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Abbreviated Title: TMZ in breast ca brain mets NIH Protocol: 17-C-0115 Version Date: 06/06/2022 NCT Number: NCT03190967 Title: Phase I/II Study of T-DM1 alone versus T-DM1 and Metronomic Temozolomide in Secondary Prevention of HER2-Positive Breast Cancer Brain Metastases Following Stereotactic Radiosurgery NCI Principal Investigator: Stanley Lipkowitz, MD, PhD, WMB, CCR, NCI
Women's Malignancies Branch National Cancer Institute Building 10, Rm 4B54 9000 Rockville Pike Bethesda, MD 20892 Phone: 240-760-6129 E-mail: lipkowis@navmed.nci.nih.gov Investigational Agents: None Commercial Agents: T-DM1, Temzolomide 1
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precis

Background:  
Breast cancer is the most common cancer in women. In the HER2+ subtype, brain metastases can occur in up to 25-40% of patients.  
The standard therapy for brain metastases continues to be surgery or stereotactic radiosurgery (SRS) and/or whole brain radiation therapy (WBRT).  
Currently, independently of localized or systemic treatment modality, once brain metastases are established, options for treatment are limited, and the disease almost invariably progresses, limiting not only survival but also quality of life in most patients.  
Preclinical literature suggests the hypothesis that preventing the formation of a metastasis by a drug may be more efficacious than attempting to shrink an established lesion.  
Our group has shown *in vitro* and *in vivo* in animal models injected with a brain tropic MGMT+ cell line, that even in very low doses temozolomide (TMZ) administered in a prophylactic, metronomic fashion can significantly prevent development of brain metastases.  
We propose a secondary-prevention clinical trial with oral TMZ given to HER2+ breast cancer patients with brain metastases after recent local treatment (SRS or surgical resection) in combination with the anti-HER2 agent T-DM1 for systemic control of disease.
Objectives:  
Phase I (run in): to identify the maximum tolerated dose (MTD) of TMZ when used in combination with T-DM1.  
Phase II: to determine if the combination regimen of T-DM1 and temozolomide improves the freedom from distant new brain metastases following stereotactic radiosurgery or surgical resection in HER2-positive breast cancer brain metastases, as compared to T-DM1 alone, guided by one-year results as an important benchmark for measuring improvement.
Eligibility:
Phase I:
Histologically confirmed HER2+ breast cancer.  
ECOG performance status 0-2 and adequate organ and marrow function. Brain metastases, treated within 12 weeks of study entry with SRS, resection or WBRT.  
Patients with leptomeningeal metastatic disease are ineligible.  
Patients that are unable to complete a brain MRI with contrast are ineligible. Patients with breast tissue expanders must have those removed before enrollment.  
HBV, HCV or HIV-positive patients are ineligible.  
Patient with impaired cardiac function or clinically significant cardiac disease are ineligible.
2  
Corticosteroids will be allowed at enrollment and during the first month of treatment with T-DM1 after SRS, up to a dose of no more than 10mg of dexamethasone daily or equivalent. Patients that need to continue corticosteroids after the initial month will not be allowed to increase the dose after that period, and will be taken off protocol.
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phase ii

Histologically confirmed HER2+ breast cancer. ECOG performance status 0-2 and adequate organ and marrow function.  
1-10 brain metastases, by contrast MRI, treated within 12 weeks of study entry with SRS
and/or resection.  
Patients with leptomeningeal metastatic disease are ineligible.  
Patients with history of WBRT are ineligible. Patients that are unable to complete a brain MRI with contrast are ineligible. Patients with breast tissue expanders must have those removed before enrollment. HBV, HCV or HIV-positive patients are ineligible.  
Patient with impaired cardiac function or clinically significant cardiac disease are ineligible.  
Corticosteroids will be allowed at enrollment and during the first month of treatment with T-DM1 after SRS, up to a dose of no more than 10mg of dexamethasone daily or equivalent. Patients that need to continue corticosteroids after the initial month will not be allowed to increase the dose after that period, and will be taken off protocol.
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design

