untitled h1
6.3.2 methods for evaluation of

All measurements should be taken and recorded in metric notation using a ruler or calipers. All baseline evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 weeks before the beginning of the treatment. The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be imaged but are assessable by clinical exam.  
Clinical lesions: Clinical lesions will only be considered measurable when they are superficial
(e.g., skin nodules and palpable lymph nodes) and 10 mm diameter as assessed using calipers
(e.g., skin nodules). In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended.  
Chest x-ray: Lesions on chest x-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung. However, CT is preferable.  
Conventional CT and MRI: This guideline has defined measurability of lesions on CT scan based on the assumption that CT slice thickness is 5 mm or less. If CT scans have slice thickness greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness. MRI is also acceptable in certain situations (e.g. for body scans). Use of MRI remains a complex issue. MRI has excellent contrast, spatial, and temporal resolution; however, there are many image acquisition variables involved in MRI, which greatly impact image quality, lesion conspicuity, and measurement. Furthermore, the availability of MRI is variable globally. As with CT, if an MRI is performed, the technical specifications of the scanning sequences used should be optimized for the evaluation of the type and site of disease. Furthermore, as with CT, the modality used at follow-up should be the same as was used at baseline and the lesions should be measured/assessed on the same pulse sequence. It is beyond the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body parts, and diseases. Ideally, the same type of scanner should be used and the image acquisition protocol should be followed as closely as possible to prior scans. Body scans should be performed with breath-hold scanning techniques, if possible. PET-CT: At present, the low dose or attenuation correction CT portion of a combined PET-CT is not always of optimal diagnostic CT quality for use with RECIST measurements. However, if the site can document that the CT performed as part of a PET-CT is of identical diagnostic quality to a diagnostic CT (with IV and oral contrast), then the CT portion of the PET-CT can be used for RECIST measurements and can be used interchangeably with conventional CT in accurately measuring cancer lesions over time. Note, however, that the PET portion of the CT introduces additional data which may bias an investigator if it is not routinely or serially performed. Ultrasound: Ultrasound is not useful in assessment of lesion size and should not be used as a method of measurement. Ultrasound examinations cannot be reproduced in their entirety for independent review at a later date and, because they are operator dependent, it cannot be guaranteed that the same technique and measurements will be taken from one assessment to the next. If new lesions are identified by ultrasound in the course of the study, confirmation by CT or MRI is advised. If there is concern about radiation exposure at CT, MRI may be used instead of CT in selected instances. Endoscopy, Laparoscopy: The utilization of these techniques for objective tumor evaluation is not advised. However, such techniques may be useful to confirm complete pathological response when biopsies are obtained or to determine relapse in trials where recurrence following complete response (CR) or surgical resection is an endpoint. Cytology, Histology: These techniques can be used to differentiate between partial responses (PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as germ cell tumors, where known residual benign tumors can remain). The cytological confirmation of the neoplastic origin of any effusion that appears or worsens during treatment when the measurable tumor has met criteria for response or stable disease is mandatory to differentiate between response or stable disease (an effusion may be a side effect of the treatment) and progressive disease. FDG-PET: While FDG-PET response assessments need additional study, it is sometimes reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in assessment of progression (particularly possible 'new' disease). New lesions on the basis of FDG-PET imaging can be identified according to the following algorithm:
a. Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of PD
based on a new lesion.  
b. No FDG-PET at baseline and a positive FDG-PET at follow-up: If the positive FDGPET at follow-up corresponds to a new site of disease confirmed by CT, this is PD. If the positive FDG-PET at follow-up is not confirmed as a new site of disease on CT, additional follow-up CT scans are needed to determine if there is truly progression occurring at that site (if so, the date of PD will be the date of the initial abnormal FDGPET scan). If the positive FDG-PET at follow-up corresponds to a pre-existing site of disease on CT that is not progressing on the basis of the anatomic images, this is not  
PD.  
c. FDG-PET may be used to upgrade a response to a CR in a manner similar to a biopsy in cases where a residual radiographic abnormality is thought to represent fibrosis or scarring. The use of FDG-PET in this circumstance should be prospectively described in the protocol and supported by disease-specific medical literature for the indication. However, it must be acknowledged that both approaches may lead to false positive CR due to limitations of FDG-PET and biopsy resolution/sensitivity.
Note: A 'positive' FDG-PET scan lesion means one which is FDG avid with an uptake greater than twice that of the surrounding tissue on the attenuation corrected image.
untitled h1
6.3.3 response criteria 6.3.3.1 evaluation

Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum of diameters. Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions). Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum of diameters while on study. 6.3.3.2 Evaluation of Non-Target Lesions Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (<10 mm short axis). Note: If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response.  
Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits.  
Progressive Disease (PD): Appearance of one or more new lesions and/or *unequivocal progression* of existing non-target lesions. *Unequivocal progression* should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase. Although a clear progression of "non-target" lesions only is exceptional, the opinion of the treating physician should prevail in such circumstances, and the progression status should be confirmed at a later time by the review panel (or Principal Investigator).
untitled h1
6.3.3.3 evaluation of best overall

The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria.  
| For Patients with Measurable Disease (i.e., Target Disease) Target  Non-Target  New  Overall  Best Overall Response when  Lesions Lesions Lesions Response Confirmation is Required* CR CR No CR >4 wks. Confirmation** CR NonCR/Non-PD No PR CR Not  No PR evaluated >4 wks. Confirmation** PR NonCR/NonPD/not  evaluated No PR SD NonCR/NonPD/not  evaluated No SD Documented at least once >4  wks. from baseline** PD Any Yes or  PD No Any PD*** Yes or no prior SD, PR or CR PD No Any Any Yes PD * See RECIST 1.1 manuscript for further details on what is evidence of a new lesion. ** Only for non-randomized trials with response as primary endpoint. *** In exceptional circumstances, unequivocal progression in non-target lesions may be accepted as  disease progression. Note: Patients with a global deterioration of health status requiring discontinuation of treatment without  objective evidence of disease progression at that time should be reported as "symptomatic  deterioration." Every effort should be made to document the objective progression even after  discontinuation of treatment.   |
|---|  
| Non-Target Lesions                                                                                                                                                                                                                             | New Lesions   | Overall Response   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------|--------------------|
| CR                                                                                                                                                                                                                                             | No            | CR                 |
| Non-CR/non-PD                                                                                                                                                                                                                                  | No            | Non-CR/non-PD*     |
| Not all evaluated                                                                                                                                                                                                                              | No            | not evaluated      |
| Unequivocal PD                                                                                                                                                                                                                                 | Yes or No     | PD                 |
| Any                                                                                                                                                                                                                                            | Yes           | PD                 |
| * 'Non-CR/non-PD' is preferred over 'stable disease' for non-target disease since SD is increasingly used  as an endpoint for assessment of efficacy in some trials so to assign this category when no lesions can be  measured is not advised |               |                    |
untitled h1
for patients with non-measurable disease

Duration of overall response: The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented. Duration of stable disease: Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements.
untitled h1
6.3.5 progression-free survival

PFS is defined as the duration of time from start of treatment to time of progression or death, whichever occurs first. 6.4 TOXICITY CRITERIA The following adverse event management guidelines are intended to ensure the safety of each patient while on the study. The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 5.0. A copy of the CTCAE version 5.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm ).  
Version Date: 06/17/2020 7 NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN  
. 7.1 DEFINITIONS
Please refer to definitions provided in Policy 801: Reporting Research Events found **here**.  
7.2 OHSRP OFFICE OF COMPLIANCE AND TRAINING / IRB REPORTING
untitled h1
7.2.1 expedited reporting

Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 802 Non-Compliance Human Subjects Research found **here**. Note: Only IND Safety Reports that meet the definition of an unanticipated problem will need to be reported per these policies.
untitled h1
7.2.2 irb requirements for pi

Please refer to the reporting requirements in Policy 801: Reporting Research Events found **here**..
untitled h1
7.3 nci clinical director reporting

Problems expeditiously reported to the OHSRP in iRIS will also be reported to the NCI Clinical Director. A separate submission is not necessary as reports in iRIS will be available to the Clinical Director. In addition to those reports, all deaths that occur within 30 days after receiving a research intervention should be reported via email to the Clinical Director unless they are due to progressive disease. To report these deaths, please send an email describing the circumstances of the death to Dr. Dahut at NCICCRQA@mail.nih.gov within one business day of learning of the death. 7.4 NIH REQUIRED DATA AND SAFETY MONITORING PLAN
untitled h1
7.4.1 principal investigator/research team

The clinical research team will meet on a regular basis weekly when patients are being actively treated on the trial to discuss each patient. Decisions about dose level enrollment and dose escalation if applicable will be made based on the toxicity data from prior patients. All data will be collected in a timely manner and reviewed by the principal investigator or a lead associate investigator. Events meeting requirements for expedited reporting as described in section 7.2.1 will be submitted within the appropriate timelines.  
The principal investigator will review adverse event and response data on each patient to ensure safety and data accuracy. The principal investigator will personally conduct or supervise the investigation and provide appropriate delegation of responsibilities to other members of the research staff.
untitled h1
8.1.1 adverse event

Any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and which does not necessarily have a causal relationship with this treatment. An adverse event (AE) can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not related to the medicinal (investigational) product (ICH E6 (R2))
untitled h1
8.1.2 serious adverse event (sae)

An adverse event or suspected adverse reaction is considered serious if in the view of the investigator or the sponsor, it results in any of the following:
- Death,  
- A life-threatening adverse event (see section **8.1.3**)
- Inpatient hospitalization or prolongation of existing hospitalization o A hospitalization/admission that is pre-planned (i.e., elective or scheduled surgery arranged prior to the start of the study), a planned hospitalization for pre-existing condition, or a procedure required by the protocol, without a serious deterioration in health, is not considered a serious adverse event.  
o A hospitalization/admission that is solely driven by non-medical reasons (e.g.,
hospitalization for patient convenience) is not considered a serious adverse event.  
o Emergency room visits or stays in observation units that do not result in admission to the hospital would not be considered a serious adverse event. The reason for seeking medical care should be evaluated for meeting one of the other serious criteria.  
- Persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions
- A congenital anomaly/birth defect. - Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse drug experience when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition.
untitled h1
8.1.3 life-threatening

