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5.3.2 tissue sampling and handling

1. Attempts will be made to obtain up to four cores if safe and feasible. These tumor core biopsies will be obtained percutaneously or per vagina through interventional radiology (for endometrial cancer cohort) as long as considered minimal surgical risk. Two 3-millimeter punch biopsies of skin will be acceptable in lieu of 18-gauge core biopsies for patients with skin involvement. Inability to get tissue with a reasonable attempt will not preclude treatment and the patient will remain eligible for all other translational components.  
a. Core 1: Electron Microscopy b. Core 2: RNA later buffer c. Cores 3: FFPE d. Cores 4 and 5: OCT
2. The use of imaging to facilitate biopsies will be decided upon by members of the interventional radiology team. Should CT scans be needed for biopsy, a limit of 10 scans for each procedure will be observed to minimize radiation exposure to the patient.  
3. The schedule for the biopsies will be made with Special Procedures (Dr. Elliott Levy).  
Members of the WMB lab will be on call to receive and embed biopsies: ozakimk@nih.gov, Phone 410-522-5661, beeper 102-11155.
4. All cores will be stored, barcoded, in the WMB lab at -80oC on site until use according to our laboratory SOP. See **Table 5** for information about specimen collection and storage.
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5.4 blood samples

The following tests will be performed on selected patients' peripheral blood samples. If collected samples are not sufficient, the correlative studies will be performed in order of priority as detailed below. These correlative studies will be done in an exploratory fashion.
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5.4.1 timing

Research blood samples will be performed at the following times: - C1D1: after consent, prior to treatment on cycle 1 day 1  
- C1D1: 6 hours after ONC201 administration (blood for PK only) - On Day 1 of each cycle starting with cycle 2: Prior to treatment on day 1 - Optional - at the time of progression
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5.4.2 sampling and handling

5. We will collect no more than 40ml of research blood at each time point specified. If there is an issue obtaining blood from a participant, samples will be prioritized as follows:
a. 8ml of blood for immune assays (citrate blue/black tiger top) b. 10 ml of blood for circulating tumor cells, cell free DNA and cell free mitochondrial DNA (cell save preservative)
c. 10ml of blood for germline DNA analysis for correlative studies (Streck tube; C1D1 only)
d. 4ml of blood for cytokine analysis (lavender top tube)
e. 6ml of blood each for serum M30/M65 and Trail expression (red top tube) f. 6ml of blood for Pharmacokinetics (lavender top tube; C1D1 only but prior to drug ingestion and then 6 hours after)
6. Members of the WMB lab will be on call to receive patient samples: ozakimk@nih.gov, Phone 410-522-5661, beeper 102-11155.
7. See **Table 5** for information about specimen collection and storage.  
8. As soon as possible after the patient is scheduled please send an email notification to the Trepel Lab, Developmental Therapeutics Branch, NCI: Jane Trepel at trepel@helix.nih.gov; Sunmin Lee at leesun@mail.nih.gov; Min-Jung Lee at min-
jung.lee@nih.gov and Akira Yuno at akira.yuno@nih.gov that the sample is scheduled. After the samples are drawn please call the Trepel lab at 240-760-6330 to communicate that the sample is ready. Keep the sample on the unit at room temperature. The sample will be picked up by a lab member, entered in a secure patient database and processed for CTC assessment and immune subset analysis.
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5.5 table 5 specimen collection