This is a Phase I/II open label study that will evaluate the potential benefit of TMZ in prevention of new brain metastases in patients with limited brain metastases from HER2+ breast cancer, previously treated with SRS or surgical resection of brain metastases.  
All patients will receive the standard second-line therapy for HER2+ metastatic breast cancer: T-DM1. During phase II patients will be randomized between T-DM1 plus TMZ versus T-DM1 alone.  
Phase I run in: T-DM1 3.6 mg/kg IV every 21 days plus TMZ 30, 40 or 50 mg/m2 daily. Phase II: T-DM1 3.6 mg/kg versus T-DM1 3.6mg/kg plus TMZ at recommended phase 2 dose (RP2D).  
Phase I will follow a standard 3+3 design. Thus, with 3 dose levels, up to 18 patients may be included in the initial safety evaluation.  
In the phase II portion of the trial, a total of 49 evaluable subjects per arm (98 total) will need to be randomized over a 3-year period and followed for an additional 2 years from the date of entry of the last patient, with occurrence of 79 total relapses in both arms combined, in order to have 80% power to compare the curves.
3  
| TABLE OF CONTENTS                                                                                                                                      |                                                                                                                                                  |                                                                                                                                                |
|--------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------|
| PRÉCIS .                                                                                                                                               |                                                                                                                                                  |                                                                                                                                                |
| TABLE OF CONTENTS ...................................................................................................................................4 |                                                                                                                                                  |                                                                                                                                                |
| STATEMENT OF COMPLIANCE .......................................................................................................................9       |                                                                                                                                                  |                                                                                                                                                |
| INTRODUCTION .                                                                                                                                         |                                                                                                                                                  |                                                                                                                                                |
| 1.                                                                                                                                                     | Study Objectives                                                                                                                                 |                                                                                                                                                |
| 1.1                                                                                                                                                    | 1.1.1                                                                                                                                            | Primary Objectives: .........................................................................................................................9 |
| 1.1.2                                                                                                                                                  | Secondary Objective(s):                                                                                                                          |                                                                                                                                                |
| 1.1.3                                                                                                                                                  | Exploratory Objective(s): ...........................................................................................................9           |                                                                                                                                                |
| 1.2                                                                                                                                                    | Background and Rationale ...........................................................................................................10           |                                                                                                                                                |
| 1.2.1                                                                                                                                                  | Brain metastasis in HER2 positive breast cancer .............................................................10                                  |                                                                                                                                                |
| 1.2.2                                                                                                                                                  | Treatment of brain metastases ..............................................................................................10                   |                                                                                                                                                |
| 1.2.3                                                                                                                                                  | Prevention of brain metastases ............................................................................................12                    |                                                                                                                                                |
| 1.3                                                                                                                                                    | Correlatives Background and Rationale                                                                                                            |                                                                                                                                                |
| 1.3.1                                                                                                                                                  | Cell free DNA (cfDNA) and primary tumor DNA sequencing                                                                                           |                                                                                                                                                |
| 1.3.2                                                                                                                                                  | MM Status ....................................................................................................................................19 |                                                                                                                                                |
| 1.3.3                                                                                                                                                  | Neuroinflammatory response and neuronal damage metrics........................................20                                                 |                                                                                                                                                |
| 1.3.4                                                                                                                                                  | EXOSOMES                                                                                                                                         |                                                                                                                                                |
| Pharmacokinetics                                                                                                                                       |                                                                                                                                                  |                                                                                                                                                |
| 1.3.5                                                                                                                                                  |                                                                                                                                                  |                                                                                                                                                |
| 2                                                                                                                                                      | ELIGIBILITY ASSESSMENT AND ENROLLMENT ..................................................................21                                       |                                                                                                                                                |
| 2.1                                                                                                                                                    | Eligibility Criteria                                                                                                                             |                                                                                                                                                |
| 2.1.1                                                                                                                                                  | Phase 1 Inclusion Criteria ..............................................................................................................21      |                                                                                                                                                |
| 2.1.2                                                                                                                                                  | Phase 1 Exclusion Criteria                                                                                                                       |                                                                                                                                                |
| 2.1.3                                                                                                                                                  | Phase 2 Inclusion Criteria ......................................................................................................24              |                                                                                                                                                |
| 2.1.4                                                                                                                                                  | Phase 2 Exclusion Criteria                                                                                                                       |                                                                                                                                                |
| 2.1.5                                                                                                                                                  | Recruitment Strategies                                                                                                                           |                                                                                                                                                |
| 2.2                                                                                                                                                    | Screening Evaluation                                                                                                                             |                                                                                                                                                |
| 2.2.1                                                                                                                                                  | Screening activities performed prior to obtaining informed consent ......................26                                                      |                                                                                                                                                |
| 2.2.2                                                                                                                                                  | Screening activities performed after a consent for screening has been signed.......27                                                            |                                                                                                                                                |
| 2.2.3                                                                                                                                                  | Pathology confirmation of diagnosis. ...................................................................................27                       |                                                                                                                                                |
| 2.2.4                                                                                                                                                  | Imaging studies: .                                                                                                                               |                                                                                                                                                |
| 2.2.5                                                                                                                                                  | Cardiology test: 2-D Echocardiogram or MUGA-scan ................................................27                                              |                                                                                                                                                |
| 2.2.6                                                                                                                                                  | Anti-HIV, anti-HCV, Hepatitis B Surface Ag..............................................................27                                       |                                                                                                                                                |
| 2.2.7                                                                                                                                                  | History and Physical examination: .....................................................................................27                        |                                                                                                                                                |
| 2.2.8                                                                                                                                                  | Laboratory Studies:                                                                                                                              |                                                                                                                                                |
| 2.2.9                                                                                                                                                  | Pregnancy Testing: ......................................................................................................................27      |                                                                                                                                                |
| 2.3                                                                                                                                                    | Participant Registration and Status Update Procedures ....................................................28                                     |                                                                                                                                                |
| 2.3.1                                                                                                                                                  | Screen Failures                                                                                                                                  |                                                                                                                                                |
| 2.4                                                                                                                                                    | Randomization (or Stratification) Procedures .......................................................................28                           |                                                                                                                                                |
| 2.5                                                                                                                                                    | Baseline Evaluation                                                                                                                              |                                                                                                                                                |
| 2.5.1                                                                                                                                                  | Clinical Evaluation: ......................................................................................................................29    |                                                                                                                                                |
| 2.5.2                                                                                                                                                  | Laboratory Evaluation: ....................................................................................................30                    |                                                                                                                                                |  
| Version Date: 06/06/2022                                                                                                                       |                                                                                                                                                  |                                                                                                                                             |
|------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------|
| 2.5.3                                                                                                                                          | Neurocognitive and quality of life assessment: ............................................................30                                    |                                                                                                                                             |
| 2.5.4                                                                                                                                          | Correlative tests evaluation: ...........................................................................................30                      |                                                                                                                                             |
| STUDY IMPLEMENTATION .............................................................................................................30           |                                                                                                                                                  |                                                                                                                                             |
| 3                                                                                                                                              | 3.1                                                                                                                                              | Study Design ............................................................................................................................30 |
| Dose Limiting Toxicity                                                                                                                         |                                                                                                                                                  |                                                                                                                                             |
| 3.1.1                                                                                                                                          |                                                                                                                                                  |                                                                                                                                             |
| 3.1.2                                                                                                                                          | Dose Escalation ..................................................................................................................31             |                                                                                                                                             |
| Drug Administration                                                                                                                            |                                                                                                                                                  |                                                                                                                                             |
| 3.2                                                                                                                                            |                                                                                                                                                  |                                                                                                                                             |
| 3.2.1                                                                                                                                          | T-DM1 ........................................................................................................................................32 |                                                                                                                                             |
| 3.2.2                                                                                                                                          | TEMOZOLOMIDE .                                                                                                                                   |                                                                                                                                             |
| Self-Administered Study Drugs                                                                                                                  |                                                                                                                                                  |                                                                                                                                             |
| 3.2.3                                                                                                                                          |                                                                                                                                                  |                                                                                                                                             |
| 3.3                                                                                                                                            | Dose Modifications and Management of Toxicities: .......................................................34                                       |                                                                                                                                             |
| T-DM1 Dose Modifications: ............................................................................................34                       |                                                                                                                                                  |                                                                                                                                             |
| 3.3.1                                                                                                                                          |                                                                                                                                                  |                                                                                                                                             |
| 3.3.2                                                                                                                                          | Temozolomide Dose Modifications ................................................................................36                               |                                                                                                                                             |
| Questionnaires .............................................................................................................................38 |                                                                                                                                                  |                                                                                                                                             |
| 3.4                                                                                                                                            |                                                                                                                                                  |                                                                                                                                             |
| 3.5                                                                                                                                            | S STUDY CALENDAR                                                                                                                                 |                                                                                                                                             |
| Cost and Compensation                                                                                                                          |                                                                                                                                                  |                                                                                                                                             |
| 3.6                                                                                                                                            |                                                                                                                                                  |                                                                                                                                             |
| 3.6.1                                                                                                                                          | COSTS ........................................................................................................................................41 |                                                                                                                                             |
| 3.6.2                                                                                                                                          | COMPENSATION ............................................................................................................................41      |                                                                                                                                             |
| REIMBURSEMENT                                                                                                                                  |                                                                                                                                                  |                                                                                                                                             |
| 3.6.3                                                                                                                                          |                                                                                                                                                  |                                                                                                                                             |
| 3.7                                                                                                                                            | Criteria for Removal from Protocol Therapy and Off Study Criteria .........................41                                                    |                                                                                                                                             |
| Criteria for removal from protocol therapy ....................................................................41                              |                                                                                                                                                  |                                                                                                                                             |
| 3.7.1                                                                                                                                          |                                                                                                                                                  |                                                                                                                                             |
| 3.7.2                                                                                                                                          | OFF-Study Criteria                                                                                                                               |                                                                                                                                             |
| 3.7.3                                                                                                                                          | Lost to Follow-up ..............................................................................................................42               |                                                                                                                                             |
| 4                                                                                                                                              | CONCOMITANT MEDICATIONS/MEASURES.....................................................................42                                          |                                                                                                                                             |
| 4.1                                                                                                                                            | T-DM1...............................................................................................................................42           |                                                                                                                                             |
| 4.1.1                                                                                                                                          | Drug Interactions .............................................................................................................42                |                                                                                                                                             |
| Temozolomide                                                                                                                                   |                                                                                                                                                  |                                                                                                                                             |
| 4.2                                                                                                                                            |                                                                                                                                                  |                                                                                                                                             |
| 4.2.1                                                                                                                                          | Drug Interactions ................................................................................................................42             |                                                                                                                                             |
| 4.2.2                                                                                                                                          | General.............................................................................................................................42           |                                                                                                                                             |
| S                                                                                                                                              | BIOSPECIMEN COLLECTION...........................................................................................................43              |                                                                                                                                             |
| Correlative Studies for Research/Pharmacokinetic Studies ..........................................43                                          |                                                                                                                                                  |                                                                                                                                             |
| 5.1                                                                                                                                            |                                                                                                                                                  |                                                                                                                                             |
| 5.2                                                                                                                                            | Sample Collection ..................................................................................................................45           |                                                                                                                                             |
| Samples will be coded, linked in the BPC and sent for final analysis to: ..............46                                                      |                                                                                                                                                  |                                                                                                                                             |
| 5.2.1                                                                                                                                          |                                                                                                                                                  |                                                                                                                                             |
| 5.2.2                                                                                                                                          | Tumor, Blood Samples and CSF samples: ......................................................................46                                   |                                                                                                                                             |
| 5.3                                                                                                                                            | Sample Storage, Tracking and Disposition ........................................................................47                              |                                                                                                                                             |
| 5.3.1                                                                                                                                          | Participant sample protections                                                                                                                   |                                                                                                                                             |
| 5.3.2                                                                                                                                          | Sample Storage, Tracking and Disposition .................................................................47                                     |                                                                                                                                             |
| Samples for Genetic/Genomic Analysis                                                                                                           |                                                                                                                                                  |                                                                                                                                             |
| 5.4                                                                                                                                            |                                                                                                                                                  |                                                                                                                                             |
| 5.4.1                                                                                                                                          | Description of the scope of genetic/genomic analysis                                                                                             |                                                                                                                                             |
| 5.4.2                                                                                                                                          | Privacy and Confidentiality of medical information/biological specimens ...........48                                                            |                                                                                                                                             |  
| Version Date: 06/06/2022                                                                                                                                     |                                                                                                                                                  |                  |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------|------------------|
| A Certificate of Confidentiality will be obtained for the study as described in                                                                              |                                                                                                                                                  |                  |
| 5.4.3                                                                                                                                                        |                                                                                                                                                  |                  |
| section 11.5.7. ..................................................................................................................................49         |                                                                                                                                                  |                  |
| 5.4.4                                                                                                                                                        | Management of Results ...................................................................................................49                      |                  |
| Genetic counseling                                                                                                                                           |                                                                                                                                                  |                  |
| 5.4.5                                                                                                                                                        |                                                                                                                                                  |                  |
| 6                                                                                                                                                            | DATA COLLECTION AND EVALUATION .............................................................................49                                   |                  |
| DATA COLLECTION                                                                                                                                              |                                                                                                                                                  |                  |
| 6.1                                                                                                                                                          | 6.1.1                                                                                                                                            | SOURCE DOCUMENTS |
| Case Report Forms .............................................................................................................................50            |                                                                                                                                                  |                  |
| 6.1.2                                                                                                                                                        |                                                                                                                                                  |                  |
| 6.1.3                                                                                                                                                        | DATA QUALITY Assurance ...............................................................................................................50         |                  |
| DATA Sharing Plans .......................................................................................................................................50 |                                                                                                                                                  |                  |
| 6.2                                                                                                                                                          |                                                                                                                                                  |                  |
| 6.2.1                                                                                                                                                        | Human Data Sharing Plan ............................................................................................................50           |                  |
| 6.2.2                                                                                                                                                        | Genomic Data Sharing Plan                                                                                                                        |                  |
| 6.3                                                                                                                                                          | Response Criteria                                                                                                                                |                  |
| 6.3.1                                                                                                                                                        | RANO-BM Criteria for Evaluation of Brain Lesions                                                                                                 |                  |
| RECIST for Systemic Evaluation                                                                                                                               |                                                                                                                                                  |                  |
| 6.3.2                                                                                                                                                        |                                                                                                                                                  |                  |
| 6.3.3                                                                                                                                                        | D DURATION OF Response                                                                                                                           |                  |
| 6.3.4                                                                                                                                                        | Relapse -Free Survival                                                                                                                           |                  |
| Validation of Disease Assessment (Safety Mechanisms) ..............................................58                                                        |                                                                                                                                                  |                  |
| 6.3.5                                                                                                                                                        |                                                                                                                                                  |                  |
| 6.4                                                                                                                                                          | Toxicity Criteria                                                                                                                                |                  |
| NIH REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN.....59                                                                                            |                                                                                                                                                  |                  |
| 7                                                                                                                                                            | 7.1                                                                                                                                              | Definitions      |
| 7.2                                                                                                                                                          | OHSRP Office of Compliance and Training / IRB  Reporting .........................................59                                             |                  |
| 7.2.1                                                                                                                                                        | Expedited Reporting                                                                                                                              |                  |
| 7.2.2                                                                                                                                                        | IRB Requirements for PI Reporting at Continuing Review ..........................................59                                              |                  |
| 7.3                                                                                                                                                          | NCI CLINICAL DIRECTOR REPORTING ...........................................................................................59                    |                  |
| 7.4                                                                                                                                                          | NIH Required Data and Safety Monitoring Plan .....................................................................59                             |                  |
| 7.4.1                                                                                                                                                        | Principal Investigator/Research Team                                                                                                             |                  |
| 7.4.2                                                                                                                                                        | Data Safety Monitoring Board (DSMB)                                                                                                              |                  |
| Corrective Action Plan ........................................................................................................60                            |                                                                                                                                                  |                  |
| 7.4.3                                                                                                                                                        |                                                                                                                                                  |                  |
| 7.4.4                                                                                                                                                        | Independent Monitoring Committee (IMC) ..................................................................60                                      |                  |
| SAFETY REPORTING CRITERIA TO THE PHARMACEUTICAL COLLABORATOR                                                                                                 |                                                                                                                                                  |                  |
| 8                                                                                                                                                            | 60                                                                                                                                               |                  |
| 9                                                                                                                                                            | STATISTICAL CONSIDERATIONS .............................................................................................61                       |                  |
| 9.1                                                                                                                                                          | Statistical hypotheses ..................................................................................................................61      |                  |
| 9.2                                                                                                                                                          | Sample size determination                                                                                                                        |                  |
| 9.3                                                                                                                                                          | Populations for analysis                                                                                                                         |                  |
| 9.4                                                                                                                                                          | Statistical analyses                                                                                                                             |                  |
| 9.4.1                                                                                                                                                        | General approach .............................................................................................................................63 |                  |
| 9.4.2                                                                                                                                                        | Analysis of the primary safety and efficacy endpoints                                                                                            |                  |
| 9.4.3                                                                                                                                                        | Analysis of the secondary efficacy endpoints ............................................................63                                      |                  |
| 9.4.4                                                                                                                                                        | Sety Analyses                                                                                                                                    |                  |
| 9.4.5                                                                                                                                                        | Baseline Descriptive Statistics                                                                                                                  |                  |  
| Version Date: 06/06/2022                                                                                                                                            |                                                                                                                                                        |                                                                                                                                 |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------|
| 9.4.6                                                                                                                                                               | Planned interim analyses ........................................................................................................63                    |                                                                                                                                 |
| 9.4.7                                                                                                                                                               | Exploratory analyses                                                                                                                                   |                                                                                                                                 |
| COLLABORATIVE AGREEMENTS ...................................................................................................64                                      |                                                                                                                                                        |                                                                                                                                 |
| 10                                                                                                                                                                  | 10.1                                                                                                                                                   | Clinical Trials Agreement (CTA)                                                                                                 |
| HUMAN SUBJECTS PROTECTIONS ............................................................................................64                                           |                                                                                                                                                        |                                                                                                                                 |
| l l                                                                                                                                                                 | 11.1                                                                                                                                                   | Rationale For Subject Selection .............................................................................................64 |
| 11.2                                                                                                                                                                | Participation Of Children .......................................................................................................65                    |                                                                                                                                 |
| 11.3                                                                                                                                                                | Participation of Subjects Unable to Give Consent .............................................................65                                       |                                                                                                                                 |
| 11.4                                                                                                                                                                | Evaluation of Benefits and Risks/Discomforts ...............................................................65                                         |                                                                                                                                 |
| 11.5                                                                                                                                                                | Risks/Benefits Analysis                                                                                                                                |                                                                                                                                 |
| 11.5.1                                                                                                                                                              | Risks ..............................................................................................................................................65 |                                                                                                                                 |
| 11.5.2                                                                                                                                                              | Research Blood Collection Risks ........................................................................................65                             |                                                                                                                                 |
| 11.5.3                                                                                                                                                              | Risks of Echocardiogram                                                                                                                                |                                                                                                                                 |
| Risks of MUGA Scans                                                                                                                                                 |                                                                                                                                                        |                                                                                                                                 |
| 11.5.4                                                                                                                                                              |                                                                                                                                                        |                                                                                                                                 |
| 11.5.5                                                                                                                                                              | Risks of Questionnaires ..........................................................................................................66                   |                                                                                                                                 |
| 11.5.6                                                                                                                                                              | Lumbar Puncture for Research Purposes ..............................................................................66                                 |                                                                                                                                 |
| 11.5.7                                                                                                                                                              | Risks of exposure to ionizing radiation .............................................................................66                                |                                                                                                                                 |
| 11.5.8                                                                                                                                                              | Risks of CT Scans                                                                                                                                      |                                                                                                                                 |
| 11.5.9                                                                                                                                                              | Risks of MRIs ....................................................................................................................................66   |                                                                                                                                 |
| 1.5.10  Other risks ..............................................................................................................................................6 |                                                                                                                                                        |                                                                                                                                 |
| 1.5.11  Non-Physical Risks of Genetic Research .....................................................................67                                              |                                                                                                                                                        |                                                                                                                                 |
| 1.5.12 Certificate of Confidentiality ...............................................................................................67                             |                                                                                                                                                        |                                                                                                                                 |
| 1.5.13 Benefits .............................................................................................................................................67     |                                                                                                                                                        |                                                                                                                                 |
| 1.5.14 Risks/Benefits Analysis .........................................................................................................67                          |                                                                                                                                                        |                                                                                                                                 |
| 11.6                                                                                                                                                                | Consent Process and Documentation ..................................................................................67                                 |                                                                                                                                 |
| 11.6.1                                                                                                                                                              | Consent Process for Adults Who Lack Capacity to Consent to Research                                                                                    |                                                                                                                                 |
| Participation                                                                                                                                                       |                                                                                                                                                        |                                                                                                                                 |
| 1.7                                                                                                                                                                 | Request for Waiver of Consent for Screening Activities ................................................68                                              |                                                                                                                                 |
| 12                                                                                                                                                                  | REGULATORY AND OPERATIONAL CONSIDERATIONS ..............................................69                                                             |                                                                                                                                 |
| 12.1                                                                                                                                                                | STUDY DISCONTINUATION AND CLOSURE ..........................................................................69                                         |                                                                                                                                 |
| 12.2                                                                                                                                                                | QUALITY ASSURANCE AND QUALITY CONTROL                                                                                                                  |                                                                                                                                 |
| 12.3                                                                                                                                                                | CONFLICT OF INTEREST POLICY .....................................................................................................70                    |                                                                                                                                 |
| 12.4                                                                                                                                                                | CONFIDENTIALITY AND PRIVACY                                                                                                                            |                                                                                                                                 |
| 13                                                                                                                                                                  | PHARMACEUTICAL INFORMATION .............................................................................................71                             |                                                                                                                                 |
| Temozolomide                                                                                                                                                        |                                                                                                                                                        |                                                                                                                                 |
| 13.1                                                                                                                                                                |                                                                                                                                                        |                                                                                                                                 |
| 13.1.1                                                                                                                                                              | SOURCE                                                                                                                                                 |                                                                                                                                 |
| 13.1.2                                                                                                                                                              | Formulation and preparation                                                                                                                            |                                                                                                                                 |
| 13.1.3                                                                                                                                                              | Storage and Stability....................................................................................................................72            |                                                                                                                                 |
| 13.1.4                                                                                                                                                              | Administration procedures                                                                                                                              |                                                                                                                                 |
| 13.1.5                                                                                                                                                              | Pharmacokinetics                                                                                                                                       |                                                                                                                                 |
| Metabolism and Elimination                                                                                                                                          |                                                                                                                                                        |                                                                                                                                 |
| 13.1.6                                                                                                                                                              |                                                                                                                                                        |                                                                                                                                 |
| 13.1.7                                                                                                                                                              | Special Populations                                                                                                                                    |                                                                                                                                 |  
Abbreviated Title: TMZ in breast ca brain mets  
| Incompatibilities and Toxicity ........................................................................................................73                  |                                                                                                                                                                    |
|------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 13.1.8                                                                                                                                                     | T-DM1.                                                                                                                                                             |
| 13.2                                                                                                                                                       |                                                                                                                                                                    |
| 13.2.1                                                                                                                                                     | SOURCE .                                                                                                                                                           |
| 13.2.2                                                                                                                                                     | Formulation and Preparation ...................................................................................................75                                  |
| 13.2.3                                                                                                                                                     | Supply and handling: ...................................................................................................................76                         |
| Formulation and preparation                                                                                                                                |                                                                                                                                                                    |
| 13.2.4                                                                                                                                                     |                                                                                                                                                                    |
| 13.2.5                                                                                                                                                     | A Administration .                                                                                                                                                 |
| 13.2.6                                                                                                                                                     | Pharmacokinetics                                                                                                                                                   |
| Distribution ...........................................................................................................................................78 |                                                                                                                                                                    |
| 13.2.7                                                                                                                                                     |                                                                                                                                                                    |
| 13.2.8                                                                                                                                                     | Metabolism and Elimination                                                                                                                                         |
| 13.2.9                                                                                                                                                     | Special populations                                                                                                                                                |
| 13.2.10  Incompatibilities and Toxicity .............................................................................................81                    |                                                                                                                                                                    |
| 14                                                                                                                                                         | REFERENCES .....................................................................................................................................................82 |
| 15                                                                                                                                                         | APPENDICES ....................................................................................................................................90                  |
| 15.1                                                                                                                                                       | Appendix A - Performance Status Criteria                                                                                                                           |
| 15.2                                                                                                                                                       | Appendix B - List Of Drugs Interacting With CYP3A4 Isoenzyme ..........................91                                                                          |
| 15.3                                                                                                                                                       | Appendix C - Oral Medication Diary .................................................................................92                                             |
| 15.4                                                                                                                                                       | Appendix D– Rano Criteria For CNS Metastasis ............................................................93                                                        |
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statement of compliance