An adverse event or suspected adverse reaction is considered "life-threatening" if, in the view of either the investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It does not include an adverse event or suspected adverse reaction that, had it occurred in a more severe form, might have caused death. (21CFR312.32)
untitled h1
8.1.4 severity

The severity of each Adverse Event will be assessed utilizing the CTCAE version v5.0.  
8.1.5 **Relationship to Study Product** All AEs will have their relationship to study product assessed using the terms: related or not related.  
- Related - There is a reasonable possibility that the study product caused the adverse event.  
Reasonable possibility means that there is evidence to suggest a causal relationship between the study product and the adverse event.  
.  
- Not Related - There is not a reasonable possibility that the administration of the study product caused the event.  
8.2 ASSESSMENT OF SAFETY EVENTS
AE information collected will include event description, date of onset, assessment of severity and relationship to study product and alternate etiology (if not related to study product), date of resolution of the event, seriousness and outcome. The assessment of severity and relationship to the study product will be done only by those with the training and authority to make a diagnosis and listed on the Form FDA 1572 as the site principal investigator or sub-investigator. AEs occurring during the collection and reporting period will be documented appropriately regardless of relationship. AEs will be followed through resolution. SAEs will be:  
- Assessed for severity and relationship to study product and alternate etiology (if not related to study product) by a licensed study physician listed on the Form FDA 1572 as the site principal investigator or sub-investigator.  
- Recorded on the appropriate SAE report form, the medical record and captured in the clinical database.  
- Followed through resolution by a licensed study physician listed on the Form FDA 1572 as the site principal investigator or sub-investigator.
For timeframe of recording adverse events, please refer to section 6.1. All serious adverse events recorded from the time of first investigational product administration must be reported to the sponsor.
untitled h1
8.3 reporting of serious adverse

Any AE that meets protocol-defined serious criteria or meets the definition of Adverse Event of Special Interest that require expedited reporting must be submitted immediately (within 24 hours of awareness) to OSRO Safety using the CCR SAE report form.  
All SAE reporting must include the elements described in section 8.2.  
SAE reports will be submitted to the Center for Cancer Research (CCR) at: OSROSafety@mail.nih.gov and to the CCR PI and study coordinator. CCR SAE report form and instructions can be found at: https://ccrod.cancer.gov/confluence/pages/viewpage.action?pageId=157942842 Following the assessment of the SAE by OSRO, other supporting documentation of the event may be requested by the OSRO Safety and should be provided as soon as possible. 8.4 SAFETY REPORTING CRITERIA TO THE PHARMACEUTICAL COLLABORATORS All events listed below must be reported in the defined timelines to CCRsafety@mail.nih.gov. The CCR Office of Regulatory Affairs will send all reports to the manufacturer as described below. Serious Adverse Event and Suspected Unexpected Serious Adverse Reaction, Medical Device Event, Potential Incident, Device Deficiency or Incident Reporting: Principal Investigator shall forward to Oncoceutics Inc, any SAE, SUSAR, Medical Device Event, Device Deficiency or Incident information, including, but not limited to, all initial and follow-up information involving any Study subject in the Study. Notification shall be in the form of a completed CIOMS I/MedWatch (or other mutually agreed upon format) within two (2) business days of learning of the information. This information shall be transmitted to Oncoceutics using the contact information provided below or such other modified contact information as provided by Oncoceutics in writing. All information shall be transmitted in the English language and contain the reporter's name and the Study subject identifier code. SUSAR information will be reported unblinded if the Study Drug has been blinded in the Study. Randomization codes for all other SAEs will be provided to Oncoceutics at end of Study if the Study Drug has been blinded in the Study. Oncoceutics may define certain Non-Serious Events of Interest. If any Non-Serious Events of Interest are defined, Oncoceutics will provide such information in writing to Principal Investigator at the time of Protocol approval, execution of the CTA or anytime thereafter. Reporting of any defined Non-Serious Events of Interest will be handled in the same manner as SAEs unless mutually agreed otherwise in writing by the parties. All reports of Study Drug exposure during pregnancy or lactation (including a female partner of a male Study subject using the Study Drug), whether associated with an AE or not, must be reported to Oncoceutics in accordance with the timelines and contact information for an SAE. Principal Investigator shall follow pregnancies to term to obtain the outcome of the pregnancy. The outcome of the pregnancy shall be forwarded to Oncoceutics. Institution and Principal Investigator shall fully comply with all of their respective reporting obligations to the applicable regulatory authorities with respect to any AE, SAE or SUSAR that arises from the Study. SAE reports and any other relevant safety information are to be forwarded to Josh Allen, PhD of Oncoceutics via email (josh.allen@oncoceutics.com) and/or facsimile number: 1-844-245-7650.  
A MedWatch form is available at http://www.fda.gov/medwatch/  
8.5 REPORTING PREGNANCY
untitled h1
8.5.1 maternal exposure

If a patient becomes pregnant during the course of the study, the study treatment should be discontinued immediately, and the pregnancy reported to the Sponsor no later than 24 hours of when the Investigator becomes aware of it. The Investigator should notify the Sponsor no later than 24 hours of when the outcome of the pregnancy becomes known, Pregnancy itself is not regarded as an SAE. However, congenital abnormalities or birth defects and spontaneous miscarriages that meet serious criteria (section **8.1.2**) should be reported as SAEs.  
The outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or congenital abnormality) should be followed up and documented.
untitled h1
8.5.2 paternal exposure

Male patients should refrain from fathering a child or donating sperm during the study and for 28 days after the last dose of ONC201. Pregnancy of the patient's partner is not considered to be an AE. However, the outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, or congenital abnormality) occurring from the date of the first dose until 28 days after the last dose should, if possible, be followed up and documented. 8.6 REGULATORY REPORTING FOR STUDIES CONDUCTED UNDER CCR-SPONSORED IND Following notification from the investigator, CCR, the IND sponsor, will report any suspected adverse reaction that is both serious and unexpected. CCR will report an AE as a suspected adverse reaction only if there is evidence to suggest a causal relationship between the study product and the adverse event. CCR will notify FDA and all participating investigators (i.e., all investigators to whom the sponsor is providing drug under its INDs or under any investigator's IND) in an IND safety report of potential serious risks from clinical trials or any other source, as soon as possible, in accordance to 21 CFR Part 312.32. All serious events will be reported to the FDA at least annually in a summary format.
untitled h1
9 clinical monitoring

As a sponsor for clinical trials, FDA regulations require the CCR to maintain a monitoring program. The CCR's program allows for confirmation of: study data, specifically data that could affect the interpretation of primary and secondary study endpoints; adherence to the protocol, regulations, ICH E6, and SOPs; and human subjects protection. This is done through independent verification of study data with source documentation focusing on: - Informed consent process  
- Eligibility confirmation
- Drug administration and accountability  
- Adverse events monitoring
- Response assessment. The monitoring program also extends to multi-site research when the CCR is the coordinating center. This trial will be monitored by personnel employed by a CCR contractor. Monitors are qualified by training and experience to monitor the progress of clinical trials. Personnel monitoring this study will not be affiliated in any way with the trial conduct.
untitled h1
10 statistical considerations 10.1 statistical

The primary objectives of this trial are to determine whether ONC201 is able to be associated with 1) potential improvement in the PFS probability of patients with refractory/recurrent metastatic HR+ breast cancer, 2) an improvement in overall response rate in patients with metastatic TNBC, or 3) an improvement in overall response rate in patients with advanced endometrial cancer.
untitled h1
primary endpoints

- 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)
Secondary Endpoints for all cohorts will include safety of ONC201, clinical benefit rate (defined as partial response + complete response + stable disease).
untitled h1
10.2 sample size determination cohort

Data from published trials indicate that the median PFS in patients with refractory/recurrent metastatic HR+ breast cancer is approximately 4 months. In this cohort, the goal is to determine if use of ONC201 is able to be associated with an 8-month median PFS probability (7, 8). With 24 evaluable patients receiving the proposed therapy, assuming accrual would take place over approximately 2 years, and that there would be at least 1 year of additional potential follow-up after the last patient has begun the therapy, there would be 80% power to determine whether there is a difference between a 4 month median PFS and an improved 8-month median PFS, with a one sided 0.10 alpha level test, using the method of Brookmeyer and Crowley (41). In practice, Kaplan-Meier curves and appropriate confidence intervals at selected time points will be provided to help interpret results relative to the expected results. As an early stopping rule, if 0 of the first 5 patients is able to be progression-free at 8 months, then no further patients will be accrued into this cohort as soon as that is able to be determined since the upper 95% one-sided confidence bound on 0/5 is 45%, and thus this indicates inconsistency with an 8-month median PFS. The study may need to pause accrual to evaluate if PFS in any of the first 5 patients is ≥ 8 months.
untitled h1
cohort 2 metastatic tnbc

Data which have been published suggest that a response rate (PR+CR) for treatment of recurrent or metastatic TNBC is approximately 30-35% (13-15). In order to establish the efficacy of this treatment in patients with metastatic TNBC, the primary objective in this cohort would be to determine if using ONC201 would rule out a 25% response rate and result in a response rate consistent with 45%. As such, this cohort will be evaluated using a single stage design in order to rule out an unacceptably low PR+CR rate of 25% (p0=0.25) in favor of an improved response rate of 45% (p1=0.45). Based on a one-sided exact binomial test, 29 patients would have 82% power to rule out 25% and result in findings consistent with a 45% PR + CR rate. At the end of the study, we would form the two-sided 80% and 95% CI about the observed percentages of both response (PR+CR) and clinical benefit (SD+PR+CR). 11 responses (PR+CR) in 29 patients (37.9%) has a two-sided 80% CI of 25.7-51.5%, which would be sufficiently interesting for further evaluation. As an early stopping rule, after 10 evaluable patients have been enrolled, if there are 0 or 1 with clinical benefit (SD+PR+CR), then the cohort would end accrual as soon as this would be determined since the 90% one-sided upper CI bound on 1/10 is 33.7%, which would be marginally too low for further interest. Both the PR+CR and SD+PR+CR rates can be formed at the interim time for descriptive purposes as well as for evaluation for early stopping. The study may need to pause accrual to evaluate if clinical benefit in the first 10 patients exceeds the early stopping rule.
untitled h1
cohort 3 advanced endometrial cancer