| Tumor Biopsy*  Correlative                                                                                                                         | Collection                                                                                                                                                       | SOP for collection                                                                                                                                                                                                                                                                                                                                                                     | Contact                                                                                      | SOP after collection   |
|----------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------|------------------------|
| Study                                                                                                                                              | Point(s)                                                                                                                                                         | Preserve the biopsy tissue sample is to  fix the tissue without delay 1. Prepare Eppendorf microfuge tube  (~1.5ml) or conical centrifuge tube  (15ml) fill with EM fixative (4%  formaldehyde + 2% glutaraldehyde  in 0.1M cacodylate buffer, ambient  temperature) and drop the tissue  into the fixative. 2. Fix for 2hrs at room temperature,  then store in 4C. 3. Obtain barcode | Fix for 2hrs at room temperature, then store in  4C.                                         |                        |
| Electron                                                                                                                                           | C1D1 prior to                                                                                                                                                    |                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                              |                        |
| Microscopy                                                                                                                                         | treatment                                                                                                                                                        | Members of the WMB labs  will be on call to receive  and embed biopsies:  ozakimk@nih.gov, Phone  410-522-5661, beeper 102- 11155.                                                                                                                                                                                                                                                     | Will be shipped in batches to Kunio Nagashima  for processing/embedding for the EM analysis. |                        |
| C2D2                                                                                                                                               | Kunio Nagashima                                                                                                                                                  |                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                              |                        |
| Optional at  time of  progression                                                                                                                  | 8560 Progress Drive Wing D, Room D2018, 2nd floor Frederick Maryland 21701 Phone: 301-846-1594 FAX: 301-846-6716 EMAIL: nagashimak@mail.nih.gov                  |                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                              |                        |
| a)                                                                                                                                                 | Prepare two or three Eppendorf  tubes which contain 1 ml of 4%  formalin and leave tubes at room  temperature.                                                   |                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                              |                        |
| b)                                                                                                                                                 | Quickly make your way to  Interventional Radiology (IR) on  the floor. Ask one of the techs or  nurses where your patient is and  prepare to receive the sample. |                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                              |                        |
| c)                                                                                                                                                 | Lay a paper towel next to your  bucket.                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                              |                        |
| d)                                                                                                                                                 | Take out the forceps and rinse/dry  them thoroughly before placing  them on the paper towel.                                                                     |                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                              |                        |
| e)                                                                                                                                                 | Put on a mask, hair net and gloves.                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                              |                        |
| Dopamine  Receptor D2                                                                                                                              | C1D1 prior to  treatment                                                                                                                                         | Oncoceutics requests: 5 unstained slides (FFPE) from each time point                                                                                                                                                                                                                                                                                                                   |                                                                                              |                        |
| Members of the WMB labs  will be on call to receive  and embed biopsies:  ozakimk@nih.gov, Phone  213-703-2032 / 410-522- 5661, beeper 102-11155.. |                                                                                                                                                                  |                                                                                                                                                                                                                                                                                                                                                                                        |                                                                                              |                        |
| And                                                                                                                                                | C2D2                                                                                                                                                             | Will later be shipped to Oncoceutics: John Coolidge                                                                                                                                                                                                                                                                                                                                    |                                                                                              |                        |
| Death  Receptors                                                                                                                                   | Optional at  time of  progression                                                                                                                                | Biorespiratory Manager- Boston SciSafe, Inc 35 Dunham Road Billerica, MA 01821  Tel. 404-242-1578                                                                                                                                                                                                                                                                                      |                                                                                              |                        |  
43 Version Date: 06/17/2020  
| f)                                       | When the radiologist is ready, take  the slide with the sample on it  while being CAREFUL NOT TO  TOUCH THE DOCTOR'S  GLOVE.                                                              |                                                                                                                                                                                                                     |                                                                                     |
|------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------|