The trial will be carried out in accordance with International Conference on Harmonisation Good Clinical Practice (ICH GCP) and the following:
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are responsible for the conduct, management, or oversight of NIH-funded clinical trials have completed Human Subjects Protection and ICH GCP Training. The protocol, informed consent form(s), recruitment materials, and all participant materials will be submitted to the Institutional Review Board (IRB) for review and approval. Approval of both the protocol and the consent form must be obtained before any participant is enrolled. Any amendment to the protocol will require review and approval by the IRB before the changes are implemented to the study. In addition, all changes to the consent form will be IRB-approved; an IRB determination will be made regarding whether a new consent needs to be obtained from participants who provided consent, using a previously approved consent form.
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1. introduction 1.1 study objectives

1.1.1 Primary Objectives: Phase I (run in):
To identify the maximum tolerated dose (MTD) of temozolomide when used in combination with T-DM1.  
Phase II:
To determine if the combination regimen of T-DM1 and temozolomide improves the freedom from distant new brain metastases following stereotactic radiosurgery or surgical resection in HER2-positive breast cancer brain metastases as compared to T-DM1 alone guided by one-year results as an important benchmark for measuring improvement.  
1.1.2 Secondary Objective(s):
To evaluate the safety and tolerability of the combination of T-DM1 and temozolomide in HER2-positive breast cancer patients, previously treated for limited brain metastases.  
To assess time to whole brain radiation, and overall patient survival when treated with the combination regimen, compared to T-DM1 alone (control arm).  
1.1.3 Exploratory Objective(s):
To assess tumor genomic and neuroinflammatory biomarkers in the CSF and serum; the effects of treatment in those biomarkers; and the ability to predict relapses using those biomarkers; as described in the correlative studies background section (Section 1.3).To evaluate MGMT methylation status in primary tumor, CSF and serum; to compare it between patients that benefit and those that don't benefit from treatment with temozolomide.  
To assess neurotoxicity and neurocognitive effects in patients treated with the combination regimen compared to the control arm.  
To assess between arm differences in symptom burden as measured by the M.D. Anderson Symptom Inventory-Brain Module (MDASI-BT). Specifically, it is hypothesized that the following subscales on the MDASI-BT (cognitive dysfunction, focal neurologic deficits and interference) will be higher in those receiving temozolomide alone due to differences in time to disease progression.  
To assess if baseline MDASI-BT subscale scores (overall symptom burden, overall interference, cognitive dysfunction and focal neurologic deficits) along with the specific items of fatigue and change in appetite will be prognostic for time to distant metastases and for OS.  
To assess if change scores over time in MDASI-BT subscale (overall symptom burden, overall interference, cognitive dysfunction, and focal neurologic deficits) along specific items (difficulty concentrating, fatigue, nausea, and headaches) were prognostic for OS, time to distant metastases, and neurocognitive decline.
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1.2 background and rationale 1.2.1

Breast cancer is the most common cancer in women 1, and the second most common cause of brain metastases in the United States 2. The incidence of brain metastases varies amongst the different molecular subtypes of breast cancer, with high incidence in the HER2-positive and triple-negative breast cancers 3,4. In the HER2-positive group, the diagnosis of brain metastases became even more frequent - up to 25-40% 5-8 - after widespread treatment with trastuzumab, a monoclonal antibody that improved survival and control of systemic disease but has low central nervous system penetrance 9,10. New agents targeting HER2 have been evaluated since the introduction of trastuzumab, and, though several are now proven effective for systemic control of the disease, a major effect in decreasing brain metastases incidence in this group is still lacking.
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1.2.2 treatment of brain metastases

Radiation therapy modalities are currently the main options for treatment of brain metastases.  
In patients with limited number of brain metastases (one to five), current treatments include surgical resection, stereotactic radiosurgery (SRS), and/or whole-brain radiotherapy (WBRT) 11.  
Most prospective trials evaluating local treatment of brain metastases included mainly NSCLC
patients and only a small proportion of breast cancer patients 12-14. Addition of WBRT to initial surgery or SRS has repeatedly demonstrated a decrease in intracranial disease recurrence but no difference in the overall survival of 7 to 10 months. However, WBRT has been reported to worsen quality of life and neurocognitive function, especially in patients with prolonged survival 15-17. In those cases, neurocognitive decline is progressive and untreatable. WBRT alone is the main indicated treatment for patients with higher numbers of brain metastatic lesions.
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1.2.2.2 systemic therapy

Systemic therapy for brain metastases, overall, has shown less efficacy than in systemic, non-CNS locations. Multiple clinical trials have documented few or no responses using agents with known activity in the metastatic setting 18-23. The reasons for this may be heterogeneous, but likely involve the blood-brain barrier (BBB), which prevents most substances from exiting the bloodstream and entering the brain parenchyma. Once a metastasis forms the BBB is transformed into a bloodtumor barrier (BTB). In general, the BTB permits greater drug penetration than the BBB, but drug levels are ~a log less than those reached in systemic metastases and therefore ineffective 24.  
In the HER2 positive breast cancer patients, HER2 targeted agents beyond trastuzumab have been also evaluated for their potential therapeutic effect in brain metastases. Lapatinib is a small molecule tyrosine-kinase inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor type 2 (HER2), able to cross the BTB to a greater extent than paclitaxel 24,25. A presurgical study documented heterogeneous levels of lapatinib in brain metastases from patients dosed pre-operatively for medically needed craniotomies26 .  
As a single agent, lapatinib showed a few responses and a small non-significant diminution in size of brain metastatic lesions 27,28. The combination lapatinib plus capecitabine produced objective responses in approximately 30% of patients in phase II trials evaluating the combination treatment in patients previously treated with WBRT 28,29. The LANDSCAPE trial evaluated the combination in a single arm phase II design as first-line treatment for low-volume brain metastases with objective CNS responses of 65.9%, median time to CNS progression of 5.5 months, and median time to WBRT of 8.5 months 30. More recently, the MA.31 trial randomized 652 patients with HER2-positive breast cancer to treatment with lapatinib plus taxane or trastuzumab plus taxane as first line treatment of metastatic disease 31. The primary endpoint was PFS. The trastuzumab combination was superior to the lapatinib combination, 9.0 months and 11.3 months, respectively (HR 1.37, p=0.001). The incidence of brain metastases as first site of progression was 28% for trastuzumab and 20% for lapatinib, with no difference in time to progression between the arms. In a brain metastasis prevention setting, a phase III trial of lapatinib plus capecitabine versus capecitabine alone in HER2-positive advanced breast cancer previously treated with an anthracycline, taxane, and trastuzumab was conducted. 4 (2%) patients developed symptomatic brain metastasis as an initial site of progression in the combination therapy arm compared to 13
(6%) patients in the monotherapy group (p=0.045) 32.  
Pertuzumab is a monoclonal antibody that targets HER2 extracellular dimerization domain and acts in synergy with trastuzumab. It was demonstrated to prolong survival when used with trastuzumab and docetaxel as first line treatment for metastatic HER2-positive breast cancer, compared to trastuzumab, docetaxel and placebo 33. An exploratory analysis of the trial evaluated the incidence of brain metastases as first site of disease progression, and found it to be similar in the pertuzumab arm and the placebo arm (13.7% and 12.6%)34.  
Trastuzumab emtansine (T-DM1) is a newer anti-HER2 therapeutic agent, with case reports describing activity in CNS metastases 35,36. T-DM1 is an antibody–drug conjugate containing emtansine (DM1), a microtubule-inhibitory agent, linked to trastuzumab. Phase III trials have shown activity of T-DM1 in HER2-positive metastatic disease after previous lines of treatment including trastuzumab and lapatinib, with an increase in PFS and OS 37,38. T-DM1 is currently the standard therapy for HER2-positive breast cancer patients with recurrence or progression of disease after treatment with trastuzumab. The EMILIA trial randomized advanced HER2-positive breast cancer patients previously treated with trastuzumab and taxanes, to receive either T-DM1 or lapatinib plus capecitabine 38. Final results showed better PFS (9.6 months with T-DM1 *versus* 6.4 months with lapatinib plus capecitabine - hazard ratio for progression or death from any cause, 0.65; 95% confidence interval [CI], 0.55 to 0.77; P<0.001), and OS (30.9 months *versus* 25.1 months; hazard ratio for death from any cause, 0.68; 95% CI, 0.55 to 0.85; P<0.001) for T-DM1. In a retrospective exploratory analysis of the EMILIA trial, the rate of CNS progression was similar between the two arms, with CNS metastases as the first site of relapse in 2% of T-DM1 treated patients and in 0.7% of lapatinib plus capecitabine treated patients, and progression of CNS disease known at baseline in 22.2% and 16%, respectively. However, in patients with treated asymptomatic CNS metastases at baseline, T-DM1 was associated with improved survival when compared to lapatinib plus capecitabine (26.8 versus 12.9 months, HR 0.38 p=0.008) 39. One report raised concerns about possible inflammatory interaction between T-DM1 and SRS-treated lesions with clinically symptomatic edema, not largely seen outside this report but it requires close monitoring 40.  
Independently of localized or systemic treatment modality, once brain metastases are established, our options for treatment are currently limited and the disease almost invariably progresses, limiting not only survival but also quality of life in most patients. 1.2.3 Prevention of brain metastases Preventing the development of brain metastases in high-risk breast cancer patients is a different way to tackle the problem clinically. Primary prevention refers to the initial development of a brain metastasis; secondary prevention refers to the development of additional brain metastases in patients with limited metastases treated with local therapy.  
The preclinical literature suggests the hypothesis that preventing the formation of a metastasis by a drug may be more efficacious than attempting to shrink an established lesion. Multiple mouse models of brain metastasis of breast cancer have been developed. Many approved drugs and candidates have been tested in a prevention mode, where they were delivered early and continuously after tumor cell injection, or in a therapeutic setting, where brain lesions were permitted to form and then treatment began. As reviewed41, several drugs have shown statistically significant efficacy in preventing the formation of brain metastases. None have shown therapeutic efficacy, in keeping with the clinical literature.  
The use of prophylactic radiation therapy, as used in small-cell lung cancer, is one approach to prevent brain metastases in high risk patients. At least two trials, one in Europe (NCT00639366) and other in Canada (NCT00916877), recruited high-risk breast cancer patients to evaluate prophylactic radiation and prevention of brain metastases and results are still maturing. The increasing evidence of neurocognitive decline related to WBRT and the lack of initial reports showing major sign of positive results are discouraging though 42. There is increasing evidence of at least transient declines in memory, executive function, and processing speed in the first few months after WBRT in up to 40-60% patients. Based on the literature, additional progressive neurocognitive complications may be expected with time.  
In an attempt to evaluate prophylactic treatment against brain metastases as first site of relapse, the phase III CEREBEL trial assessed 501 women that were randomized to lapatinib plus capecitabine *versus* trastuzumab plus capecitabine 43. The incidence of brain metastases was overall low, 3% and 5% respectively, with no significant difference between the two arms.  
Considering the different subtypes of breast cancer, a retrospective analysis of 154 patients
(56% HER2-positive) treated with SRS, evaluated their outcome in terms of new brain metastases, overall survival and death ultimately caused by progression in brain 44. Median overall survival was 16.7 months for HER2-positive patients and 8.4 months for HER2-negative patients (p<0.001). Brain metastasis progression was the cause of death in 46% of HER2-positive and 31% of HER2-negative patients (p=0.066). Freedom from distant brain failure at 1 year was 58% in HER2-positive as opposed to 40% in HER2-negative patients (p=0.034), however, the majority of those patients had also received WBRT in the past. Such analysis reinforces the findings that, once brain metastases are found, HER2-positive patients may have a better overall survival, but they are more prone to develop and to die from brain metastasis progression. Most interestingly, since brain metastatic disease frequently recurs after initial SRS, a secondary prevention strategy could be helpful at this point.
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temozolomide in brain metastases