Published data from several trials report response rates of 8 to 15% for second line treatment in patients with advanced endometrial cancer (18-21). As such, it would be desirable if new treatments are associated with at least a 12-15% response rate for further consideration. In patients with advanced endometrial cancer, the primary objective of this study would be to determine if using ONC201 would rule out a 5% response rate and result in a response rate consistent with 25%. As such, the trial will be conducted using a single stage design (42) in order to rule out an unacceptably low PR+CR rate of 5% (p0=0.05) in favor of an improved response rate of 25%
(p1=0.25). Based on a one-sided exact binomial test, 25 patients would have 90% power to rule out 5% and result in findings consistent with a 25% PR + CR rate. At the end of the study, we would form the two-sided 80% and 95% CI about the observed percentages of both response (PR+CR) and clinical benefit (SD+PR+CR). 3 responses (PR + CR) in 25 patients (12.0%) has a two-sided 80% CI of 4.5-24.8%, which is potentially worthy of further consideration, while 4 responses in 25 (16.0%) has a two-sided 80% CI of 7.2-29.5%, which would be sufficiently interesting for further evaluation. As an early stopping rule, after 13 evaluable patients have been enrolled, if there are 0 patients with clinical benefit (SD+PR+CR), then the cohort would end accrual since the 90% onesided upper CI bound on 0/13 is 16.2%, which would be marginally too low for further interest.  
Both the PR+CR and SD+PR+CR rates can be formed at the interim time for descriptive purposes as well as for evaluation for early stopping. The study may need to pause accrual to evaluate if clinical benefit in the first 13 patients exceeds the early stopping rule. Any secondary evaluations performed will be done in an exploratory fashion, with results presented without any formal adjustment for multiple comparisons. It is expected that approximately 1 patient per month may enroll onto this trial in the ER+ breast cancer cohort, and that 6 to 8 patients per year may enroll in each of the other two cohorts. Thus, it is expected that 3 to 4 years may be required in order to enroll up to 24+29+25=78 evaluable patients. In order to allow for a small number of unevaluable patients, the accrual ceiling will be set at 90 patients. 10.3 POPULATIONS FOR ANALYSES Evaluable for toxicity: All patients will be evaluable for toxicity from the time of their first treatment with ONC201. Evaluable for objective response: Only those patients who have measurable disease present at baseline, have received at least one cycle of therapy, and have had their disease re-evaluated will be considered evaluable for response. These patients will have their response classified according to the definitions stated below. (Note: Patients who exhibit objective disease progression prior to the end of cycle 1 will also be considered evaluable.) Evaluable Non-Target Disease Response: Patients who have lesions present at baseline that are evaluable but do not meet the definitions of measurable disease, have received at least one cycle of therapy, and have had their disease re-evaluated will be considered evaluable for non-target disease. The response assessment is based on the presence, absence, or unequivocal progression of the lesions. Evaluable for Progression Free Survival: In all three cohorts, all participants who took at least one cycle of study drug and have adequate follow-up to be included in a PFS analysis (cohort 1) or to have response and clinical benefit evaluated (cohorts 2 and 3) will be included in the analyses. 10.4 STATISTICAL ANALYSES
untitled h1
10.4.1 general approach

Analyses performed will be descriptive, reporting the outcome measure indicated along with twotailed 80% and 95% confidence intervals for PFS (cohort 1), and 80% and 95% confidence intervals about the response rate and clinical benefit rate in cohorts 2 and 3.
untitled h1
10.4.2 analysis of the primary

Cohort 1: Kaplan-Meier curves for PFS using all patients who receive treatment and are eligible for evaluation will be included, and appropriate confidence intervals (80% and 95% two sided confidence intervals) at selected time points will be provided to help interpret results relative to the expected results. Cohort 2 and 3: At the end of the trial we will form the two-sided 80% and 95% CI about the observed percentages of both response (PR+CR) and clinical benefit (SD+PR+CR).
untitled h1
10.4.3 analysis of the secondary

- Cohort 1 HR+BC - Safety—AEs will be tabulated, ORR—response rate will be reported along with 95% two-sided confidence interval, clinical benefit rate (CBR; CR + PR + SD by RECIST) clinical benefit rate will be reported along with 95% two-sided confidence interval  
- Cohort 2 TNBC - Safety-AEs will be tabulated CBR, clinical benefit rate will be reported along with 95% two-sided confidence interval; PFS by Kaplan-Meier analysis
- Cohort 3 Endometrial Cancer - Safety, CBR clinical benefit rate will be reported along with 95% two-sided confidence interval, PFS by Kaplan-Meier analyses
untitled h1
10.4.4 safety analyses

The safety of ONC201 tabulations be evaluated by descriptive statistics of each adverse event type and grade.
untitled h1
10.4.6 planned interim analyses

The interim analyses are described for each cohort in the sample size determination paragraph.
untitled h1
10.4.9 exploratory analyses

Blood samples at baseline and Day 1 of each subsequent cycle, as well as biopsy specimens at baseline and cycle 2 will yield a set of biomarkers, such as markers of apoptosis, indications of mitochondrial damage and immune subsets. The difference of the values at cycle 1 vs. baseline and cycle 2 vs. baseline will be determined, and these differences will be used as a covariate for analysis of PFS in each cohort to see if the level of change in biomarkers may be associated with PFS. These analyses will be done in an exploratory fashion using Kaplan-Meier curves and log rank tests. The changes in the biomarkers will also be compared between responders and nonresponders in the three cohorts using an exact form of a Wilcoxon rank sum test. All of these explorations will be done only if adequate patient data are available. Results will be presented without adjustment for multiple comparisons.
untitled h1
11 collaborative agreements 11.1 clinical

The study agent, ONC201 is provided by Oncoceutics., Inc. under a Clinical Trial Agreement (NCI CTA \#1035). Results or study data may be communicated to CTA partner, according to the terms of the NIH- Advanced Accelerator Applications SA CTA.
untitled h1
12 human subjects protections 12.1

Subjects from all racial and ethnic groups are eligible for this trial if they meet the eligibility criteria. Efforts will be made to extend the accrual to a representative population. If differences in outcome that correlate to racial or ethnic identity are noted, accrual may be expanded or additional studies may be performed to investigate those differences more fully.
untitled h1
12.2 participation of children

The age group for enrollment on this trial is 18 or more years of age. Because no dosing or adverse event data are currently available on the use of ONC201 in participants < 18 years of age, children are excluded from this study.
untitled h1
12.3 participation of subjects unable

Adults unable to give consent are excluded from enrolling in the protocol. However, re-consent may be necessary and there is a possibility, though unlikely, that subjects could become decisionally impaired. For this reason and because there is a prospect of direct benefit from research participation (section **12.5**), all subjects will be offered the opportunity to fill in their wishes for research and care, and assign a substitute decision maker on the "NIH Advance Directive for Health Care and Medical Research Participation" form so that another person can make decisions about their medical care in the event that they become incapacitated or cognitively impaired during the course of the study. Note: The PI or AI will contact the NIH Ability to Consent Assessment Team (ACAT) for evaluation as needed for the following: an independent assessment of whether an individual has the capacity to provide consent; assistance in identifying and assessing an appropriate surrogate when indicated; and/or an assessment of the capacity to appoint a surrogate. For those subjects that become incapacitated and do not have pre-determined substitute decision maker, the procedures described in NIH HRPP SOP 14E for appointing a surrogate decision maker for adult subjects who are (a) decisionally impaired, and (b) who do not have a legal guardian or durable power of attorney, will be followed.
untitled h1
12.4 evaluation of benefits and

ONC201 is an investigational agent for use in metastatic breast cancer and in advanced endometrial cancer. The protocol provides for detailed and careful monitoring of all patients to assess for toxicity. Toxicity data will be collected and reviewed to ensure that there were no severe toxicities that would preclude further patient enrollment. Patients will be treated with therapeutic intent and response to the therapy will be closely monitored.  
Version Date: 06/17/2020
untitled h1
12.5 risks/benefits analysis 12.5.1 benefits

The potential benefit to a patient on this study is a reduction or stability in breast cancer or endometrial cancer, as well as potentially the prevention or delay in development of new metastatic lesions, which may or may not have favorable impact on symptoms and/or survival.
untitled h1
12.5.2 risks 12.5.2.1 study drug

Risks include the possible occurrence of any of a range of side effects which are listed in the Consent Document or in section **13.1.2** of this protocol document. Frequent monitoring for adverse effects will help to minimize the risks associated with administration of the study agents.  
12.5.2.2 Radiation Risks The study will involve radiation from the following sources:
- Up to 3 CT scans for the collection of biopsies  
- Up to 8 CT scans per year for disease assessment
- 1 technetium-99 bone scan for disease assessment
Subjects may be exposed to approximately 11.59 rem. This amount is more than would be expected from everyday background radiation. Being exposed to excess radiation can increase the risk of cancer. The risk of getting cancer from the radiation exposure in this study is 1.2 out of 100 (1.2 %) and of getting a fatal cancer is 0.6 out of 100 (0.6%). 12.5.2.3 Risks from Blood Draws: Side effects of blood draws include pain and bruising, lightheadedness, and rarely, fainting. 12.5.2.4 Risks of CT Scans: In addition to the radiation risks discussed above, risks associated with CT scans are allergic reaction to and kidney damage from the contrast dye, nausea, vomiting, and anxiety. 12.5.2.5 Risks of Technetium-99 Bone Scans: In addition to the radiation risks described above, risks associated with technetium-99 bone scans are pain, bruising, injection site infection, and allergic reaction to the contrast agent. 12.5.2.6 Specimen Collection Risks Risks include those associated with specimen collection including pain, bleeding and the possibility of infection at the sampling site. All care will be taken to minimize risks that may be incurred by tumor sampling. However, there are procedure-related risks (such as bleeding, infection and visceral injury) that will be explained fully during informed consent. If patients suffer any physical injury as a result of the biopsy, immediate medical treatment is available at the NIH's Clinical Center in Bethesda, Maryland. Although no compensation is available, any injury will be fully evaluated and treated in keeping with the benefits or care to which patients are entitled under applicable regulations.
untitled h1
12.5.3 risks/benefits analysis