| g)                                       | Use the forceps to gently pickup  and place the sample in the  Eppendorf tube.                                                                                                            |                                                                                                                                                                                                                     |                                                                                     |
| h)                                       | Leave the Eppendorf tube at room  temperature overnight.                                                                                                                                  |                                                                                                                                                                                                                     |                                                                                     |
| i)                                       | Next day carefully remove 4%  formalin from the Eppendorf tube  and replace 1 ml of 70% ethanol.  Leave the tube at room  temperature.                                                    |                                                                                                                                                                                                                     |                                                                                     |
| j)                                       | Call AmericanHistolab (Tel: 301- 330-1200) to pick up the sample.                                                                                                                         |                                                                                                                                                                                                                     |                                                                                     |
| k)                                       | Cost for paraffin embedding and  H&E staining: $7.95/block.  Catalogue Number: 1001                                                                                                       |                                                                                                                                                                                                                     |                                                                                     |
| l)                                       | The sample labelling with a  random code and barcoding should  be same as OTC preserved  samples collected at the same day                                                                | Email: jfc@scisafe.com  At the time of shipment, the laboratory must  complete the Specimen Shipping Form ( section  15.6.5) and email it to the following: 1. Rohinton.tarapore@oncoceutics.com a) jfc@scisafe.com |                                                                                     |
| -                                        | Perform core needle biopsy.                                                                                                                                                               |                                                                                                                                                                                                                     |                                                                                     |
| -                                        | Pick the core from the biopsy  needle onto a sterile glass slide.                                                                                                                         |                                                                                                                                                                                                                     |                                                                                     |
| -                                        | Fill cryomold about 1/3 full with  OCT. Place the cryomold in dry ice  to partially freeze the optimal  cutting temperature (OCT). The  OCT should be jelly-like, not  completely frozen. |                                                                                                                                                                                                                     |                                                                                     |
| -                                        | Carefully lift the core biopsy by  both ends with sterile forceps. Do  not stretch the biopsy or it will  break.                                                                          | 3                                                                                                                                                                                                                   | Frozen sections should be cut at 5-8um on  plain, uncoated glass microscope slides. |
| 4                                        | The tissue section should be placed as  close as possible to the center of the slide.  Do not place the frozen section at the end  of the slide.                                          |                                                                                                                                                                                                                     |                                                                                     |
| 5                                        | Two tissue sections from the same biopsy  may be placed on the same glass slide if  space permits.                                                                                        |                                                                                                                                                                                                                     |                                                                                     |
| 6                                        | Do not allow the tissue section to air on  the slide. Freeze immediately on dry ice or                                                                                                    |                                                                                                                                                                                                                     |                                                                                     |
| Whole Exome  Sequencing and  RNAseq done | C1D1 prior to  treatment                                                                                                                                                                  | Members of the WMB labs  will be on call to receive  and embed biopsies:  ozakimk@nih.gov, Phone  410-522-5661, beeper 102- 11155.                                                                                  |                                                                                     |
| C2D2 Optional at  time of  progression   |                                                                                                                                                                                           |                                                                                                                                                                                                                     |                                                                                     |  
| -                                                                         | Lay the biopsy as straight as  possible in the OCT. Once the  sample touches the OCT, you  cannot reposition it or the sample  will break apart.   |                   |
|---------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------|-------------------|
| -                                                                         | Quickly add OCT on top of the  biopsy, completely covering the  sample.                                                                            |                   |
| -                                                                         | Ensure the sample is level and  freeze immediately in dry ice.                                                                                     |                   |
| -                                                                         | Obtain barcode                                                                                                                                     | at– 80 degrees C. |
| Store wrapped in aluminum foil or in a  50ml Falcon tube at 70 degrees C. |                                                                                                                                                    |                   |  
*Biopsies may be not performed on the specific dates and times due to the following reasons, including but not limited to, delayed recovery of hematologic toxicities, delayed clinic schedule, or national holidays.
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plasma/serum