Temozolomide (TMZ) is a cytotoxic alkylating agent with almost 100% oral bioavailability, known to penetrate the BBB and currently FDA approved for treatment of glioblastoma multiforme and recurrent anaplastic astrocytoma. Although not proven effective as single agent to treat established metastatic breast cancer 45, TMZ has been extensively evaluated in the treatment of established brain metastases in combination with other agents and/or radiation. therapy, showing mixed responses and good tolerability 46-56.  
Table 1. Prevention of experimental brain metastases by TMZ over a wide dose range.  
Experiment: Dose  
  
1 0 2.0 63.3 2 0 2.6 86.4 3 0 6.5 143.3 4 0 2.3 101.1 Mice were injected with brain tropic MDAMB231-BR cells and treated from day 3 post-injection with the indicated dose of TMZ. At necropsy large (>300 um) and micrometastases were quantified in step sections through one brain hemisphere. Experiments 1-3, p< 0.01. Results of four experiments are shown to demonstrate the efficacy of TMZ dose.
Our group has shown in models developed with brain tropic HER2–positive JIMT-1-BR3 (in vitro) and triple-negative 231-BR-EGFP (*in vitro* and *in vivo* in animal models) sublines, that even in very low doses TMZ administered in a prophylactic fashion can prevent development of brain metastases (**Table 1**) 57. The brain tropic tumor cells were injected into left cardiac ventricle and on day 3 the mice were randomized to vehicle *versus* TMZ given by oral gavage for 5 days every week. At necropsy, brain metastases were quantified in step sections through one hemisphere, and Abbreviated Title: TMZ in breast ca brain mets doses of TMZ from 5 to 50mg/kg significantly prevented brain metastases formation. TMZ was ineffective in the treatment (shrinking) of established brain metastases.  When mice injected with brain-tropic breast cancer cells were treated with a course of TMZ and then allowed to live untreated, TMZ extended survival, with early treatment better than later (Figure 1).  The ability of TMZ to prolong life more effectively when given earlier is consistent with its ability to prevent metastases but inability to treat established metastases.  
  
  
temozolomide at 10, 50 or 100 µM final concentrations. Colonies were fixed, stained with crystal violet and counted at 14 days posttreatment. Colony counts are shown as mean +/- SEM; **P=.008, ***P
O6-Methylguanine-DNA methyltransferase (MGMT) is critical for repairing pro-mutagenic DNA  
 bases and known to be contributory to tumor resistance to alkylating agents 58,59. Tumor response to TMZ has been shown to depend on low MGMT expression both in glioblastomas and in our preclinical breast cancer models as well (**Figure 2**) 57,60. The correlation between primary breast tumor expression of MGMT and metastatic tissue expression of MGMT has not been largely investigated. Our group also evaluated 62 primary breast cancer tumor samples and their patientmatched brain metastases (**Figure 3**). Primary breast tumor MGMT expression was not a reliable predictor of brain MGMT status, with 40% discordance between sites. In our sample, 60% of patients with breast cancer brain metastases showed low MGMT expression (**Table 2**), indicating that these patients are potentially susceptible to TMZ prevention of brain metastasis. A strong trend was observed for an association of HER2 overexpression and MGMT negativity in the HER2 positive brain metastases (P = 0.089), suggesting the eligibility of this subset of patients for potential clinical trials (**Table 3**) 57.  
Table 2. Cross-tabulation of MGMT IHC staining in matched primary breast tumors and resected brain  
  
TMZ has been administered in at least two doses and schedules. In its activity repairing O6methylguanine lesions, MGMT transfers the alkyl group from guanine to a cysteine moiety in its active site, thereby repairing the DNA. MGMT is irreversibly inactivated in this process and new MGMT protein synthesis is needed to restore MGMT activity 61,62. In an attempt to overcome MGMT activity, trials evaluating the metronomic use of temozolomide were developed mainly for treatment of melanoma and brain tumors 63,64. MGMT enzyme activity was assayed in peripheral blood monocytes (PBMCs) collected at various time points during treatment and shown to be progressive and cumulatively decreased 65,66. More recently, at least three phase II clinical trials evaluated the continuous daily use of temozolomide (50mg/m2) in heavily pretreated brain tumors and demonstrated activity and safety of the regimen, even in non-methylated MGMT tumors 54,67,68.  
To our knowledge, the only published trial evaluating the combined use of temozolomide with an anti-HER2 agent in breast cancer brain metastases was the LAPTEM trial 50, combining temozolomide with lapatinib for treatment of established brain metastases, not prevention. The regimen showed a favorable toxicity profile, with main side effects being fatigue, diarrhea and constipation. Temozolomide was used in three dose levels: 100, 150 and 200 mg/m2/day, days 1–5 with cycles of 28 days; lapatinib was given orally, once a day also at three dose levels: 1000, 1250, 1500 mg/day. The MTD was not reached, per protocol. No cardiac toxicity events were observed. Of the 15 evaluable patients, 10 achieved stabilization of disease, with estimated median survival time of 10.94 months (95% CI: 1.09–20.79), and median progression-free survival time of 2.60 months [95% CI: 1.82–3.37].  
We propose a secondary-prevention clinical trial with oral temozolomide for HER2-positive breast cancer patients with 1 to 3 brain metastases after recent local treatment (SRS or surgical resection), with the goal to decrease incidence of new metastatic lesions in the brain at 1 year. Temozolomide will be combined to the anti-HER2 agent T-DM1 for systemic control of disease.  
Table 3Associations of primary tumor and brain metastasis MGMT expression with patient characteristics
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1.3 correlatives background and rationale

Studies will address aspects of TMZ resistance, molecular alterations in brain metastatic tumor cells, the neuro-inflammatory response, and markers of cognition.
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1.3.1 cell free dna (cfdna)

Few molecular tools exist to analyze brain metastasis progression other than MRI scans and neurocognitive testing. Recent work from Harvard demonstrated that brain metastases show unique mutational patterns as compared to primary tumors 69. An example is shown on **Figure 4.**
These data were based on craniotomy samples, which will generally be unavailable in the current clinical study. Detecting DNA alterations in brain metastasis from patients' systemic samples will likely be compromised by the contributions of systemic disease. We propose sequencing of cell free tumor DNA in the CSF as an alternative endpoint.  
Recently several publications have reported the sequencing of cell free DNA (cfDNA) in CSF
from patients with brain cancers and leptomeningeal disease from melanoma 70,71. The primary tumor tissue was subjected to DNA sequencing to identify specific alterations from a preselected targeted panel. In one report four CSF samples were analyzed by whole exome sequencing without prior knowledge of specific mutations; two of the four revealed mutant allele frequencies >10%
that could be tracked 70. The presence or absence of cfDNA in CSF, or its levels and alterations was a correlate of clinical course and more informative than serum cfDNA71. Based on these data it is hypothesized that: (a) patients will have mutations that can be identified in the CSF in the secondary brain metastasis prevention trial; (b) alterations in the pattern or prevalence of mutations will correlate with clinical outcome in the secondary brain metastasis prevention trial and (c) that alterations in the pattern or prevalence of mutations or cfDNA levels in CSF will predict relapse before MRI imaging. In addition, the data will provide a landscape of the genetic mutations from cfDNA in CSF for brain metastasis of breast cancer, and a portrait of its evolution through progression. The CSF cfDNA sequence data will be compared to that of the primary tumor where available and serum cfDNA. Comparison with the primary tumor may provide a portrait of mutations that are shared between the primary tumor and the brain metastasis as evidenced in cfDNA from CSF. Comparison with serum may identify events distinct between systemic and CNS progression.  
  
This approach has been pioneered by Drs. Carter and Brastianos and is potentially groundbreaking. Drs. Brastianos and Carter have already subjected nearly 100 matched brain metastases, primary tumors and normal tissues to whole exome sequencing and demonstrated that in 53% of cases, oncogenic drivers were detected in the brain metastases that were not present in the clinically sampled primary tumor69. A case of a resected HER2+ brain metastasis is presented in **Figure 4**,
showing that the brain metastasis was genetically divergent from the primary tumor biopsy and that multiple regions of the brain metastasis were relatively genetically homogeneous. Now they have developed techniques to analyze cfDNA in CSF69,72-74.  
  