The potential benefits from this therapy are stabilization or shrinkage of the tumor and a reduction in chances of developing new lesions, with decrease of symptoms caused be progressive disease. Given the efforts to minimize risk with the administration of this combination, this protocol involves greater than minimal risk, but presents the potential for direct benefit to individual subjects.
untitled h1
12.6 consent process and documentation

The informed consent document will be provided to the participant or consent designee(s) (e.g., the legally authorized representative [LAR] if participant is an adult unable to consent) for review prior to consenting. A designated study investigator will carefully explain the procedures and tests involved in this study, and the associated risks, discomforts and benefits. In order to minimize potential coercion, as much time as is needed to review the document will be given, including an opportunity to discuss it with friends, family members and/or other advisors, and to ask questions of any designated study investigator. A signed informed consent document will be obtained prior to entry onto the study. The initial consent process as well as re-consent, when required, may take place in person or remotely (e.g., via telephone or other NIH approved remote platforms) per discretion of the designated study investigator and with the agreement of the participant/consent designee(s). Whether in person or remote, the privacy of the subject will be maintained. Consenting investigators (and participant/consent designee, when in person) will be located in a private area (e.g., clinic consult room). When consent is conducted remotely, the participant/consent designee will be informed of the private nature of the discussion and will be encouraged to relocate to a more private setting if needed. For the optional biopsy in the protocol, the patient will consent at the time of the procedure. If the patient refuses the optional biopsy at that time, the refusal will be documented in the medical record and in the research record.
untitled h1
12.6.1 request for waiver of

Prior to the subject signing the consent for this study pre-screening activities listed in section **2.5.1**
may be performed. We request a waiver of consent for these activities as they involve only minimal risk to the subjects. A waiver will not adversely affect the rights and welfare of the subjects given that the activities are only intended to determine suitability for screening for participation in research protocols. These activities could not practicably be carried out without the wavier as central recruiting services, utilized in the NIH Clinical Center, perform pre-screening activities for multiple studies and obtaining consent for each one is beyond their resources. The subjects will be provided with additional pertinent information after participation as they will be informed whether or not they are eligible to sign a consent for additional screening. 13 PHARMACEUTICAL INFORMATION 13.1 DRUG ONC201 (IND \# 136939)
untitled h1
13.1.1 source

ONC201 is an investigational drug supplied under a Clinical Trials Agreement by Oncoceutics INC.
untitled h1
13.1.2 toxicity

Generally, ONC201 is well tolerated at all of the tested dose levels. To date, over 70 patients have taken at least one dose of ONC201 and no Grade IV events have been reported. One grade II
allergic reaction occurred after the 5th dose of ONC201 625mg (q3 week schedule) that was managed by standard over the counter medications. One grade III neutropenia was transiently observed after the 5th dose of ONC201. The dose was reduced to 500mg q3weeks without recurrence of the neutropenia (**Table 6**).(2, 4)
Table 6: All Grade I AEs attributed (possibly related) to ONC201 on every 3-week dosing schedule for the Phase I trial.  
| ONC201 Dose      | 125mg   | 250mg   | 375mg   | 500mg   |    |
|------------------|---------|---------|---------|---------|----|
| No. of Patients  | 1       | 1       | 1       | 1       |    |
| Pyrexia          | 1       | 0       | 0       | 0       | 0  |
| Fatigue          | 1       | 0       | 0       | 0       | 0  |
| Elevated Amylase | 0       | 0       | 0       | 0       | 2  |
| Emesis           | 0       | 0       | 0       | 0       | 1  |
| Nausea           | 0       | 0       | 0       | 0       | 1  |  
625mg  
| 24   |
|------|  
  
* Note: No drug-related AE > Grade 1 reported on the Phase I solid tumor trial While human exposure to ONC201 is still somewhat limited at this time, side effects seen in animals included the following:  
- Nausea
- Salivation
- Vomiting
- Abnormal breathing
- Twitching
- Abnormal walking or standing  
  
While side effects are minimal, based on pharmacokinetic data, it would be reasonable to dose reduce if more significant side effects did occur. Based on the mean AUC and Cmax data from the phase I study, a dose reduction to 500mg weekly with two further reduction to 375mg weekly and then 250mg weekly would be reasonable and would likely not significantly affect clinical benefit
(**Figure 5**).  
Figure 5: Mean AUC and Cmax for each dose cohort following first dose of ONC201. *Image* adapted *from Stein et al, 2015* (2).  
The metabolism and excretion of ONC201 is still unknown at this time. However, it is suspected to be excreted by the kidneys. This is supported by preliminary observations in rats (unpublished data, communication with Oncoceutics, Inc). The effects of ONC201 on the developing human fetus are unknown. For this reason, women of childbearing potential and men must agree to use adequate contraception (hormonal or barrier method of birth control; abstinence) prior to study entry and for the duration of study participation. Should a woman become pregnant or suspect she is pregnant while she or her partner is participating in this study, she should inform her treating physician immediately. An assessment should be made to ensure the potential benefit would justify a potential risk to the fetus. 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 2 months after the last dose.
untitled h1
13.1.3 formulation and preparation

The investigational drug product is a hydroxypropyl methylcellulose (HPMC) capsule filled with ONC201 dihydrochloride, intended for oral administration. Each capsule of drug product contains the equivalent 125mg of anhydrous ONC201 free base with or without microcrystalline cellulose. This corresponds to ~150mg of drug substance that corrects.  
| dihydrochloride salt. ONC201 dihydrochloride is a white to off-white solid, freely soluble in water.   |                                          |
|--------------------------------------------------------------------------------------------------------|------------------------------------------|
| Compound Code(s)                                                                                       | ONC201•2HCI                              |
| ONC201                                                                                                 |                                          |
| TIC10                                                                                                  |                                          |
| MLS003171082                                                                                           |                                          |
| NC-350625                                                                                              |                                          |
| 41276-02-2                                                                                             |                                          |
| AC1L7JLY NCIStruc1_00799                                                                               |                                          |
| NCIStruc2_001940                                                                                       |                                          |
| Aternative Name(s)                                                                                     | CG-37418                                 |
| NCGC00014802                                                                                           |                                          |
| NCI350625                                                                                              |                                          |
| NCGC00014802-02                                                                                        |                                          |
| NCGC00097903-01                                                                                        |                                          |
| NCI60_003126                                                                                           |                                          |
| SMR001874991                                                                                           |                                          |
| Chemical Name(s)                                                                                       | 7-benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9- |
| hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(4H)-                                                   |                                          |
| one•2HCl                                                                                               |                                          |
| Chemical Abstracts Service                                                                             | 41276-02-2                               |
| (CAS) number                                                                                           | C24H26N40 (free base)                    |
| Molecular Formula                                                                                      | C24H26N4O•2HCl (salt)                    |
| 36.49 (free base)                                                                                      |                                          |
| Molecular Weight                                                                                       | 459.41 (salt)                            |
| Molecular Structure                                                                                    |                                          |
untitled h1
13.1.4 stability and storage

The capsules are to be stored in a closed container at room temperature (15 to 30°C).  
Based on the current stability data room temperature (25°C/60%RH) will be used for the drug product storage. No shelf life has been established for this product at this point. However, representative clinical trial batches will be placed on stability. Any batches that are out of specifications will be removed from use.  
The product is stored in a multi-dose container. The capsules are packaged in high-density polyethylene (HDPE) white opaque bottles, closed with an induction seal and capped with a white ribbed SecuRx® polypropylene (PPE) cap. The capsules are to be stored in the original closed container at room temperature (15 to 30°C).
untitled h1
administration procedures

ONC201 is taken by mouth every 7 days by the patient. Pharmacy can dispense the exact number of capsules needed for cycle, as prescribed by the physician, by transferring them to a white, opaque, HDPE container with child-proof closures. This type of container is the same as the one the drug is initially supplied in. Remaining capsules will be kept in the original bottle and will be dispensed for the subsequent prescription/subject.
untitled h1
13.1.5 incompatibilities

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 induced of the CYP450 system (CYP1A2, 2B6, 2B6, and 3A4). ONC201 was observed to be a mild inhibitor of the CYP450 enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 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 ONC201; 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.
untitled h1
14 references