| Correlative                                                                                                                                                                                                                                         | Collection               | SOP for collection                                                                                                           | Contact                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               | SOP after collection   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------|------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------|
| Study                                                                                                                                                                                                                                               | Point(s)                 | 1. Collect 6ml of blood into a  Lavender top, vacutainer tube 2. Obtain barcode                                              | 1. Centrifuge the filled lavender top tube at  1500 g (3000 rpm) for 15 minutes until the  plasma and the cells are well separated.  2. Label 2 blue cap transfer vials lengthwise  with the appropriate barcode labels  indicating "plasma (blue)".  3. Clearly print the study number "Study  XXX", patient ID number, date and time of  the blood draw.  4. Transfer ~1.5 mL of plasma into each of the  2 blue top transfer vials labeled "plasma  (blue)" 5. Store sample in a freezer with a minimum  -20ºC or preferable -70ºC |                        |
| Pharmacokinetics                                                                                                                                                                                                                                    | C1D1 prior to  treatment | Members of the WMB labs will be on call to receive  study samples:  ozakimk@nih.gov, Phone  410-522-5661, beeper 102- 11155. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                        |
| Blood must be centrifuged, separated  and frozen within 2 hours of  collection. Specimens must be stored  frozen at -70ºC (preferred) or -20ºC  (acceptable) at each study site until  shipped on dry ice to the designated  analytical laboratory. |                          |                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                        |
| C1D1 6hr  after drug Optional at  time of  progression                                                                                                                                                                                              |                          |                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |                        |  
45 Version Date: 06/17/2020  
| Correlative   | Collection   | SOP for collection   | Contact   | SOP after collection   |
|---------------|--------------|----------------------|-----------|------------------------|
| Study         | Point(s)     |                      |           |                        |  
| Will later be shipped to Oncoceutics: Sample Receiving Officer XenoBiotic Laboratories, Inc 107 Morgan Lane Plainsboro, NJ 08536 Tel. 609-799-2295 x252 Email: sro@xbl.com At the time of shipment, the laboratory must  complete the Specimen Shipping Form (15.6.5 Appendix E) and email it to the following: m) Rohinton.tarapore@oncoceutics.com n) sro@xbl.com   |                                                                                                                                                                                                                                                     |                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 4.Collect 6ml of blood into a Red  top, vacutainer tube 5. Obtain barcode                                                                                                                                                                                                                                                                                             |                                                                                                                                                                                                                                                     |                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |
| Serum M30 and                                                                                                                                                                                                                                                                                                                                                         | C1D1 prior to                                                                                                                                                                                                                                       |                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |
| M65                                                                                                                                                                                                                                                                                                                                                                   | treatment                                                                                                                                                                                                                                           | Members of the WMB labs will be on call to receive  study samples:  ozakimk@nih.gov, Phone  410-522-5661, beeper 102- 11155. | 1. Centrifuge the filled red top tube at 1500 g  (3000 rpm) for 15 minutes until the plasma  and the cells are well separated.  2. Label 2 yellow cap transfer vials lengthwise  with the appropriate barcode labels  indicating "serum (red)".  3. Clearly print the study number "Study  XXX", patient ID number, date and time of  the blood draw.  4. Transfer ~1.5 mL of serum into each of the  2 yelloq top transfer vials labeled "serum  (red)" 5. Store sample in a freezer with a minimum  -20ºC or preferable -70ºC |
| Day 1 of each  subsequent  cycle                                                                                                                                                                                                                                                                                                                                      | Blood must be centrifuged, separated  and frozen within 2 hours of  collection. Specimens must be stored  frozen at -70ºC (preferred) or -20ºC  (acceptable) at each study site until  shipped on dry ice to the designated  analytical laboratory. |                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |
| Optional at  time of  progression                                                                                                                                                                                                                                                                                                                                     |                                                                                                                                                                                                                                                     |                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |  
46 Version Date: 06/17/2020  
| Correlative   | Collection   | SOP for collection   | Contact   | SOP after collection   |
|---------------|--------------|----------------------|-----------|------------------------|
| Study         | Point(s)     |                      |           |                        |  
| See above Pharmocokinetics section for  shipping information to Oncoceutics.   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 |                                                                                                                                                                                                                                             |                                                                                                                              |                             |
|--------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------|-----------------------------|
| 1. Collect 6ml of blood into a Red  top, vacutainer tube 2. Obtain barcode     | 2. Centrifuge the filled red top tube at 1500 g  (3000 rpm) for 15 minutes until the plasma  and the cells are well separated.  3. Label 2 yellow cap transfer vials lengthwise  with the appropriate barcode labels  indicating "serum (red)".  4. Clearly print the study number "Study  XXX", patient ID number, date and time of  the blood draw.  5. Transfer ~1.5 mL of serum into each of the  2 yelloq top transfer vials labeled "serum  (red)" 6. Store sample in a freezer with a minimum  -20ºC or preferable -70ºC |                                                                                                                                                                                                                                             |                                                                                                                              |                             |
| TRAIL                                                                          | C1D1 prior to                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |                                                                                                                                                                                                                                             |                                                                                                                              |                             |
| Expression                                                                     | treatment                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | Members of the WMB labs will be on call to receive  study samples:  ozakimk@nih.gov, Phone  410-522-5661, beeper 102- 11155.                                                                                                                |                                                                                                                              |                             |