DNA will be subjected to solution-phase hybrid capture followed by Illumina sequencing to >100x mean coverage. Mutations and copy-number changes will be determined within the Firehose pipeline environment at the Broad Institute using a suite of existing tools, which include the published MuTect, Indelocator, GISTIC and MutSig algorithms72,73,75-79. To understand inter- and intratumoral heterogeneity, we will use computational tools developed by Dr. Carter to perform an integrative analysis of somatic mutations and copy-number alterations. This analysis, termed Absolute80, estimates the cancer-cell fraction (CCF) of each mutation, allowing parsimonious resolution of the clonal architecture of bulk cancer tissue samples. Dr. Carter also developed a computational tool called Phylogic, which automates the inference of phylogenetic trees relating all cancer subclones identified in the cancer tissue samples of each individual patient69. Phylogic operates by analyzing the modeled absolute copy-numbers and mutation CCF estimates provided by ABSOLUTE. **Figure 5** presents preliminary data developed by Drs. Brastianos and Carter, showing copy number alterations in the CSF of a HER2+ brain metastasis patient.  
We will characterize cfDNA from CSF samples and blood at diagnosis and during treatment, and bulk DNA from primary tumors using targeted and whole-exome sequencing (WES). All samples will be subjected to targeted sequencing and selected cases of adequate quality will be subjected to WES. This will allow us to characterize somatic point mutations, insertion-deletion events, and copy-number changes.
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1.3.2 mgmt status

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that confers resistance to TMZ. MGMT expression and activity have been determined by several methods in clinical tissues, most often by the DNA methylation status of its promoter, including methylation specific PCR (MSP), pyrosequencing and semi-quantitative MSP. Other techniques quantify total MGMT expression including IHC and QRT-PCR 81. When tumor tissue was assessed for MGMT
status using MSP, low expression of MGMT generally correlated with response or better outcome to TMZ therapy in GBM82, TMZ combination therapy studies in GBM83,84, and other primary brain tumors85,86. MGMT studies have been conducted in other cancer types when TMZ was incorporated into the therapy, including lung cancer87, aerodigestive and colorectal cancers88, and melanoma89. These studies used tumor samples from biopsy or surgery.  
It is noteworthy that MGMT alterations occur in tissues other than tumor. Alterations were detected in stroma around tumor in breast cancer90. Similar findings have been reported for other cancers91.The data suggest that the microenvironment may also play a role in drug sensitivity or resistance.  
MGMT methylation has been measured in serum and compared to that in FFPE tumor blocks for glioma92. Circulating DNA was extracted from plasma using the QIAamp DNA Blood Mini kit. After bisulfite modification, an end point PCR assay, using primers specific to either methylated or unmethylated DNA, was used to determine MGMT promoter methylation status
(Qiagen). The frequency of MGMT methylation was 60% in tumor and 41% in serum. Serum MGMT changed from baseline to 12 months. Both measurements predicted response to TMZ.  
Other studies have reported similar approaches93-95. CSF as a source of MGMT methylation data has only been reported in abstract form96 and a literature search revealed no analysis of MGMT in CTCs.  
In preliminary studies in the Steeg laboratory, MGMT expression was determined by IHC in FFPE blocks of matched sets of primary breast tumors and resected brain metastases (n=62)97.  
Samples were dichotomized into low expression (<5% of tumor cells+) and high expression (>5%) categories by a pathologist. Overall concordance of primary tumor and brain metastasis MGMT expression was weak. Low MGMT expression was prevalent, in 58% of brain metastasis specimens. A strong trend was observed in brain metastases for an association of HER2 overexpression and MGMT negativity (P = 0.089), suggesting the eligibility of this subset for potential clinical trials.  
The hypothesis to be tested in this trial is that MGMT status, either at baseline or changes over time, will reflect secondary preventive activity of TMZ. A second hypothesis posits that alterations in MGMT may be an early sign of resistance to therapy and precede progression detectable on MRI. In addition, we will determine the concordance of MGMT methylation from multiple sources (tissue, serum, CSF) and by two assays (methylation and IHC). MGMT status will be assayed from patient serum at baseline and on study, the primary FFPE tumor block as available, and CSF as available in this trial. MGMT methylation will be used for all samples. For the FFPE block, MGMT IHC will also be conducted as previously reported. 1.3.3 Neuroinflammatory response and neuronal damage metrics.  
Brain metastases elicit a prominent neuro-inflammatory response including activated astrocytes, identified by GFAP positivity, and microglia98. Multiple preclinical studies indicate that the neuro-inflammatory milieu conducts cross-talk with tumor cells to augment metastatic colonization99. The hypothesis to be tested is that the extent of neuro-inflammatory response, either at baseline or through treatment, will correlate with secondary prevention efficacy of TMZ. Other goals of these studies are to determine the prevalence, magnitude and kinetics of the neuroinflammatory response in brain metastasis progression, which has not been addressed in the clinical literature.  
The neurocognitive sequelae of brain metastases derive from both the lesions and the treatments100-103. In preclinical studies brain metastases of breast cancer caused neuronal cell death 104, which would be a contributor to neurocognitive dysfunction. Regional or systemic tests for neuro-inflammatory responses and neurocognitive sequelae have not been meaningfully incorporated into clinical research on brain metastasis, other than mental function testing. Several leads have been developed for other brain pathologies and will be tested in this trial:
a. The traumatic brain injury (TBI) field, another disease of the brain and BBB, has pioneered blood-based assays. Blood markers of neuro-inflammation (GFAP) and neuronal injury (UCH-L1)
have been measured in patients with mild-severe TBI over time105-107. Serum levels were measured in duplicate using an ELISA assay (Banyan Biolarmers Inc.). Levels of each protein, or a ratio of the two were reported. Normal controls or trauma patients without TBI showed low levels of each marker. Serum levels were elevated in TBI patients and correlated with several indicators of outcome, need for treatments, and disease severity. The same markers have been infrequently reported in CSF108. Similar results have been reported for stroke109.  
Quantification of GFAP and UCH-L1 will be performed in serum and CSF samples from all patients on the trial. Validation experiments and the serum measurements from the trial will be conducted in the Steeg laboratory. The questions to be answered include: (a) what is the variation in baseline levels of brain metastasis patients? (b) what are the changes in time with and without progression? (c) Do changes in either level, or the ratio, correlate with preventive efficacy of TMZ? (d) Do changes predict clinical progression earlier than MRI?  
b. MicroRNA-34c (miR-34c) has been studied in Alzheimer's disease (AD) as a biological covariate of cognitive function110. miR-34c was identified as a miR enriched in the hippocampus of mice. It targets genes involved in axonogenesis, neuron projection neurogenesis, cell communication, neuronal activities, synaptic transmission, channel activity. Injection of miR-34c functionally improved learning in a mouse model of AD110. MiR-34c has been quantified in plasma of AD and healthy controls111. Of 110 AD and 123 normal control patients, RNA of sufficient integrity was extracted from 78 and 85, respectively. MiRNA extraction was completed using plasma samples stored in RNAlater, subjected to Trizol/chloroform extraction and centrifugation. A Taqman miRNA reverse transcription kit was used to quantify miR-34c, using QPCR. A 30 ml blood sample was used, it is not known if smaller amounts can be used. Levels of miR-34c were elevated in AD patients compared to normal, or normal elderly controls, and correlated with mental function on Mini-Mental Status Examinations111.  
Preclinical work will be performed in the Steeg lab to assay validate this technique, and to determine if an amplification will permit quantification of miR-34c from smaller plasma samples. If sufficient plasma is available from patients, miR-34c levels will be determined to answer the same questions posed in (a). If sufficient miRNA is obtained, a full miRNA profile may be obtained on the samples using a miRNA array112.
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1.3.4 exosomes

Currently there is a lack of rapid and reliable methods for cancer detection, metastatic prediction and treatment response assessment. Dr. Lyden's laboratory is developing non-invasive methods to predict patient outcome for site-specific metastasis and that can be used to monitor treatment responses. These methods are based on a simple blood test and rely on the isolation and characterization of tumor-derived exosomes from the plasma of cancer patients.  
Exosomes are small membrane vesicles (30-100 nm in diameter) of endocytotic origin and secreted by most cells constitutively. These microvesicles contain cell type-specific proteins and genetic materials, including mRNAs, miRNAs and DNA, and can exert a functional influence once uptaken by recipient cells, therefore representing novel mediators of intercellular communication. Importantly, exosomes are shed from cancers and are readily isolated from the peripheral circulation. A growing body of evidence has shown that tumor-derived exosomes not only have a crucial role in regulating tumor growth and metastasis, but also provide a "treasure box" of novel non-invasive biomarkers for different types of cancers113.  
We propose to use this "liquid biopsy" in this trial to evaluate the possibility of rapidly screening for metastatic disease detection, as well as for metastasis prediction and treatment response monitoring. Analysis of exosomes in blood samples will be performed in the Lyden lab at Weill Cornell Medical College.
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1.3.5 pharmacokinetics

Both temozolomide and T-DM1 are FDA approved drugs with established information on their pharmacokinetics. Since we are using temozolomide in new dose and schedule for this study, we will sample a cohort of patients in order to determine temozolomide exposure.
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2 eligibility assessment and enrollment

2.1.1 Phase 1 Inclusion Criteria 2.1.1.1 Participants must have histologically confirmed HER2-positive breast cancer for which standard curative measures do not exist or are no longer effective. HER2 testing must have been performed in a laboratory accredited by the College of American Pathologists (CAP) or another accrediting entity.  
2.1.1.2 Participants must have brain metastases, treated within 12 weeks of study entry with SRS,
resection or WBRT. A minimum interval of 3 weeks between completion of brain SRS and/or resection and 6 weeks for WBRT and the start of treatment in this trial will be observed to allow proper healing. The presence of concomitant extracranial metastatic disease is allowed for enrollment.  
2.1.1.3 Corticosteroids will be allowed at enrollment and during the first month of treatment with T-DM1 after SRS, up to a dose of no more than 10mg of dexamethasone daily or equivalent. Participants that need to continue corticosteroids after the initial month will be allowed to continue in the protocol treatment if no further increase in dose is necessary. Participants that need increase in dose of corticosteroid after initial month will be taken off protocol treatment.  
2.1.1.4 Age >18 years. Because breast cancer is not commonly found in pediatric population and no dosing or adverse event data are currently available on the use of temozolomide in combination with T-DM1 in participants <18 years of age, children are excluded from this study, but will be eligible for future pediatric trials.  
2.1.1.5 ECOG performance status <2 (Karnofsky >60%, see **Appendix A**). 2.1.1.6 Participants must have adequate organ and marrow function as defined below:
leukocytes >3,000/mcL  
total bilirubin within normal institutional limits  
AST(SGOT)/ALT(SGPT) <3.0 X institutional upper limit of normal OR
creatinine clearance >60 mL/min/1.73 m2 for participants with creatinine levels above institutional normal.  
2.1.1.7 Alkylating agents as well as other therapeutic agents used in this trial are known to be teratogenic, women of child-bearing potential and men must agree to use adequate contraception (hormonal or barrier method of birth control; abstinence) prior to study entry, for the duration of study participation and for 7 months (women) or 4 months (men) after treatment completion. Should a woman become pregnant or suspect she is pregnant while she or her partner is participating in this study, she should inform her treating physician immediately.  
2.1.1.8 Ability of subject to understand and the willingness to sign a written informed consent document.  
2.1.2 Phase 1 Exclusion Criteria 2.1.2.1 Patients who are receiving any other investigational agents. 2.1.2.2 Patients unable to speak or understand English, since they cannot complete neurocognitive evaluation.  
|  absolute neutrophil count   | >1,000/mcL   |
|-------------------------------|--------------|  
platelets >100,000/mcL creatinine up to 1.5 upper institutional limits  
2.1.2.3 Patients with known leptomeningeal metastatic disease. 2.1.2.4 Patients with major symptoms or impairments related to brain metastases, such as aphasia or severe confusion, will be excluded per PI discretion when those symptoms preclude proper neurocognitive evaluation during the study treatment.  
2.1.2.5 Patients who have received previous treatment with T-DM1 and had systemic progression of disease while on it, are ineligible. Patients receiving treatment with T-DM1 whose only site of disease progression was brain are allowed to enroll in this trial.  
2.1.2.6 Patients who have received chemotherapy in the previous 3 weeks (6 weeks for nitrosoureas or mitomycin).  
2.1.2.7 HBV (HBs Ag positive) or HCV (anti-HCV positive) patients are ineligible because of potential reactivation of hepatitis virus with temozolomide use.  
2.1.2.8 Grade ≥3 peripheral neuropathy according to (NCI CTCAE) version 4.0. 2.1.2.9 Cerebral Vascular Accident (CVA) or Transitory Ischemic Attack (TIA) in the year before enrollment, or presence of residual symptoms from CVA that happened more than a year before.  
2.1.2.10 Pulmonary Embolism (PE) in the 3 months before enrollment. Anticoagulation is permitted.  
2.1.2.11 Impaired cardiac function or clinically significant cardiac disease including the following:
- New York Heart Association grade III or IV congestive heart failure.  
- Myocardial infarction within the last 12 months.  
- Subjects with impaired LVEF (<50%).
2.1.2.12 Patients with inability to complete brain MRI studies with contrast. 2.1.2.13 Patients with breast tissue expanders must have those removed before enrollment.  
2.1.2.14 History of allergic reactions attributed to compounds of similar chemical or biologic composition to temozolomide or T-DM1. Patients who are tolerating TDM1 successfully with premedications and slower infusion will be allowed.  
2.1.2.15 Patients receiving medications that are strong CYP3A4 inhibitors or inducers are ineligible. In vitro studies indicate that DM1, the cytotoxic component of T-DM1, is metabolized mainly by CYP3A4 and to a lesser extent by CYP3A5. Concomitant use of strong CYP3A4 inhibitors with T-DM1 should be avoided due to the potential for an increase in DM1 exposure and toxicity. Consider an alternate medication with no or minimal potential to inhibit CYP3A4. Lists including medications and substances known or with the potential to interact with CYP3A4 isoenzymes are provided in **Appendix B.**
2.1.2.16 Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, that would limit compliance with study requirements.  
2.1.2.17 Pregnant women are excluded from this study because temozolomide is an alkylating agent with the potential for teratogenic or abortifacient effects. Because there is an
unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with temozolomide and/or T-DM1, breastfeeding should be discontinued if the mother is treated with either of those agents. These potential risks may also apply to other agents used in this study.  
2.1.2.18 HIV-positive patients are excluded because these patients are at increased risk of lethal infections when treated with marrow-suppressive therapy.  
2.1.2.19 Patients with any other concomitant invasive malignancies are ineligible. Prior invasive cancers treated with curative intent, and with no evidence of recurrent disease, may be eligible after PI evaluation. Patients with treated limited stage basal cell or squamous cell carcinoma of the skin or carcinoma in situ of the breast or cervix are eligible.  
2.1.3 Phase 2 Inclusion Criteria 2.1.3.1 Participants must have histologically confirmed HER2-positive breast cancer for which standard curative measures do not exist or are no longer effective. HER2 testing must have been performed in a laboratory accredited by the College of American Pathologists (CAP) or another accrediting entity.  
2.1.3.2 Participants must have 1-10 brain metastases, by contrast MRI, treated within 12 weeks of study entry with SRS and/or resection. A minimum interval of 3 weeks between completion of brain SRS and/or resection and the start of treatment in this trial will be observed to allow proper healing. The presence of concomitant extracranial metastatic disease is allowed for enrollment.  
2.1.3.3 Corticosteroids will be allowed at enrollment and during the first month of treatment with T-DM1 after SRS, up to a dose of no more than 10mg of dexamethasone daily or equivalent. Participants that need to continue corticosteroids after the initial month will be allowed to continue in the protocol treatment if no further increase in dose is necessary. Participants that need increase in dose of corticosteroid after initial month will be taken off protocol treatment.  
2.1.3.4 Age >18 years. Because breast cancer is not commonly found in pediatric population and no dosing or adverse event data are currently available on the use of temozolomide in combination with T-DM1 in participants <18 years of age, children are excluded from this study, but will be eligible for future pediatric trials.  
2.1.3.5 ECOG performance status <2 (Karnofsky >60%, see **Appendix A**). 2.1.3.6 Participants must have normal organ and marrow function as defined below:
leukocytes >3,000/mcL  
|  absolute neutrophil count   | >1,000/mcL   |
|-------------------------------|--------------|  
platelets >100,000/mcL total bilirubin within normal institutional limits creatinine up to 1.5 upper institutional limits  
AST(SGOT)/ALT(SGPT) <3.0 X institutional upper limit of normal
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or