1. Allen JE, Krigsfeld G, Mayes PA, Patel L, Dicker DT, Patel AS, Dolloff NG, Messaris E, Scata KA, Wang W, Zhou JY, Wu GS, El-Deiry WS. Dual inactivation of Akt and ERK by TIC10 signals Foxo3a nuclear translocation, TRAIL gene induction, and potent antitumor effects. Sci Transl Med. 2013;5(171):171ra17. doi: 10.1126/scitranslmed.3004828. PubMed PMID: 23390247; PMCID: PMC4535715. 2. Stein MN, Chan N, Silk AW, Fang B, Kaufman H, Haffty BG, Saunders T, Najmi S, Zheng L, Stogniew M, Allen JE, Oster W, Bertino JR, Mehnert JM. First-in human trial of ONC201 in patients with refractory solid tumors. J Clin Oncol. 2016;34(suppl; abstract 2514). 3. Endo Greer Y, Lipkowitz S. ONC201: Stressing tumors to death. Sci Signal.  
2016;9(415):fs1. doi: 10.1126/scisignal.aad7955. PubMed PMID: 26884598.  
4. Stein MN, Bertino JR, Kaufman HL, Mayer T, Moss R, Silk A, Chan N, Malhotra J, Rodriguez-Rodriguez L, Aisner J, Aiken RD, Haffty BG, DiPaola RS, Saunders T, Zloza A, Damare S, Beckett Y, Yu B, Najmi S, Gabel C, Dickerson S, Zheng L, El-Deiry WS, Allen JE, Stogniew M, Oster W, Mehnert JM. First-in-human Clinical Trial of Oral ONC201 in Patients with Refractory Solid Tumors. Clin Cancer Res. 2017. Epub 2017/03/22. doi: 10.1158/1078-0432.CCR16-2658. PubMed PMID: 28331050.  
5. Ryerson AB, Eheman CR, Altekruse SF, Ward JW, Jemal A, Sherman RL, Henley SJ, Holtzman D, Lake A, Noone AM, Anderson RN, Ma J, Ly KN, Cronin KA, Penberthy L, Kohler BA. Annual Report to the Nation on the Status of Cancer, 1975-2012, featuring the increasing incidence of liver cancer. Cancer. 2016;122(9):1312-37. doi: 10.1002/cncr.29936. PubMed PMID: 26959385; PMCID: PMC4840031. 6. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin. 2017;67(1):730. Epub 2017/01/05. doi: 10.3322/caac.21387. PubMed PMID: 28055103. 7. Colleoni M, Sun Z, Price KN, Karlsson P, Forbes JF, Thürlimann B, Gianni L, Castiglione M, Gelber RD, Coates AS, Goldhirsch A. Annual Hazard Rates of Recurrence for Breast Cancer During 24 Years of Follow-Up: Results From the International Breast Cancer Study Group Trials I to V. J Clin Oncol. 2016;34(9):927-35. doi: 10.1200/JCO.2015.62.3504. PubMed PMID: 26786933. 8. Metzger-Filho O, Sun Z, Viale G, Price KN, Crivellari D, Snyder RD, Gelber RD, Castiglione-Gertsch M, Coates AS, Goldhirsch A, Cardoso F. Patterns of Recurrence and outcome according to breast cancer subtypes in lymph node-negative disease: results from international breast cancer study group trials VIII and IX. J Clin Oncol. 2013;31(25):3083-90. doi: 10.1200/JCO.2012.46.1574. PubMed PMID: 23897954; PMCID: PMC3753700. 9. Reinert T, Barrios CH. Optimal management of hormone receptor positive metastatic breast cancer in 2016. Ther Adv Med Oncol. 2015;7(6):304-20. doi: 10.1177/1758834015608993. PubMed PMID: 26557899; PMCID: PMC4622303. 10. Carrick S, Parker S, Thornton CE, Ghersi D, Simes J, Wilcken N. Single agent versus combination chemotherapy for metastatic breast cancer. Cochrane Database Syst Rev. 2009(2):CD003372. Epub 2009/04/15. doi: 10.1002/14651858.CD003372.pub3. PubMed PMID: 19370586. 11. Robert NJ, Diéras V, Glaspy J, Brufsky AM, Bondarenko I, Lipatov ON, Perez EA,
Yardley DA, Chan SY, Zhou X, Phan SC, O'Shaughnessy J. RIBBON-1: randomized, doubleblind, placebo-controlled, phase III trial of chemotherapy with or without bevacizumab for firstline treatment of human epidermal growth factor receptor 2-negative, locally recurrent or metastatic breast cancer. J Clin Oncol. 2011;29(10):1252-60. doi: 10.1200/JCO.2010.28.0982. PubMed PMID: 21383283. 12. Gray R, Bhattacharya S, Bowden C, Miller K, Comis RL. Independent review of E2100: a phase III trial of bevacizumab plus paclitaxel versus paclitaxel in women with metastatic breast cancer. J Clin Oncol. 2009;27(30):4966-72. Epub 2009/08/31. doi: 10.1200/JCO.2008.21.6630. PubMed PMID: 19720913; PMCID: PMC2799052. 13. Brenton JD, Carey LA, Ahmed AA, Caldas C. Molecular classification and molecular forecasting of breast cancer: Ready for clinical application? Journal of Clinical Oncology. 2005;23(29):7350-60. doi: 10.1200/jco.2005.03.3845. PubMed PMID: WOS:000232546200012. 14. Bauer KR, Brown M, Cress RD, Parise CA, Caggiano V. Descriptive analysis of estrogen receptor (ER)negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype - A population-based study from the California Cancer Registry. Cancer. 2007;109(9):1721-8. doi: 10.1002/cncr.22618. PubMed PMID: WOS:000245937000004. 15. Tutt A, Ellis P, Kilburn L, Gilett C, Pinder S, Abraham J, Barrett S, Barrett-Lee P, Chan S, Cheang M, Dowsett M, Fox L, Gazinska P, Grigoriadis A, Gutin A, Harper-Wynne C, Hatton M, Kernaghan S, Lanchbury J, Morden J, Owen J, Parikh J, Parker P, Rahman N, Roylance R, Shaw A, Smith I, Thompson R, Timms K, Tovey H, Wardley A, Wilson G, Harries M, Bliss J. The TNT trial: A randomized phase III trial of carboplatin (C) compared with docetaxel (D) for patients with metastatic or recurrent locally advanced triple negative or BRCA1/2 breast cancer (CRUK/07/012). Cancer Research. 2015;75(9):2. doi: 10.1158/1538-7445.sabcs14-s3-01. PubMed PMID: WOS:000356730200018. 16. Ueda SM, Kapp DS, Cheung MK, Shin JY, Osann K, Husain A, Teng NN, Berek JS, Chan JK. Trends in demographic and clinical characteristics in women diagnosed with corpus cancer and their potential impact on the increasing number of deaths. Am J Obstet Gynecol.  
2008;198(2):218.e1-6. doi: 10.1016/j.ajog.2007.08.075. PubMed PMID: 18226630. 17. Win AK, Lindor NM, Winship I, Tucker KM, Buchanan DD, Young JP, Rosty C, Leggett B, Giles GG, Goldblatt J, Macrae FA, Parry S, Kalady MF, Baron JA, Ahnen DJ, Marchand LL, Gallinger S, Haile RW, Newcomb PA, Hopper JL, Jenkins MA. Risks of colorectal and other cancers after endometrial cancer for women with Lynch syndrome. J Natl Cancer Inst. 2013;105(4):274-9. Epub 2013/02/05. doi: 10.1093/jnci/djs525. PubMed PMID: 23385444; PMCID: PMC3576323. 18. Ray M, Fleming G. Management of advanced-stage and recurrent endometrial cancer. Semin Oncol. 2009;36(2):145-54. doi: 10.1053/j.seminoncol.2008.12.006. PubMed PMID: 19332249. 19. Humber CE, Tierney JF, Symonds RP, Collingwood M, Kirwan J, Williams C, Green JA. Chemotherapy for advanced, recurrent or metastatic endometrial cancer: a systematic review of Cochrane collaboration. Ann Oncol. 2007;18(3):409-20. doi: 10.1093/annonc/mdl417. PubMed PMID: 17150999. 20. Sorbe B, Andersson H, Boman K, Rosenberg P, Kalling M. Treatment of primary advanced and recurrent endometrial carcinoma with a combination of carboplatin and paclitaxel-long-term follow-up. Int J Gynecol Cancer. 2008;18(4):803-8. doi: 10.1111/j.1525-1438.2007.01094.x. PubMed PMID: 17944917. 21. Dellinger TH, Monk BJ. Systemic therapy for recurrent endometrial cancer: a review of North American trials. Expert Rev Anticancer Ther. 2009;9(7):905-16. doi: 10.1586/era.09.54. PubMed PMID: 19589030. 22. Allen JE, Kline CL, Prabhu VV, Wagner J, Ishizawa J, Madhukar N, Lev A, Baumeister M,
Zhou L, Lulla A, Stogniew M, Schalop L, Benes C, Kaufman HL, Pottorf RS, Nallaganchu BR,
Olson GL, Al-Mulla F, Duvic M, Wu GS, Dicker DT, Talekar MK, Lim B, Elemento O, Oster W, Bertino J, Flaherty K, Wang ML, Borthakur G, Andreeff M, Stein M, El-Deiry WS. Discovery and clinical introduction of first-in-class imipridone ONC201. Oncotarget. 2016. doi: 10.18632/oncotarget.11814. PubMed PMID: 27602582. 23. Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters SA, Blackie C, Chang L, McMurtrey AE, Hebert A, DeForge L, Koumenis IL, Lewis D, Harris L, Bussiere J, Koeppen H, Shahrokh Z, Schwall RH. Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest. 1999;104(2):155-62. doi: 10.1172/JCI6926. PubMed PMID: 10411544; PMCID: PMC408479. 24. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, Sutherland GR, Smith TD, Rauch C, Smith CA, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity. 1995;3(6):673-82. PubMed PMID: 8777713. 25. Rahman M, Pumphrey JG, Lipkowitz S. The TRAIL to targeted therapy of breast cancer. Adv Cancer Res. 2009;103:43-73. doi: 10.1016/S0065-230X(09)03003-6. PubMed PMID: 19854352; PMCID: PMC3538140. 26. Kischkel FC, Lawrence DA, Chuntharapai A, Schow P, Kim KJ, Ashkenazi A. Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity. 2000;12(6):611-20. PubMed PMID: 10894161. 27. Ashkenazi A. Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer. 2002;2(6):420-30. doi: 10.1038/nrc821. PubMed PMID: 12189384. 28. Ashkenazi A. Targeting the extrinsic apoptotic pathway in cancer: lessons learned and future directions. J Clin Invest. 2015;125(2):487-9. doi: 10.1172/JCI80420. PubMed PMID: 25642709; PMCID: PMC4319431. 29. Dine JL, O'Sullivan CC, Voeller D, Greer YE, Chavez KJ, Conway CM, Sinclair S, Stone B, Amiri-Kordestani L, Merchant AS, Hewitt SM, Steinberg SM, Swain SM, Lipkowitz S. The TRAIL receptor agonist drozitumab targets basal B triple-negative breast cancer cells that express vimentin and Axl. Breast Cancer Res Treat. 2016;155(2):235-51. doi: 10.1007/s10549-015-3673-z. PubMed PMID: 26759246; PMCID: PMC4753803. 30. Rahman M, Davis SR, Pumphrey JG, Bao J, Nau MM, Meltzer PS, Lipkowitz S. TRAIL induces apoptosis in triple-negative breast cancer cells with a mesenchymal phenotype. Breast Cancer Res Treat. 2009;113(2):217-30. doi: 10.1007/s10549-008-9924-5. PubMed PMID: 18266105; PMCID: PMC2615075. 31. Yang A, Wilson NS, Ashkenazi A. Proapoptotic DR4 and DR5 signaling in cancer cells: toward clinical translation. Curr Opin Cell Biol. 2010;22(6):837-44. doi: 10.1016/j.ceb.2010.08.001. PubMed PMID: 20813513. 32. Ishizawa J, Kojima K, Chachad D, Ruvolo P, Ruvolo V, Jacamo RO, Borthakur G, Mu H, Zeng Z, Tabe Y, Allen JE, Wang Z, Ma W, Lee HC, Orlowski R, Sarbassov dD, Lorenzi PL, Huang X, Neelapu SS, McDonnell T, Miranda RN, Wang M, Kantarjian H, Konopleva M, Davis RE, Andreeff M. ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies. Sci Signal. 2016;9(415):ra17. doi: 10.1126/scisignal.aac4380. PubMed PMID: 26884599; PMCID: PMC4815038. 33. Kline CL, Van den Heuvel AP, Allen JE, Prabhu VV, Dicker DT, El-Deiry WS. ONC201 kills solid tumor cells by triggering an integrated stress response dependent on ATF4 activation by specific eIF2α kinases. Sci Signal. 2016;9(415):ra18. doi: 10.1126/scisignal.aac4374. PubMed PMID: 26884600. 34. Markianos M, Hatzimanolis J, Lykouras L. Neuroendocrine responsivities of the pituitary dopamine system in male schizophrenic patients during treatment with clozapine, olanzapine, risperidone, sulpiride, or haloperidol. Eur Arch Psychiatry Clin Neurosci. 2001;251(3):141-6. PubMed PMID: 11697576. 35. Greer YE, Porat-Shliom N, Nagashima K, Stuelten C, Crooks D, Koparde VN, Gilbert SF, Islam C, Ubaldini A, Ji Y, Gattinoni L, Soheilian F, Wang X, Hafner M, Shetty J, Tran B, Jailwala P, Cam M, Lang M, Voeller D, Reinhold WC, Rajapakse V, Pommier Y, Weigert R, Linehan WM, Lipkowitz S. ONC201 kills breast cancer cells in vitro by targeting mitochondria. Oncotarget. 2018;9(26):18454-79. doi: 10.18632/oncotarget.24862. PubMed PMID: 29719618; PMCID: PMC5915085. 36. Graves PR, Aponte-Collazo LJ, Fennell EMJ, Graves AC, Hale AE, Dicheva N, Herring LE, Gilbert TSK, East MP, McDonald IM, Lockett MR, Ashamalla H, Moorman NJ, Karanewsky DS, Iwanowicz EJ, Holmuhamedov E, Graves LM. Mitochondrial Protease ClpP is a Target for the Anticancer Compounds ONC201 and Related Analogues. ACS Chem Biol. 2019. doi:
10.1021/acschembio.9b00222. PubMed PMID: 31021596. 37. Ishizawa J, Zarabi SF, Davis RE, Halgas O, Nii T, Jitkova Y, Zhao R, St-Germain J, Heese LE, Egan G, Ruvolo VR, Barghout SH, Nishida Y, Hurren R, Ma W, Gronda M, Link T, Wong K, Mabanglo M, Kojima K, Borthakur G, MacLean N, Ma MCJ, Leber AB, Minden MD, Houry W,
Kantarjian H, Stogniew M, Raught B, Pai EF, Schimmer AD, Andreeff M. Mitochondrial ClpPMediated Proteolysis Induces Selective Cancer Cell Lethality. Cancer Cell. 2019. doi:
10.1016/j.ccell.2019.03.014. PubMed PMID: 31056398. 38. Hur KY, Lee MS. New mechanisms of metformin action: Focusing on mitochondria and the gut. J Diabetes Investig. 2015;6(6):600-9. doi: 10.1111/jdi.12328. PubMed PMID: 26543531; PMCID: PMC4627534. 39. Wagner J, Kline CL, Zhou L, Zloza A, Chesson C, Newman J, Kaufman H, Bertino J, Stein M, El-Deiry W, editors. Imipridone ONC201promotes intra-tumoral accumulation of CD3+/NK+ cells that contribute to its anti-tumor efficacy. AACR; 2017; Washington, DC. 40. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Dancey J, Arbuck S, Gwyther S, Mooney M, Rubinstein L, Shankar L, Dodd L, Kaplan R, Lacombe D, Verweij J. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45(2):228-47. Epub 2008/12/23. doi: 10.1016/j.ejca.2008.10.026. PubMed PMID: 19097774. 41. Brookmeyer R, Crowley J. A confidence interval for the median survival time. Biometrics. 1982;38:29-41. 42. Simon R. Controlled Clinical Trials. 1989. p. 1-10.
untitled h1
15 appendices