| Day 1 of each  subsequent  cycle                                               | Blood must be centrifuged, separated  and frozen within 2 hours of  collection. Specimens must be stored  frozen at -70ºC (preferred) or -20ºC  (acceptable) at each study site until  shipped on dry ice to the designated  analytical laboratory.                                                                                                                                                                                                                                                                             |                                                                                                                                                                                                                                             |                                                                                                                              |                             |
| Optional at  time of  progression                                              | See above Pharmocokinetics section for  shipping information to Oncoceutics.                                                                                                                                                                                                                                                                                                                                                                                                                                                    |                                                                                                                                                                                                                                             |                                                                                                                              |                             |
| Germline gDNA                                                                  | C1D1                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | 1. Collect 10mL of blood into BCT  Streck tubes ( Label the tube with  appropriate Study/Patient Number  Identifiers. Record the time of  collection and whether the blood  was drawn using a peripheral  venous access device (eg. cannula | Members of the WMB labs will be on call to receive  study samples:  ozakimk@nih.gov, Phone  410-522-5661, beeper 102- 11155. | See Appendix D for full SOP |  
47 Version Date: 06/17/2020  
| Correlative                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Collection                                                                                                                                                                                                                                                                           | SOP for collection                                                                                                                                                                                                                                                                                                                | Contact   | SOP after collection   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------|------------------------|
| Study                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       | Point(s)                                                                                                                                                                                                                                                                             | or butterfly) or a central venous  access device (CVAD).                                                                                                                                                                                                                                                                          |           |                        |
| 2. After collection, gently invert  tubes 8-10 times to mix and leave  tubes upright prior to  centrifugation. 3. Obtain barcode 1. Either one 10-mL CellSave  Preservative Tube OR a 10-mL  Streck tube, PLUS one 10-mL  CPT tube for each time point will  be collected from each patient.  Please, make sure tubes are full. 2. Immediately after collection,  invert the blood tubes 3-4 times. 3. Plasma from these samples will be  stored at Trepel lab after CTCs are  harvested. 4. Obtain barcode | As soon as possible after the patient is scheduled please send email notification  to the Trepel lab: Jane Trepel at trepel@helix.nih.gov; Sunmin Lee at  leesun@mail.nih.gov; Min-Jung Lee at min-jung.lee@nih.gov; Akira Yuno at  akira.yuno@nih.gov that the sample is scheduled. |                                                                                                                                                                                                                                                                                                                                   |           |                        |
| Circulating  Tumor Cells, Cell  free DNA, and  Cell free  mitochondrial  DNA                                                                                                                                                                                                                                                                                                                                                                                                                                | C1D1 prior to  treatment Day 1 of each  subsequent  cycle                                                                                                                                                                                                                            | As soon as the sample is drawn, please call the Trepel lab at 240-760-6330 to  communicate that the sample is ready. Keep the sample on the unit at room  temperature. The sample will be picked up by the lab and processed for CTC  enumeration.                                                                                |           |                        |
| Optional at  time of  progression                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | 3. Collect 4ml of venous blood in a  venous blood BD Vacutainer  spray-coated K2EDTA (lavender  top) tube.  4. Gently invert 10 times to mix  blood and anticoagulant.  5. Store tubes at room temperature  until centrifugation.                                                    | As soon as possible after the patient is scheduled please send email notification  to the Blood Processing Core for processing and stoage of the blood sampled  (EDTA plasma) at NCIBloodcore@mail.nih.gov - at least 24 hours before  transporting samples (the Friday before is preferred). For sample pickup, page  102-11964. |           |                        |
| Cytokine                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | C1D1 prior to                                                                                                                                                                                                                                                                        |                                                                                                                                                                                                                                                                                                                                   |           |                        |
| Analysis                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | treatment Day 1 of each  subsequent  cycle                                                                                                                                                                                                                                           | For immediate help, call 240-760-6180 (main blood processing core number)  or, if no answer, 240-760-6190 (main clinical pharmacology lab number). For  questions regarding sample processing, contact NCIBloodcore@mail.nih.gov                                                                                                  |           |                        |
| Optional at  time of  progression                                                                                                                                                                                                                                                                                                                                                                                                                                                                           | As soon as the sample is drawn, please call the BPC to communicate that the  sample is ready. Keep the sample on the unit at room temperature.                                                                                                                                       |                                                                                                                                                                                                                                                                                                                                   |           |                        |  
48 Version Date: 06/17/2020  
| Correlative   | Collection   | SOP for collection                                                                                   | Contact                                                                                                                                       | SOP after collection   |
|---------------|--------------|------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------|------------------------|
| Study         | Point(s)     | It is generally preferred and often  necessary to process the samples  within 5 hours of collection. | Plasma will be separated and aliquoted according to the BPC SOPs and stored,  barcoded, in the CPC repository until use (See Section 15.6.5). |                        |
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peripheral blood mononuclear cells (pbmcs)