creatinine clearance >60 mL/min/1.73 m2 for participants with creatinine levels above institutional normal.  
2.1.3.7 Alkylating agents as well as other therapeutic agents used in this trial are known to be teratogenic, women of child-bearing potential and men must agree to use adequate contraception (hormonal or barrier method of birth control; abstinence) prior to study entry, for the duration of study participation and for 7 months (women) or 4 months (men) after treatment completion. Should a woman become pregnant or suspect she is pregnant while she or her partner is participating in this study, she should inform her treating physician immediately.  
2.1.3.8 Ability of subject to understand and the willingness to sign a written informed consent document.  
2.1.4 Phase 2 Exclusion Criteria 2.1.4.1 Patients who are receiving any other investigational agents. 2.1.4.2 Patients unable to speak or understand English, since they cannot complete neurocognitive evaluation.  
2.1.4.3 Patients with known leptomeningeal metastatic disease. 2.1.4.4 Patients previously treated with whole brain radiation therapy (WBRT). 2.1.4.5 Patients with major symptoms or impairments related to brain metastases, such as aphasia or severe confusion, will be excluded per PI discretion when those symptoms preclude proper neurocognitive evaluation during the study treatment.  
2.1.4.6 Patients who have received previous treatment with T-DM1 and had systemic progression of disease while on it, are ineligible. Patients receiving treatment with T-DM1 whose only site of disease progression was brain are allowed to enroll in this trial.  
2.1.4.7 Patients who have received chemotherapy in the previous 3 weeks (6 weeks for nitrosoureas or mitomycin).  
2.1.4.8 HBV (HBs Ag positive) or HCV (anti-HCV positive) patients are ineligible because of potential reactivation of hepatitis virus with temozolomide use.  
2.1.4.9 Grade ≥3 peripheral neuropathy according to (NCI CTCAE) version 4.0. 2.1.4.10 Cerebral Vascular Accident (CVA) or Transitory Ischemic Attack (TIA) in the year before enrollment, or presence of residual symptoms from CVA that happened more than a year before.  
2.1.4.11 Pulmonary Embolism (PE) in the 3 months before enrollment. Anticoagulation is permitted.  
2.1.4.12 Impaired cardiac function or clinically significant cardiac disease including the following:
- New York Heart Association grade III or IV congestive heart failure. - Myocardial infarction within the last 12 months.
- Subjects with impaired LVEF (<50%).  
2.1.4.13 Patients with inability to complete brain MRI studies with contrast.  
2.1.4.14 Patients with breast tissue expanders must have those removed before enrollment. 2.1.4.15 History of allergic reactions attributed to compounds of similar chemical or biologic composition to temozolomide or T-DM1. Patients who are tolerating TDM1 successfully with premedications and slower infusion will be allowed.  
2.1.4.16 Patients receiving medications that are strong CYP3A4 inhibitors or inducers are ineligible. In vitro studies indicate that DM1, the cytotoxic component of T-DM1, is metabolized mainly by CYP3A4 and to a lesser extent by CYP3A5. Concomitant use of strong CYP3A4 inhibitors with T-DM1 should be avoided due to the potential for an increase in DM1 exposure and toxicity. Consider an alternate medication with no or minimal potential to inhibit CYP3A4. Lists including medications and substances known or with the potential to interact with CYP3A4 isoenzymes are provided in **Appendix B.**
2.1.4.17 Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, that would limit compliance with study requirements.  
2.1.4.18 Pregnant women are excluded from this study because temozolomide is an alkylating agent with the potential for teratogenic or abortifacient effects. Because there is an unknown but potential risk for adverse events in nursing infants secondary to treatment of the mother with temozolomide and/or T-DM1, breastfeeding should be discontinued if the mother is treated with either of those agents. These potential risks may also apply to other agents used in this study.  
2.1.4.19 HIV-positive patients are excluded because these patients are at increased risk of lethal infections when treated with marrow-suppressive therapy.  
2.1.4.20 Patients with any other concomitant invasive malignancies are ineligible. Prior invasive cancers treated with curative intent, and with no evidence of recurrent disease, may be eligible after PI evaluation.  
2.1.5 Recruitment Strategies
This protocol may be abstracted into a plain language announcement posted on NIH websites and on NIH social media platforms. Participants will be recruited from the current patient population at NIH. The study may be listed in patient advocacy websites such as the Army of Women.
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2.2 screening evaluation

2.2.1 Screening activities performed prior to obtaining informed consent Minimal risk activities that may be performed before the subject has signed a consent include the following:
Email, written, in person or telephone communications with prospective subjects  
Review of existing medical records to include H&P, laboratory studies, etc.  
Review of existing MRI, x-ray, or CT images Review of existing photographs or videos Review of existing pathology specimens/reports from a specimen obtained for diagnostic purposes
A waiver of consent for these activities has been requested in Section **11.7**.  
2.2.2 Screening activities performed after a consent for screening has been signed The following activities will be performed only after the subject has signed the study consent OR the consent for study 01-C-0129 (provided the procedure is permitted on that study) on which screening activities may also be performed. Assessments performed at outside facilities or on another NIH protocol within the timeframes below may also be used to determine eligibility once a participant has signed the consent. Screening evaluations or tests will be done prior to enrollment within the following time frames:
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anytime 2.2.3 pathology confirmation of

Pathological confirmation of diagnosis and ER, PR and HER2 expression in the Laboratory of Pathology at NIH Clinical Center or Walter Reed National Military Medical Center at Bethesda. However, if no pathologic specimen is available, participants may enroll with a pathologist's report showing a histologic diagnosis of HER2-positive breast cancer in a College of American Pathologists (CAP) accredited laboratory and a clinical course consistent with the disease.
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within 30 days

2.2.4 Imaging studies:
CT of chest/abdomen /pelvis (MRI may be substituted at investigator's discretion)  
Bone scan Contrast enhanced brain MRI
2.2.5 Cardiology test: 2-D Echocardiogram or MUGA-scan 2.2.6 Anti-HIV, anti-HCV, Hepatitis B Surface Ag
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within 10 days

2.2.7 History and Physical examination: Complete medical history (including prior therapy) and physical examination (including height, weight, vital signs, ECOG performance status and complete neurological exam). 2.2.8 Laboratory Studies:  
CBC with differential, Biochemical profile: sodium, potassium, chloride, carbon dioxide, BUN, creatinine or measured creatinine clearance, glucose, AST, ALT, total bilirubin, calcium, total protein, albumin, alkaline phosphatase, magnesium.  
2.2.9 Pregnancy Testing:
For female subjects of childbearing potential, urine or serum HCG will be performed. Participants who are postmenopausal (age-related amenorrhea for 12 or more consecutive months, or documented FSH > 40 mIU/mL / Estradiol < 20mIU/mL), or who had undergone hysterectomy or bilateral oophorectomy are exempt from pregnancy testing. For baseline evaluations, please see Section 2.5.
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2.3 participant registration and status

Registration and status updates (e.g., when a participant is taken off protocol therapy and when a participant is taken off-study) will take place per CCR SOP ADCR-2, CCR Participant Registration & Status Updates found here. 2.3.1 Screen Failures Screen failures are defined as participants who consent to participate in the clinical trial but are not subsequently assigned to the study intervention or entered in the study. A minimal set of screen failure information is required to ensure transparent reporting of screen failure participants, to meet the Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to respond to queries from regulatory authorities. Minimal information includes demography, screen failure details, eligibility criteria, and any serious adverse event (SAE).
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2.4 randomization (or stratification) procedures

| Number   | Name   | Description   |
|----------|--------|---------------|  
1 Phase I Cohort Subjects on phase I portion of study 2 Phase II Cohort Subjects on phase II portion of study  
1 Phase I Phase I: T-DM1 + TMZ dose escalation  
| Arms Letter   | Name   | Description   |
|---------------|--------|---------------|  
2A Phase II arm A Phase II: T-DM1 alone 2B Phase II arm B Phase II: T-DM1 + TMZ
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stratifications randomization and arm assignment

There are two stratification factors in the randomization: number of brain lesions (1 vs. 2-5 vs >5) and status of extracranial lesions (none/controlled. vs. progressing). This results in six stratification groups:  
| Name                           | Distinct Options   |                    |
|--------------------------------|--------------------|--------------------|
| Number of CNS lesions          |                   | 1                  |
|                               | 2-5                |                    |
|                               | >5                 |                    |
| Status of extracranial lesions |                   | None or controlled |
|                               | Progressing        |                    |  
1. One brain lesion with no or controlled extracranial lesions 2. One brain lesion with progressing extracranial lesions 3. Two - three to five brain lesions with no or controlled extracranial lesions 4. Two - three to five brain lesions with progressing extracranial lesions 5. More than five brain lesions with no or controlled extracranial lesions 6. More than five brain lesions with progressing extracranical lesions
Within each of these six groups participants will be randomized at study entry to either arm 2A or arm 2B. Arm 2A will receive treatment with T-DM1 alone and Arm 2B will receive treatment with T-DM1 plus temozolomide. The Central Registration Office will be contacted via encrypted email at: ncicentralregistration-l@mail.nih.gov to determine the appropriate assignment for each participant. Once the NCI Central Registration Office receives subject stratification group assignment, randomization to treatment arms will be performed by the CRO.  
  