| 15.1                                                                                                                                                                                       | APPENDIX A-PERFORMANCE STATUS CRITERIA ECOG Performance Status Scale   | Karnofsky Performance Scale                                  |             |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------|--------------------------------------------------------------|-------------|
| Grade                                                                                                                                                                                      | Descriptions                                                           | Percent                                                      | Description |
| 100                                                                                                                                                                                        | Normal, no complaints, no  evidence of disease.                        |                                                              |             |
| Normal activity. Fully active,                                                                                                                                                             |                                                                        |                                                              |             |
| 0                                                                                                                                                                                          | able to carry on all pre-disease  performance without restriction.     | Able to carry on normal activity;                            |             |
| 90                                                                                                                                                                                         | minor signs or symptoms of  disease. Normal activity with effort;      |                                                              |             |
| 80                                                                                                                                                                                         | some signs or symptoms of  disease.                                    |                                                              |             |
| Symptoms, but ambulatory.  Restricted in physically strenuous  activity, but ambulatory and able  to carry out work of a light or  sedentary nature (e.g., light  housework, office work). |                                                                        |                                                              |             |
| 1                                                                                                                                                                                          | Cares for self, unable to carry on                                     |                                                              |             |
| 70                                                                                                                                                                                         | normal activity or to do active  work.                                 |                                                              |             |
| In bed <50% of the time.  Ambulatory and capable of all  self-care, but unable to carry out  any work activities. Up and  about more than 50% of waking  hours.                            | Requires occasional assistance,                                        |                                                              |             |
| 60                                                                                                                                                                                         | but is able to care for most of  his/her needs.                        |                                                              |             |
| 2                                                                                                                                                                                          | 50                                                                     | Requires considerable assistance  and frequent medical care. |             |
| 40                                                                                                                                                                                         | Disabled, requires special care  and assistance.                       |                                                              |             |
| In bed >50% of the time.  Capable of only limited self-care,  confined to bed or chair more  than 50% of waking hours.                                                                     |                                                                        |                                                              |             |
| 3                                                                                                                                                                                          | Severely disabled,                                                     |                                                              |             |
| 30                                                                                                                                                                                         | hospitalization indicated. Death  not imminent.                        |                                                              |             |
| 20                                                                                                                                                                                         | Very sick, hospitalization  indicated. Death not imminent.             |                                                              |             |
| 100% bedridden. Completely  disabled. Cannot carry on any  self-care. Totally confined to bed  or chair.                                                                                   |                                                                        |                                                              |             |
| 4                                                                                                                                                                                          | 10                                                                     | Moribund, fatal processes  progressing rapidly.              |             |
| 5                                                                                                                                                                                          | Dead.                                                                  | 0                                                            | Dead.       |  
15.2 APPENDIX B- METHOD FOR PREPARING PBMC SAMPLES FROM BLOOD FOR
PHARMACODYNAMIC (PD) STUDIES
1. Using a BD Vacutainer CPT, draw the blood sample. Assure that the anticoagulant being used (sodium citrate or sodium heparin) is correct for the intended use of the specimen and that the tube size is correct for the blood draw volume required by the protocol.  
2. Mix the sample by inverting the tube for 5-8 times and centrifuge the sample at 1500 g for 30min at 18-25°C, without break.  
3. After centrifuge, remove two thirds of plasma and transfer whitish PBMC layer into the labeled 15-ml conical tube using a 3-ml Falcon transfer pipet.  
4. Discard the Vacutainer CPT tube in biohazard waste container. 5. Add Plasma-Lyte A USP to the 15-ml tube to bring total volume to 14 ml, cap and mix by gentle inversion 5-8 times.  
6. Centrifuge the sample at 430 g for 10 min at 18-25°C, without break. 7. Using a new transfer pipet, aspirate as much supernatant as possible without disturbing the cell pellet and discard the supernatant into biohazardous liquid waste.  
8. Re-suspend the cell pellet in 2 ml of Plasma-Lyte A USP by gently flicking the bottom of the tube with the index finger and then gently pipet up and down a few times.  
9. Immediately after resuspending the cell pellet, transfer 10 ul of sample into a cell count to count the number of cells.  
10. The calculated volume required to make a suspension /ml.  
Transfer 1 mL aliquots of the PBMC cell suspension into individual 1.5-ml cryovials. Centrifuge the cryovials in Eppendorf centrifuge at 10000 rpm for 10 minutes at 4-10°C. Remove and discard as much supernatant as possible without disturbing the cell pellet.  
11. For viable cells storage, the cell pellet should be suspended in FBS with 10% DMSO at this stage and then stored at -80°C until use.  
15.3 APPENDIX C - SOP FOR CYTOKINE COLLECTION Appendix C -
untitled h1
time points of the sampling

- Venous blood should be collected from all patients enrolled in the protocols at the beginning of therapy, prior to the administration of the investigational agent. The pre-cycle 1 samples will serve as the baseline sample. Additional time points should be defined in the clinical protocol.
untitled h1
blood collection

- Collect 4 mL of blood in a venous blood BD Vacutainer® spray-coated K2EDTA
tube (example BD Cat\# 367861; http://www.bd.com/us/contact/). Gently invert 10 times to mix blood and anticoagulant.  
- Store the tubes at room temperature until centrifugation. It is generally preferred and often necessary to process the samples within 5 hours.
untitled h1
blood sample processing