| Correlative                                                        | Collection               | SOP for collection                                                                                                                                                                                                                                                                                                                            | Contact   | SOP after collection   |
|--------------------------------------------------------------------|--------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------|------------------------|
| Study                                                              | Point(s)                 | 6. One 8-ml BD Vacutainer Cell  Preparation Tubes (CPT citrate  blue/black tiger top) will be collected  from each patient. 7. Immediately after collection, mix the  blood sample by gentle inversion  several times. The date and exact time  of each blood draw should be recorded  on the tube.                                           |           |                        |
| Immune Assays                                                      | C1D1 prior to  treatment | As soon as possible after the patient is scheduled please send email  notification to the Trepel lab at trepel@helix.nih.gov; Sunmin Lee at  leesun@mail.nih.gov; Min-Jung Lee at min-jung.lee@nih.gov; Akira  Yuno at akira.yuno@nih.gov and call the Trepel lab at 240-760-6330 to  arrange for immediate pick-up when the sample is drawn. |           |                        |
| Day 1 of each  subsequent  cycle Optional at  time of  progression |                          |                                                                                                                                                                                                                                                                                                                                               |           |                        |  
5.6 SAMPLE STORAGE, TRACKING AND DISPOSITION Samples collected at NCI will be ordered in CRIS and tracked through a Clinical Trial Data Management system. Should a CRIS screen not be available, the CRIS downtime procedures will be completed and will accompany the specimen and be filed in the medical record. Samples will not be sent outside NIH without appropriate approvals and/or agreements, if required. At the end of the protocol, samples will be stored for potential further analysis as new information becomes available (only for those subjects who consented to future optional studies). Any new use of identified or coded samples, specimens, or data will undergo prospective and continuing IRB review and approval.
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5.6.1 patient sample protections