Participants in cohort 1 will be directly assigned to arm 1. Participants in cohort 2 will be randomized between arms 2A and 2B, stratified for number of CNS lesions and status of extracranial lesions.
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2.5 baseline evaluation

The following procedures will be completed within 7 days before initiating of the treatment. Tests done at Screening do not need to be repeated on Baseline if performed in designated time frame.  
2.5.1 Clinical Evaluation:
Review of concurrent medications Review of baseline adverse events Physical exam (including complete neurological exam, vital signs, weight and ECOG
performance status)
2.5.2 Laboratory Evaluation:  
CBC with differential and platelet count Biochemical profile: (sodium, potassium, chloride, carbon dioxide, BUN, creatinine or measured creatinine clearance, glucose, AST, ALT, bilirubin, calcium, total protein, albumin, alkaline phosphatase, magnesium)
PT/INR/PTT
TSH
Urinalysis Pregnancy test for women of childbearing potential.  
2.5.3 Neurocognitive and quality of life assessment:
Baseline MD Anderson Symptom Inventory-Brain Tumor Module (MDASI-BT) (**Error!**
Reference source not found.).  
Application of Patient-Reported Outcomes Measurement Information System
(PROMIS®) cognitive questionnaire (**Error! Reference source not found.).**
2.5.4 Correlative tests evaluation:
Blood samples for correlative studies (will be collected at Day 1 of Cycle 1).  
Lumbar puncture by Interventional Radiology for collection of CSF sample.  
Primary tumor paraffin block and /or fresh frozen tissue from previous surgery will be requested from participants at baseline to perform comparative genomic analysis as part of correlative studies planned for this clinical trial.
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3 study implementation 3.1 study

This is an open label Phase I/II trial, accruing initially one cohort to determine the safety and RP2D of Temozolomide combined to T-DM1 in participants with HER2-positive breast cancer metastatic to the brain after SRS or resection of those brain metastases (Phase I); and to examine the safety and clinical activity of the Temozolomide plus T-DM1 combination versus T-DM1 alone in the same group of participants (Phase II). In the Phase II portion, participants will be randomized between the two treatment arms. Participants will receive T-DM1 every 21-days, as per standard use recommendation. Administration of Temozolomide will be in daily dose by mouth. T-DM1 can be given up to 3 days earlier or delayed up to 14 days due to holidays, inclement weather, conflicts, or similar reasons. The timing of subsequent administrations is then adjusted to maintain a 21 day-interval. Phase I: 3.1.1 Dose Limiting Toxicity The DLT period is one cycle, 21 days. DLT is defined as any of the following: Non-Hematologic dose-limiting toxicity:
Grade 3 or higher adverse events (AEs), excluding:  
Grade 3 hypertension controlled with anti-hypertensive therapy Grade 3 asymptomatic electrolytes imbalance with optimal and continuing repletion that downgrades to grade 1 or better within 3 days after onset of the event.  
Grade 3 endocrinopathy that is managed with or without hormone replacement therapy and the participant is asymptomatic.  
Grade 3 asymptomatic increase in AST (SGOT), ALT (SGPT), that downgrades to grade 1 or better within 7 days after onset of the event.  
Transient (lasting less than<48 hrs.) nausea/emesis/diarrhea if corrected with conservative measures within 24-48 hours.
Persistent (>14 days) non-hematologic Grade 2 AEs despite optimal medical management and temozolomide treatment delay > 14 days.  
Hematologic Toxicity:
Grade 4 neutropenia of ≥ 7 days duration, grade 3 or 4 neutropenia of any duration with fever or documented infection (febrile neutropenia).  
Grade ≥ 3 thrombocytopenia.  
All other grade 4 hematologic toxicities, excluding:
Grade 4 lymphopenia, or leukopenia in the absence of grade 3 or higher neutropenia.
Note: Participants in use of corticosteroids that require increase in dose of corticosteroid after initial month of treatment in this clinical trial will be taken off protocol treatment given potential of tumor necrosis post SRS and will not be considered as DLT.
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3.1.2 dose escalation

Dose escalation will follow the classical '3+3' trial design. If none of the first three participants in a cohort experiences a dose-limiting toxicity during one cycle of treatment, another three participants will be treated at the next higher dose level. However, if one of the first three participants experiences a dose-limiting toxicity during first cycle of treatment, three more participants will be treated at the same dose level. Dose escalation continues until the final dose level meets safety criteria or at least two participants among a cohort of three to six participants experience dose-limiting toxicities (i.e., ≥33% of participants with a dose-limiting toxicity at that dose level).  
| Dose Escalation Schedule   |                              |                             |
|----------------------------|------------------------------|-----------------------------|
| Dose Level                 | T-DM1 mg/kg IV every 21 days | Temozolomide mg/m2 PO daily |
| Level 1                    | 3.6                          | 30                          |
| Level 2                    | 3.6                          | 40                          |  
| Number of Participants  with DLT at a Given Dose Level                    | Escalation Decision Rule                                                                                                                                                                                                                                                                                                                                                                                                 |
|---------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 0 out of 3                                                                | Enter up to 3 participants at the next dose level                                                                                                                                                                                                                                                                                                                                                                        |
| > 2                                                                       | Dose escalation will be stopped. This dose level will  be declared the maximally administered dose  (highest dose administered). Up to 3 additional  participants will be entered at the next lowest dose  level if only 3 participants were treated previously at  that dose.                                                                                                                                           |
| 1 out of 3                                                                | Enter up to 3 more participants at this dose level.  If 0 of these 3 participants experience DLT,  proceed to the next dose level.  If 1 or more of this group suffer DLT, then dose  escalation is stopped, and this dose is declared  the maximally administered dose. Up to 3  additional participants will be entered at the next  lowest dose level if only 3 participants were  treated previously at that dose. |
| <1 out of 6 at highest dose  level below the maximally  administered dose | This is the MTD and is generally the recommended  Phase II dose. At least 6 participants must be entered  at the recommended Phase II dose.                                                                                                                                                                                                                                                                              |  
Dose escalation will follow the rules outlined in the Table below. During the Phase I DLT time period (first 21 days), no decrease or delay for toxicity reasons in dose of T-DM1 is allowed. If participants develop side effects considered mainly related to TDM1 that would require dose reductions leading to discontinuation of that drug, they should be taken off protocol treatment and substituted by another participant. During the Phase II portion of this trial, dose reductions should be done as described in **3.3.1**.
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phase ii

The phase II portion of this trial will randomize participants between treatment with T-DM1 alone at standard recommended dose (3.6 mg/kg IV every 21 days) or the combination of T-DM1 at standard dose and Temozolomide. The Temozolomide recommended dose for phase II trial will be considered the maximum tolerated dose determined from the lead-in phase I trial.
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3.2 drug administration 3.2.1 t-dm1

See section **13.2.4** for preparation instructions. T-DM1 will be administered intravenously to all participants in the study. T-DM1 will be administered at standard recommended dose (3.6 mg/kg), once every 21 day cycle until disease progression, participant withdrawal or toxicities. The infusion site will be closely monitored for possible subcutaneous infiltration during drug administration. First infusion will be administered approximately 90 minutes. Participants should be observed during the infusion and for at least 90 minutes following the initial dose for fever, chills, or other infusion related reactions. Subsequent infusions will be administered approximately 30 minutes if prior infusions were well tolerated. Participants should be observed during the infusion and for at least 30 minutes after infusion. If the infusion is interrupted, the reason for interruption will be documented in the clinical record. Administration of T-DM1 may be given 3 days earlier or delayed up to 14 days due to holidays, inclement weather, conflicts, or similar reasons. The timing of subsequent administrations is then adjusted to maintain a 21 days-interval. Participants will be monitored before and after the infusion with assessment of vital signs at the times specified in the Study Calendar (Section **3.5)**. In the event of a ≤grade 2 infusion-related reactions (IRR), the infusion rate of study drug may be decreased by 50% or interrupted until resolution of the event (up to 4 hours) and re-initiated at 50% of the initial rate until completion of the infusion. For subjects with a ≤grade 2 infusion related reaction, subsequent infusions may be administered at 50% of the rate at which the IRR occurred. Acetaminophen and/or an antihistamine (e.g., diphenhydramine) or equivalent medications may be administered at the discretion of the investigator. T-DM1 will be given until disease progression, participant withdrawal or toxicities. The start of the first cycle will be scheduled within 7 days of registration. In the event of G3 or G4 IRR, treatment should be stopped and not repeated in the future.
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3.2.2 temozolomide

During Phase I all participants will receive temozolomide and T-DM1. During Phase II only participants randomized to the combination treatment arm, will receive temozolomide and T-DM1. Temozolomide at the appropriate dose level will be given orally continuously once a day, every day during the 21 cycle. Temozolomide will be given in combination with T-DM1 until disease progression, participant withdrawal or toxicities. Temozolomide will be dispensed at the start of each cycle. Participants will be provided with a pill diary (**Appendix C**), instructed in its use, and asked to bring it with them to each appointment. The dose will be determined using the body surface area (BSA) calculated at the beginning of each cycle unless significant (> 3 kg) weight loss or gain is observed. The BSA will be calculated from the height obtained at the pretreatment visit and from the weight obtained at the visit before each odd cycle. Capsules of temozolomide will be available in 5, 20, and 100 mg for this study. The daily dose will be rounded to the nearest 5 mg. The exact dose administered should be recorded in the CRF. Each daily dose should be given with the least number of capsules. Prior to each treatment cycle with temozolomide a complete blood count (CBC) will be obtained (within 72 hours prior to dosing).
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3.2.3 self-administered study drugs

The Temozolomide used in this study is a self-administered investigational agent. Such agents are dispensed from the pharmacy to a participant or to a Patient Care Unit for self-medication and a record of the dispensed investigational agent is generated and kept by the dispensing pharmacy. Participants will be instructed to fast at least 2 hours before and 1 hour after temozolomide administration. Water is allowed during the fast period. Participants will be instructed to swallow the capsules whole, in rapid succession, without chewing them. If vomiting occurs during the course of treatment, no re-dosing of the participant is allowed before the next scheduled dose. Antiemetic prophylaxis with a 5-HT3 antagonist is strongly recommended and should administered 30 to 60 minutes before temozolomide administration. As indicated above, participants will be asked to keep a medication diary and bring it with them on each study visit. Participants will also bring any remaining pills to each study visit. The Research Nurse reviews and validates the completeness and accuracy of the participant's diary with the participant. If a participant goes off study while at home, the Research Nurse will ensure and document the return of the unused oral investigational agents from the participant. Unused investigational agent will be destroyed per dispensing pharmacy procedure.
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3.3 dose modifications and management