- Centrifuge at 1300 RCF for 10 min in a swing-head centrifuge, or 15 min in a fixed angle centrifuge (balance the tube in the centrifuge). Do not use brake to stop centrifuge.  
- Carefully aspirate the supernatant (plasma) into a new centrifuge tube.  
- Inspect serum for turbidity. Turbid samples should be centrifuged and aspirated again to remove insoluble matter.
untitled h1
sample storage

- Carefully aliquot each sample into two cryovials with printed labels with a sample identifier and store them at -80oC.
untitled h1
shipping information

- One of the two sets of plasma samples should be sent to Dr. Liang Cao at the National Cancer Institute at the end of the study on dry ice. A separate sample sheet with at least a patient number and a sample identifier for each sample should be provided via secured electronic mail. A separate email with tracking information should be sent to the recipient at the time of shipping to ensure the receiving of the samples package on time. See Storage **Instructions** in Section 15.6 Appendix F for information regarding the second set of plasma samples.  
- Ship information is as follows:
Liang Cao, Ph.D. Genetics Branch Version Date: 06/17/2020 Center for Cancer Research National Cancer Institute 37 Convent Dr. MSC 4265 Bldg 37, Rm 6134 Bethesda, MD 20892-1906 Phone: (301) 435-9039 E-mail: caoli@mail.nih.gov
untitled h1
data collection and transmission

- Data will be generated in a table format.  
- Data will be transmitted via email to the PI of the studies in an Excel document.  
15.4 APPENDIX D- STANDARD OPERATING PROCEDURE FOR PLASMA AND BUFFY COAT COLLECTION
FOR CIRCULATING TUMOR DNA
Ref 1: Plasma protocol from Brenton Laboratory SOP at Cancer Research UK Cambridge Institute. Ref2: Manual for cell free DNA BCT® tubes from Streck labs for buffy coat isolation. Required consumables - Streck tubes (Cell free DNA BCT tubes, Streck Inc) with freshly drawn patient blood.. - 2ml screw-capped micro-tubes (Sarstedt: 72.694.006), Dnase/Rnase free. - 2ml non-screw-capped Non-Stick DNase/RNase-free Microfuge Tubes (Applied Biosystems: AM12475) - Transfer pipette (Fisher Scientific: 2655116) Procedure 1: Recommended procedure for the Plasma collection, processing, storage and transport of plasma for ctDNA and/or circulating nucleic acid studies IMPORTANT: Blood samples should be centrifuged as soon as possible after collection to avoid fragmentation, degradation and leukocyte lysis. Samples should be spun within 1 hour of collection.  
1. Collect 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 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. Within 1 hour of collection, centrifuge samples at 1600g for 10 minutes at room temperature using a "swing out" rotor. (NB. Centrifuge with the brake off). Record the time of centrifugation.  
Note: If adapters are not available, place the streck tubes into 50 mL conical tubes and then spin  
4. Transfer 1ml aliquots of plasma to sterile 2ml Microfuge tubes (DNase/RNase-free tubes can be used at this stage) and leave about 0.5 ml over the buffy coat (Refer figure on next page).
Take care to avoid any buffy coat layer in this step. See Procedur 2 below for separate collection of buffy coat for genomic DNA  
Version Date: 06/17/2020 5. Centrifuge the plasma aliquots in a bench top centrifuge at 14,000rpm (10,000 xg) for 10 minutes to pellet any remaining cellular debris.  
6. Carefully transfer 1ml aliquots of supernatant to sterile 2ml screw-capped micro-tubes, and discard the pellet.  
7. Label tubes with appropriate barcode labels as required by the Study. Freeze aliquots in a -
800C freezer in appropriately labeled storage boxes.  
Procedure 2. Preparation of buffy coat isolate from blood sample for isolation of total genomic DNA (gDNA).  
1. Following Step 4 in the plasma collection protocol 1 above, carefully transfer the buffy coat layer into a separate sterile screw capped tube using a transfer pipette or standard pipette. It may help to use a gentle swirling action with the tip to remove the buffy coat layer, taking care to avoid plasma and the red blood cell layer wherever possible. Volume should not exceed 0.5 -1 ml.  
2. Label tubes with appropriate barcode labels as required by the Study. Freeze tubes in a -
800C freezer in appropriately labelled storage boxes.  
Note: The buffy coat is going to contain erythrocytes and some plasma. This may be frozen directly in -80C freezer for months before being used for gDNA preparation. Qiagen blood DNA mini kits may be used and this requires about 200 ul of buffy coat. ** Electronically record all appropriate collection details (study and anonymized patient  
 identifier), collection date, sample volume, and sample/biorepository identifiers into study log records, as defined by study requirements. Record box number/row/column position, and if there were any sample handling or quality issues (e.g. if samples were hemolyzed).  
Figure 1. Streck tubes after centrifugation (step 3)  
  
Version Date: 06/17/2020 15.5 APPENDIX E- ORAL MEDICATION DIARY
untitled h1
oral medication diary for onc201

Patient's Name: __________________________________ Date: __________________ Patient Study ID: _________________________________ Cycle #: ________________
untitled h1
instructions to the patient

1. Complete one form for each cycle (28 days).  
2. You will take ___ (number) ____ mg (dosage) capsules each Tuesday Morning (white spaces) +/- 1 day (light gray space). You may take the capsules one hour before or two hours after eating, as you prefer. If you forget to take the pills on your assigned day, you can take the missed dose up to 1 day after (light gray space). Do not take the weekly dose if more than 1 day late after scheduled dose (dark gray spaces). 3. Record the date, the number of pills you took, and when you took them.  
4. If you have any comments or notice any side effects, please record them in the Comments column.  
5. Please bring your pill bottle and this form to your physician when you go for your next appointment.
untitled h1
general comments

| # pills and when taken:                                                            | # pills and when taken:   |      |        |            |      |          |
|------------------------------------------------------------------------------------|---------------------------|------|--------|------------|------|----------|
| Day                                                                                | ONC201                    | Day  | ONC201 |            |      |          |
| Comments                                                                           |                           |      |        |            |      |          |
| Date                                                                               | # Capsules                | Time | Date   | # Capsules | Time | Comments |
| 1                                                                                  | 15                        |      |        |            |      |          |
| 2                                                                                  | 16                        |      |        |            |      |          |
| 3                                                                                  | 17                        |      |        |            |      |          |
| 4                                                                                  | 18                        |      |        |            |      |          |
| 5                                                                                  | 19                        |      |        |            |      |          |
| 6                                                                                  | 20                        |      |        |            |      |          |
| 7                                                                                  | 21                        |      |        |            |      |          |
| 8                                                                                  | 22                        |      |        |            |      |          |
| 9                                                                                  | 23                        |      |        |            |      |          |
| 10                                                                                 | 24                        |      |        |            |      |          |
| 11                                                                                 | 25                        |      |        |            |      |          |
| 12                                                                                 | 26                        |      |        |            |      |          |
| 13                                                                                 | 27                        |      |        |            |      |          |
| 14                                                                                 | 28                        |      |        |            |      |          |
| Patient's Signature: ___________________________________Date: ____________________ |                           |      |        |            |      |          |  
Version Date: 06/17/2020 Study Team will complete this section:  
| 7.   | Date patient was removed from study _____________________     |
|------|---------------------------------------------------------------|
| 8.   | Patient's planned daily dose______________________________    |
| 9.   | Total number of pills taken this month ______________________ |  
Physician/Nurse's Signature ____________________________________________________________________
15.6 APPENDIX F- ONCOCEUTICS SPECIMEN COLLECTION, PROCESSING, STORAGE AND SHIPPING
3624 MARKET STREET; SUITE 5E  
  
UNIVERSITY CITY SCIENCE CENTER
PHILADELPHIA, PA 19104 SPONSOR CONTACT:
ROHINTON TARAPORE, PHD
TELEPHONE: 215-966-6115 E-MAIL: ROHINTON.TARAPORE@ONCOCEUTICS.COM
untitled h1
summary of laboratory studies

Laboratory studies on specimens obtained from patients receiving ONC201 treatment will be performed to characterize ONC201 exposure and its biological activity in advanced cancer patients. Blood for laboratory analysis will be drawn to assess plasma ONC201 drug concentration and serum tumor biomarkers.  
If available, as described in the protocol, archival tumor tissue will be accessed at the time of enrollment for subjects enrolled in this study to conduct future correlative assays to measure biomarkers of therapeutic response to ONC201 including molecular markers involved in the mechanism of action of ONC201. All blood and tissue specimens will be collected and prepared for shipping by the clinical sites.
untitled h1
blood sample collection and processing

At the specified time points, approximately 6 mL of blood will be collected in the specified tube type(s), to be processed, aliquoted into duplicate transfer vials and frozen, to be shipped to the designated analytical laboratory. Refer to the study protocol for blood sample collection time points. 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.
untitled h1
supplies for collection of blood

Plasma Sample Supplies Plasma samples are to be collected and aliquoted using the following commercially available tubes. For plasma blood collection Becton-Dickinson Plastic Whole Blood tube with spray-coated K2EDTA Lavender top, Vacutainer Catalogue 367525 Tube Size: 16x100 Draw Volume: 10mL For plasma aliquots Wheaton 2mL Ext FS CryoElite Blue Cap Patch Sterile Catalogue W985868
untitled h1
15.6.1 serum sample supplies

Serum samples are to be collected and aliquoted using the following commercially available tubes. For serum blood collection Serum Tube, Increased Silica Act Clot Activator, Silicone-Coated Interior Red Top Vacutainer BD Catalogue 366430 Version Date: 06/17/2020 Tube Size: 16 X 100 Draw Volume: 10mL For serum aliquot storage Wheaton 2mL Ext FS CryoElite Yellow Cap Patch Sterile Wheaton Catalogue W985866
untitled h1
collection of blood