Each patient sample set will be coded with a unique patient identifier. No patient specific information is encoded in this identifier. The protocol scientific investigators handling samples will be blinded as to as to the patient identification, patient data and outcome. Tissue and blood samples obtained by and stored through WMB will be labeled with unique barcodes. Blood samples obtained by and stored through the Clinical Pharmacology Core and Trepel Lab will be labeled with unique barcodes obtained by Dr. Figg's laboratory barcode system. The amount of blood that may be drawn from adult patients (i.e., those persons 18 years of age or older) for research purposes shall not exceed 10.5 mL/kg or 550 mL, whichever is smaller, over any eight week period. If a patient needs to have a malignant effusion or ascites tapped for diagnostic or therapeutic purposes, a sample will be collected for research.
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5.6.2 sample storage tracking and

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

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

Samples will be processed immediately by the Trepel laboratory. Biospecimens will be collected and processed using validated SOPs that will ensure both specimen quality and patient confidentiality. Using a computerized inventory system and a backup hardcopy process, all specimen collection and processing steps will be documented and the specific location of each specimen will be tracked. Each new specimen collected will be assigned a unique barcode identifier that can be linked to the original specimen collected and other relevant information within the inventory system. Specimen labels will indicate: protocol number, order in which the patient enrolled on the trial, type of sample, collection time, and total volume collected, as appropriate. The inventory process contains other security provisions sufficient to safeguard patient privacy and confidentiality. Access to the inventory system and associated documents will be restricted to appropriate individuals. Requests to use specimens stored in the repository must be approved. SOPs ensure that any changes in informed consent made by a patient and relayed to the PI will be reflected in the inventory system to ensure that specimens are destroyed as appropriate. All laboratory personnel will be trained to adhere to SOPs and will be monitored for high-quality performance.
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5.6.4 procedures for storage of

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

At the end of the protocol, samples will be stored for potential further analysis as new information becomes available (only for those subjects who consented to future optional studies). Any new use of identified or coded samples, specimens, or data will undergo prospective and continuing IRB review and approval. The study will remain open so long as sample or data analysis continues. Samples, and associated data, will be stored in the locations listed above until they are no longer of scientific value, or unless the patient withdraws consent for their continued use. If researchers have samples remaining once they have completed all studies associated with the protocol, they must be returned to the WMB laboratory or to the BPC repository. Samples can only be saved at the completion of the study for future use if subjects consented. Access to these samples will only be granted following IRB approval of an additional protocol, granting the rights to use the material.
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5.7 samples for genetic/genomic analysis

If not previously evaluated for germline mutations in BRCA1/2 or Lynch syndrome, patients will undergo evaluation upon enrollment on trial. If the research findings are verified in the CLIA certified lab, the subject will be offered the opportunity to come to NIH (at our expense) to have genetic education and counseling with the NCI Genetics Branch to explain this result. If the subject does not want to come to NIH, a referral to a local genetic healthcare provider will be provided (at their expense). This is the only time during the course of the study that incidental findings will be returned. No interrogations regarding clinically actionable findings will be made after the primary analysis. Subjects will be contacted if a clinically actionable gene variant is discovered. Clinically actionable findings for the purpose of this study are defined as disorders appearing in the American College of Medical Genetics and Genomics recommendations for the return of incidental findings that is current at the time of primary analysis. (A list of current guidelines is maintained on the CCR intranet: https://ccrod.cancer.gov/confluence/display/CCRCRO/Incidental+Findings+Lists). Subjects will be contacted at this time with a request to provide a blood sample to be sent to a CLIA certified laboratory which has an FDA approved test for that mutation. If the research findings are verified in the CLIA certified lab, the subject will be referred to the NIH Genetics counseling service for the disclosure of the results. This is the only time during the course of the study that incidental findings will be returned. No interrogations regarding clinically actionable findings will be made after the primary analysis.
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5.7.1 certificate of confidentiality

As part of study efforts to provide confidentiality of subject information, this study will obtain a Certificate of Confidentiality which helps to protect personally identifiable research information. The Certificate of Confidentiality allows investigators on this trial to refuse to disclose identifying information related to the research participants, should such disclosure have adverse consequences for subjects or damage their financial standing, employability, insurability or reputation. The informed consent includes the appropriate coverage and restrictions of the Certificate of Confidentiality.
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6 data collection and evaluation

The PI will be responsible for overseeing entry of data into an in-house password protected electronic system (C3D) and ensuring data accuracy, consistency and timeliness. The principal investigator, associate investigators/research nurses and/or a contracted data manager will assist with the data management efforts. All data obtained during the conduct of the protocol will be kept in secure network drives or in approved alternative sites that comply with NIH security standards. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant. All adverse events, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until return to baseline or stabilization of event.  
Document AEs from the first study intervention, Study Day 1, through 30 days after the study treatment was last administered. Beyond 30 days after the last intervention, only adverse events which are serious and related to the study intervention need to be recorded. An abnormal laboratory value will be considered an AE if the laboratory abnormality is characterized by any of the following:
- Results in discontinuation from the study - Is associated with clinical signs or symptoms - Requires treatment or any other therapeutic intervention - Is associated with death or another serious adverse event, including hospitalization. - Is judged by the Investigator to be of significant clinical impact - If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the patient's outcome.  
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH
Intramural Records Retention Schedule as applicable.  
Loss or destruction of data: Should we become aware that a major breach in our plan to protect subject confidentiality and trial data has occurred, this will be reported expeditiously per requirements in section **7.2.1**.
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6.1.1 source documents

Source documents are defined as original documents, data and records. This may include hospital records, clinical and office charts, laboratory data/information, patients' diaries or evaluation checklists, pharmacy dispensing and other records, recorded data from automated instruments, microfiches, photographic negatives, microfilm or magnetic media, X-rays. The investigator will permit trial-related monitoring, audits, IRB review, and regulatory inspection(s), providing direct access to source documents.
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6.1.2 case report forms

Data may be entered from the source documents directly into eCRFs in C3D for each patient enrolled in this study. The principal investigator or research nurse will review the eCRFs for completeness and accuracy. Independent audits may also be conducted by NCI personnel to ensure completeness and accuracy of data in C3D.
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6.2 data sharing plans 6.2.1

I will share human data generated in this research for future research as follows:  
1. Coded, linked data in an NIH-funded or approved public repository. 2. Coded, linked data in BTRIS (automatic for activities in the Clinical Center) 3. Identified or coded, linked data with approved outside collaborators under appropriate agreements.
Data will be shared through:  
1. An NIH-funded or approved public repository. Insert name or names:_clinicaltrials.gov, dbGaP.  
2. BTRIS (automatic for activities in the Clinical Center) 3. Approved outside collaborators under appropriate individual agreements. 4. Publication and/or public presentations.  
Data will be shared:
a) Before publication. b) At the time of publication or shortly thereafter.
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6.2.2 genomic data sharing plan

Unlinked genomic data will be deposited in public genomic databases such as dbGaP in compliance with the NIH Genomic Data Sharing Policy. 6.3 RESPONSE CRITERIA For the purposes of this study, patients should be re-evaluated for response every 8 weeks +/- 4 days with imaging modalities determined appropriate by the investigators. In addition to a baseline scan, confirmatory scans should also be obtained 4 weeks +/- 4 days following initial documentation of objective response. Response and progression will be evaluated in this study using the new international criteria proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline
(version 1.1).(40) Changes in the largest diameter (unidimensional measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph nodes are used in the RECIST criteria.
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6.3.1 disease parameters

Measurable disease: Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as: 1. By chest x-ray: >20 mm; 2. By CT scan:
1. Scan slice thickness 5 mm or under: as >10 mm 2. Scan slic thickness > 5 mm: double the slice thickness 3. With calipers on clinical exam: >10 mm. All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters). Malignant lymph nodes. To be considered pathologically enlarged and measurable, a lymph node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). At baseline and in follow-up, only the short axis will be measured and followed. Non-measurable disease. All other lesions (or sites of disease), including small lesions (longest diameter <10 mm or pathological lymph nodes with ≥10 to <15 mm short axis), are considered non-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by CT or MRI), are considered as non-measurable. Note: Cystic lesions that meet the criteria for radiographically defined simple cysts should not be considered as malignant lesions (neither measurable nor non-measurable) since they are, by definition, simple cysts. 'Cystic lesions' thought to represent cystic metastases can be considered as measurable lesions, if they meet the definition of measurability described above. However, if non-cystic lesions are present in the same patient, these are preferred for selection as target lesions. Target lesions. All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, should be identified as **target lesions** and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. It may be the case that, on occasion, the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected. A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease. Non-target lesions. All other lesions (or sites of disease) including any measurable lesions over and above the 5 target lesions should be identified as **non-target lesions** and should also be recorded at baseline. Measurements of these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each should be noted throughout follow-up.