Check the expiration dates on the vacutainer tubes before collecting blood. If the tubes have expired, discard them. Do not substitute another brand of collection tube. Prepare the blood collection tubes by affixing the barcode labels to each tube before collecting specimens from the subject. Using standard aseptic venipuncture techniques, draw the blood sample into the specified collection tube. For time points requiring both plasma and serum blood draws, the plasma sample should be drawn first. Remove tourniquet as soon as blood flow into the first vacutainer tube is established; excessive use of tourniquets can lead to hemoconcentration and falsely elevated results. Ensure each vacutainer is full to provide accurate blood:additive ratios. Mix blood tubes by gently inverting each tube 5 times. On the barcode label, clearly print with a black permanent marking pen the study number "XXX", patient ID number, the date and time of the blood draw (hours and minutes). Enter the information on the Patient-Specific Specimen Collection Log and maintain the log in the Patient source records. Place the full blood collection tube(s) into a resealable clear biohazard bag. Arrange prompt pick up by, or delivery to, the local laboratory since blood must be processed and frozen within 2 hours of collection.
untitled h1
processing of blood specimens 15.6.2

Centrifuge the filled lavender top tube at 1500 g (3000 rpm) for 15 minutes until the plasma and the cells are well separated. Label 2 blue cap transfer vials lengthwise with the appropriate barcode labels indicating "plasma (blue)". Clearly print the study number "Study XXX", patient ID number, date and time of the blood draw. Transfer ~1.5 mL of plasma into each of the 2 blue top transfer vials labeled "plasma (blue)."
untitled h1
15.6.3 serum processing

After filling the tubes, let the tubes stand at room temperature until a clot has formed in the red top tube (approximately 30 minutes). Centrifuge the tube at 1500 g (3000 rpm) for 15 minutes until the serum and the cells are well separated. Label 2 yellow cap transfer vials lengthwise with the appropriate barcode labels indicating "serum (red)". Clearly print the study number "Study XXX", the patient ID number, date and time of the blood draw. Transfer ~1.5 mL of serum into each of the 2 yellow top transfer vials labeled "serum (red)".
untitled h1
obtaining archived tumor tissue sections

Archived tumor blocks obtained prior to treatment with ONC201 are to be obtained as a baseline sample. If multiple blocks are available, preference will be given to the most recently obtained tissue. A total of 5 slides should be prepared from this archival tissue. The archival tissue should be returned to its original storage location. Using black permanent marking pen, clearly print the study number "Study XXX", patient ID number and the date of collection (i.e., biopsy) on each slide.
untitled h1
storage instructions

The clinical site laboratory is responsible for proper storage of the samples. Do NOT store plasma/serum specimens in a frost-free refrigerator-top freezer--results may be affected by temperature variation during the freezer cycle. Store in a freezer with minimum -20ºC or preferably -70ºC. Take all precautions to ensure the specimens remain frozen at the study site. Duplicate aliquots of plasma and serum specimens will be prepared. One set of aliquots is to be kept stored frozen at the clinical site laboratory, and the second set is to be shipped according to this procedures manual.
untitled h1
shipping instructions

All shipments must be authorized by the Sponsor. Before preparing the shipment, the clinical site personnel preparing the shipment must contact the Sponsor and e-mail a copy of the completed Specimen Shipping Form to the Sponsor Project Office: rohinton.tarapore@oncoceutics.com.
untitled h1
preparation of the dry ice

The frozen plasma/serum aliquots must be shipped on dry ice to the designated analytical laboratory. Ensure that specimens remain frozen during preparation of the shipment. Specimens must be shipped by overnight courier service on dry ice on a Monday, Tuesday or Wednesday only. Shipments are not to be initiated between Thursday and Sunday as it is not possible to ensure specimens will remain frozen if shipment is delayed, or upon receipt over weekends or on a holiday. All packaging must conform to IATA Packaging Instruction 650 for shipment of non-infectious human specimens. Samples from participants known to be infectious (Hepatitis B virus, HIV-I and HIV-II non-cultured) are not to be shipped. Ensure sufficient dry ice is used to keep the specimens solidly frozen until they arrive at the designated analytical laboratory. Place half of the dry ice in the bottom of the shipping box. Place the resealable bag containing the specimens in transfer vials, filter paper and absorbent on top of the dry ice. Do not put dry ice inside the plastic resealable bags. Cover and surround the specimens with the remaining dry ice. Place the lid on the Styrofoam portion of the shipper. DO NOT TAPE BOX SHUT. Ensure the Hazardous Goods label for Dry Ice is affixed to the box (diamond with upper half stripped and '9' at bottom). Record the amount of dry ice (in Kg or pounds) added to the box on the small label with a permanent black marking pen. Prepare shipment documentation for over-night shipping of the package (FedEx is preferred). Affix the clear, resealable plastic courier envelope to the side of the dry ice box. Please ask your local courier for a supply of these plastic courier envelopes. Prepare a Specimen Shipping Form and include it in the plastic courier envelope. Ask your courier for a version of the airway bill that allows you to indicate the shipment is packed with dry ice. Meticulous attention to labeling will ensure prompt delivery of dry ice shipments. The "Nature and Quantity of Goods" box of the air waybill must show "UN 3373", the text "BIOLOGICAL SUBSTANCE, CATEGORY B" and the number of packages. In addition, since the package contains dry ice, you also need to write "Dry Ice - UN1845" and the number of packages and the net quantity of dry ice per package. Finally, these markings must be accompanied by the Class 9 label for Miscellaneous Dangerous Goods.
untitled h1
preparing tumor sections for shipping

The tumor section slides should be shipped at room temperature (RT) to the Tumor Specimen Repository. Samples on glass or plastic slides must be adequately cushioned. Place the slides in a primary receptacle such as a slide box. Place this primary receptacle into a secondary leakproof sturdy outer container or flexible-envelope packaging. The shipment must be packaged in compliance with IATA Packaging Instruction 650 for shipment of non-infectious human specimens. Use of the FedEx Clinical Pak is an option. Indicate the \# of boxes shipped, and their total weight. Select 'Overnight Service', or other appropriate service assuring delivery within 24 hours. One aliquot of plasma should be shipped to XenoBiotic Laboratories while the duplicate aliquot is shipped to Scisafe. Both the serum aliquots and tumor slides should be shipped to Scisafe.
untitled h1
15.6.4 arranging for shipments

- In the Ship From section: Enter name and address of person responsible for the shipment from the clinical site.  
- In the Ship To section: Enter name and address of designated laboratory as below:
Ship **PLASMA** Samples to:
Sample Receiving Officer XenoBiotic Laboratories, Inc. 107 Morgan Lane Plainsboro, NJ 08536 Tel. 609-799-2295 X252 Email: sro@xbl.com Ship **SERUM** Samples to:
John Coolidge, Biorepository Manager - Boston SciSafe, Inc. 35 Dunham Road Billerica, MA 01821 Tel. 404-242-1578 Email: jfc@scisafe.com Ship **TUMOR** Slides to:
John Coolidge Biorepository Manager - Boston SciSafe, Inc. 35 Dunham Road Billerica, MA 01821 Tel. 404-242-1578 Email: jfc@scisafe.com Sign and date the waybill. Tear off "Senders Copy" for retention. It is recommended that all shipments be tracked to ensure timely delivery.
untitled h1
notification of shipment

At the time of shipment, the laboratory must e-mail the completed Specimen Shipping Form to the following: a) Sponsor Project Office at rohinton.tarapore@oncoceutics.com Version Date: 06/17/2020 b) **Plasma** Pharmacokinetics Laboratory at sro@xbl.com OR
Serum Pharmacodynamic Laboratory at jfc@scisafe.com OR
Tumor Tissue Repository at jfc@scisafe.com c) Local study nurse or coordinator
untitled h1
specimen collection log - patient

| Type of Specimen and  Protocol-Specified Time  Point   | Date / Time of Collection   | Notes   |
|--------------------------------------------------------|-----------------------------|---------|
| (mm/dd/yyyy) ; (mm:hh)                                 |                             |         |
| □Tumor Tissue Archival sample at time of  enrollment. □Plasma:  Cycle 1 = Baseline □Plasma:  Cycle 1 = 6 hrs (±15  minutes) postadministration of ONC201 □Serum:  Cycle 1 = Baseline; predose □Serum:  Cycle 1 = 6 hours after 1st dose  □Serum:  Cycle 2 = Day 1                                                        |                             |         |
specimen collection log patient specific
patient id#

| Type of Specimen and   |                                |                           |       |
|------------------------|--------------------------------|---------------------------|-------|
| Cycle                  | Protocol-Specified Time  Point | Date / Time of Collection | Notes |
| (mm/dd/yyyy) ; (mm:hh) |                                |                           |       |
| □ Serum                |                                |                           |       |
| 2                      | Pre-Dose □ Serum               |                           |       |
| 3                      | Pre-Dose □ Serum               |                           |       |
| 4                      | Pre-Dose □ Serum               |                           |       |
| 5                      | Pre-Dose □ Serum               |                           |       |
| 6                      | Pre-Dose □ Serum               |                           |       |
| 7                      | Pre-Dose                       |                           |       |
| 8                      | □ Serum                        |                           |       |  
| Pre-Dose □ Serum   |                  |
|--------------------|------------------|
| 9                  | Pre-Dose □ Serum |
| 10                 | Pre-Dose         |  
98
specimen collection log patient specific
15.6.5 oncoceutics specimen shipping form

(Check a single applicable sample type; use additional forms as needed)
□ Plasma □ Serum □ Tissue
specimen collection log patient specific
date

Total Number of Specimens in Shipment: ______ Courier and Tracking Number: _________________________________ Contact Information for Person Preparing Shipment:  
| Name: _______________________________________________________ Institution: ___________________________________________________ Phone Number: ___________________________ E-mail: ___________________________________ Date / Time of Collection Patient ID# Packed By:   | Received By: Initials/Date   |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------|
| (mm/dd/yyyy) ; (mm:hh)                                                                                                                                                                                                                                                  | Initials/Date                |
| 1 2 3 4 5 6 7 8 9 10 11                                                                                                                                                                                                                                                 |                              |  
Abbreviated Title: ONC201 in advanced cancers
Version Date: 06/17/2020  
| 12   |
|------|
| 13   |
| 14   |
| 15   |
| 16   |
| 17   |
| 18   |
| 19   |
| 20   |