In the case of toxicity, appropriate medical treatment should be used (including anti-emetics, anti-diarrheals, etc.).  
Once a participant has a dose reduction for toxicity, the dose will not be increased.  
Participants continuing to experience toxicity at the off-study visit will be contacted for additional assessments until the toxicity has resolved or is deemed irreversible. Participants must remain on the study to have additional assessment.  
For AEs that are unrelated to the study drugs, study drug may be held for up to 14 days at the discretion of the PI.  
3.3.1 T-DM1 Dose Modifications:
If a planned dose is delayed or missed, administer as soon as possible; do not wait until the next planned cycle. Adjust the schedule of administration to maintain a 3-week interval between doses. Administer the infusion at the dose and rate the participant tolerated in the most recent infusion. Management of increased serum transaminases, hyperbilirubinemia, left ventricular dysfunction, thrombocytopenia, pulmonary toxicity or peripheral neuropathy may require temporary interruption, dose reduction or treatment discontinuation of T-DM1 as per guidelines provided in the tables below.  
| Dose level        | Dose         |
|-------------------|--------------|
| Starting dose     | 3.6 mg/kg    |
| Dose reduction -1 | 3.0 mg/kg    |
| Dose reduction -2 | 2.4 mg/kg    |
| Stop treatment    | No treatment |  
Recommended dose reductions for AEs during Phase II part of the trial.  
| Dose Modification Guidelines for Infusion Related Reactions Grade ≤ 2 Grade ≥3 Reduce rate of infusion to 50% of the rate at  Permanently discontinue T-DM1  which the toxicity occurred   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| Dose Modification Guidelines for Increased Serum Transaminases (AST/ALT) Grade 2 (< 3 to ≤ 5 X ULN) Grade 3 (< 5 to ≤ 20 X ULN) Grade 4 (>20 X ULN) Treat at same dose level Do not administer T-DM1 until  AST/ALT recovers to Grade ≤ 2, and then reduce one dose  level.  Permanently discontinue TDM1   |
|---|  
ALT = alanine transaminase; AST = aspartate transaminase; ULN = upper limit of normal.  
| Dose Modification Guidelines for Hyperbilirubinemia Grade 2 (>1.5 to ≤ 3 X ULN) Grade 3 (>3 to ≤ 10 X ULN)   | Grade 4 (>10 X ULN)                                                                                     |    |
|--------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------|----|
| Do not administer T-DM1 until  total bilirubin recovers to Grade  ≤ 1, and then treat at same dose  level.   | Do not administer T-DM1 until  total bilirubin recovers to Grade  ≤ 1, and then reduce one dose  level. | Permanently discontinue TDM1.    |  
Permanently discontinue T-DM1 treatment in participants with serum transaminases > 3 x ULN and concomitant total bilirubin > 2 X ULN. Permanently discontinue T-DM1 in participants diagnosed with nodular regenerative hyperplasia (NRH).  
| Dose Modifications for Left Ventricular Dysfunction                       | LVEF 40% to ≤ 45% and  decrease is <  10% points from  baseline   |                                          |    |    |
|---------------------------------------------------------------------------|-------------------------------------------------------------------|------------------------------------------|----|----|
| LVEF 40% to ≤ 45% and  decrease is ≥ 10% points from  baseline            |                                                                   |                                          |    |    |
| Symptomatic  CHF                                                          | LVEF <40%                                                         | LVEF >45%                                |    |    |
| Discontinue TDM1                                                                           | Do not administer  T-DM1.                                         | Do not administer  T-DM1.                | Continue  treatment with TDM1.    | Continue  treatment with TDM1    |
| Repeat LVEF  assessment within  3 weeks. If LVEF  < 40% is  confirmed,  discontinue TDM1.                                                                           | Repeat LVEF  assessment within  3 weeks. If the  LVEF has not  recovered to  within 10% points  from baseline,  discontinue TDM1.                                                                   | Repeat LVEF  assessment within  3 weeks. |    |    |
| CHF = Congestive Heart Failure; LVEF = Left Ventricular Ejection Fraction |                                                                   |                                          |    |    |  
| Dose Modification Guidelines for Thrombocytopenia Grade 3 Grade 4 PLT 25.000/L to <50.000/L PLT <25.000/L Do not administer T-DM1 (hold up to 4 weeks)  until platelet count recovers to ≤ Grade 2  (50,000/L to < 75,000/L) , and then treat at same  dose level, or reduce one dose level at the  discretion of the investigator. If not resolved after  4 weeks, then stop. Do not administer T-DM1 (hold up to 4 weeks)  until platelet count recovers to ≤ Grade 2  (50,000/L to < 75,000/L), and then reduce one  dose level. If not resolved after 4 weeks, then  stop. PLT = platelets   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Dose modification for Interstitial Lung Disease: Monitor participants for signs and symptoms of pneumonitis or ILD (new onset or worsening shortness of breath or cough). Participants should be evaluated with imaging and/or pulmonary function tests including other diagnostic procedures as described below. Initial work-up may include clinical evaluation, monitoring of oxygenation via pulse oximetry (resting and exertion), laboratory work-up and high-resolution CT scan. Permanently discontinue T-DM1 in participants diagnosed with interstitial lung disease (ILD) or pneumonitis. Dose modification for Peripheral Neuropathy: Temporarily discontinue T-DM1 in participants experiencing Grade 3 or 4 peripheral neuropathy until resolution to ≤ Grade 2. 3.3.2 Temozolomide Dose Modifications Dosing is based on adverse events (AEs) during the prior treatment cycle. If multiple AEs are seen, the dose administered should be based on the dose reduction required for the most severe grade of any single AE.  
| Dose Level   | Dose mg/m2 daily   | Comments                               |
|--------------|--------------------|----------------------------------------|
| 0            | RP2D               | Starting dose for cycle 1 of  phase II |
| -1           | RP2D -10           | Reduction if prior AE                  |
| -2           | RP2D -20           | Reduction if prior AE                  |  
Temozolomide will be started at a dose recommended for phase 2 after completion of phase 1 portion of this study.
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planned delay

On day 1 of each cycle (within the prior 72 hours), if ANC ≤ 1.0 x 109 /L, platelet count ≤ 75 x 109 /L and until all grade 2, 3 or 4 non-hematologic AEs (except for alopecia, nausea, and vomiting) have resolved to grade ≤ 1. If AEs persists, treatment should be delayed by 1 week for up to 4 consecutive weeks. If, after 4 weeks of delay, all treatment related AEs have still not resolved (to grade ≤ 1): then any further treatment with temozolomide should be stopped.  
Dose reductions: If any treatment related non-hematologic AE observed was grade > 2 (except alopecia, lymphopenia, nausea and vomiting) and/or if platelets < 50 x 109 /L and/or ANC < 1 x 109 /L, then the dose should be reduced by one dose level. Participants who require more than two dose reductions will have treatment stopped. If any treatment-related non-hematologic AE observed was grade 4 (except alopecia, lymphopenia, nausea and vomiting) then temozolomide treatment should be stopped. Any dose reductions of temozolomide will be determined according to: (1) non-hematologic AE during the preceding treatment cycle, as well as (2) the lowest ANC and platelets observed. No dose escalation should be attempted. Important: If the dose was reduced or delayed for AEs, there will be no dose re-escalation in subsequent treatment cycles. Summary of Dose Modifications or Discontinuation for Temozolomide-Related Adverse Events Worst Treatment-Related Non-Hematologic AE (except for alopecia, nausea, and vomiting) During the Previous Cycles  
| Grade   | Dose modification                                                                                                      |
|---------|------------------------------------------------------------------------------------------------------------------------|
| 0-2     | No dose modifications for non-hematologic AEs. Dose reductions based on ANC and  platelet counts are applicable.       |
| 3       | Reduce by one dose level (except alopecia, nausea, and vomiting).                                                      |
| 4       | Stop (except alopecia, nausea, and vomiting). Dose modifications based on ANC and  platelet counts are not applicable. |  
| Platelets                                                                                     |                |                |                      |               |
|-----------------------------------------------------------------------------------------------|----------------|----------------|----------------------|---------------|
| Worst AE                                                                                      | ≥75.000 /L     | 50 - 75.000 /L | < 50.000 /L          |               |
| ≥ 1.5 x 109 /L                                                                                | Dose unchanged | Dose unchanged | Reduce 1 dose  level |               |
| ≥1 & ≥ 1.5 x 109  /L                                                                          | Dose unchanged | Dose unchanged | Reduce 1 dose  level |               |
| ANC                                                                                           | < 1 x 109  /L  | Reduce 1 dose  | Reduce 1 dose        | Reduce 1 dose |
| level                                                                                         | level          | level          |                      |               |
| Note: A complete blood count must be performed on days 14 and 28 (± 72 hours) after the first |                |                |                      |               |  
| Hematologic AE on Day 1 of Each Cycle (within the prior 72 hours before Day 1) AE Delay ANC< 1.000/L Delay up to 4 weeks until resolved. If not resolved after 4 weeks, then  stop. If resolved, dose delay/reductions based on non-hematologic AEs  are applicable.  Delay up to 4 weeks until resolved to grade 2 or better (≥ 50,000 /L),  then continue with same dose, or ability to dose reduce per  investigator discretion. If resolved, dose delay/reductions based on  non-hematologic AEs are applicable. If not resolved after 4 weeks,  then stop. Platelet count < 50,000 /L Non-Hematologic AE (except for alopecia, nausea, and vomiting) On Day 1 of Each Cycle (within the  prior 72 hours) Grade Delay Delay up to 4 weeks until all resolved (to grade ≤ 1). If not resolved  2-4 after 4 weeks, then stop. If resolved, dose delay/reductions based on  ANC and platelets are applicable.   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Instructions on Missed Doses: Participants should be instructed to take the medication as soon as it is remembered. The next due dose should be taken no sooner than 12 hours after the previous dose, and the time of administration of the subsequent doses should be adjusted until it is back on its regular "am or pm" schedule.
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3.4 questionnaires

The MDASI-BT (**Error! Reference source not found.**) and the PROMIS® (**Error! Reference** source not found.) will be utilized for this portion of the study. Full instruments are provided in the appendix. Both questionnaires will be filled by the participants at the same timepoint where they have scheduled brain MRIs scheduled, before the appointment with the physician. In addition, information regarding demographics and treatment history will be collected as part of the larger study and used in this analysis. The MDASI-BT consists of 23 symptoms rated on an 11-point scale (0 to 10) to indicate the presence and severity of the symptom, with 0 being "not present" and 10 being "as bad as you can imagine." Each symptom is rated at its worst in the last 24 hours. Symptoms included on the instrument include those commonly associated with cancer therapies, those associated with increased intracranial pressure, and those related to focal deficits. The questionnaire also includes ratings of how much symptoms interfered with different aspects of a participant's life in the last 24 hours. These interference items include: general activity, mood, work (includes both work outside the home and housework), relations with other people, walking, and enjoyment of life. The interference items are also measured on 0 - 10 scales. The average time to complete these instruments is 5 minutes. The MDASI-BT or SP has been translated into 18 languages 114. The PROMIS® assess patient-perceived functional abilities with regard to cognitive tasks including the perception that one's cognitive ability with regard to the domain of inquiry (e.g.  
concentration, memory) has not changed. (http://www.healthmeasures.net/explore-measurementsystems/promis) The applied cognition abilities is universal rather than disease specific. Each question has five response options ranging in value from one to five. To find the total raw score for a short form with all questions answered, sum the values of the response to each question.  
Using rigorous methods, PROMIS® has developed item banks (a collection of items that assess a specific trait, e.g., pain). These instruments are available to researchers free of cost. PROMIS® can be applied as computerized tests or on paper and have been translated into languages other than English 115.  
| 3.5                                                                          | STUDY CALENDAR   | Day 1 of   |      |    |
|------------------------------------------------------------------------------|------------------|------------|------|----|
| Procedure                                                                    | Screening 15     | Baseline   | each |    |
| Cycle13, 15                                                                  | End of           |            |      |    |
| treatment  visit14 Long Term  Follow-up                                      |                  |            |      |    |
| T-DM11                                                                       | X                |            |      |    |
| Temozolomide2                                                                | X                |            |      |    |
| History                                                                      | X                |            |      |    |
| Pathology confirmation                                                       | X                |            |      |    |
| Height                                                                       | X                |            |      |    |
| PE3                                                                          | X                | X          | X    |    |
| NIH Advanced Directives Form4                                                | X                |            |      |    |
| HIV, HCV, HepB                                                               | X                |            |      |    |
| Cardiology Assessments at screening  and every 3 months (ECHO)5              | X                | X          |      |    |
| CT of chest/abdomen /pelvis and Brain  MRI6                                  | X                | X          |      |    |
| Bone scan                                                                    | X                |            |      |    |
| CBC with differential                                                        | X                | X          | X    |    |
| Biochemical profile 7                                                        | X                | X          | X    |    |
| CD4 T cell count                                                             | X                |            |      |    |
| PT/INR/PTT 17                                                                | X                | X          |      |    |
| TSH                                                                          | X                | X          |      |    |
| Urinalysis                                                                   | X                | X          |      |    |
| Pregnancy testing8                                                           | X                | X          | X    | X  |
| Concomitant Medications                                                      | X                | X          |      |    |
| Adverse events                                                               | X                | X          | X    |    |
| MDASI-BT assessment9                                                         | X                | X          |      |    |
| PROMIS9                                                                      | X                | X          |      |    |
| Blood samples for Correlative Research  Studies                              | X16              |            |      |    |
| Tumor block10                                                                | X                |            |      |    |
| Fresh frozen tissue11                                                        | X                | X          |      |    |
| CSF sample / lumbar puncture by IR12                                         | X                | X          |      |    |
| Phone call or e-mail for survival/new  cancer treatments every 3 months once | X                |            |      |    |
| off treatment                                                                |                  |            |      |    |  
1 Participants will receive 3.6 mg/kg T-DM1 IV on Day 1 of each cycle. One cycle is 21 days.  
Allowance for scheduling changes: Brief interruption and delay in the 21-day cycle (- 3/ +14 days) may occasionally be required due to travel delays, airport closure, inclement weather, family responsibilities, security alerts and government holidays, etc. This can also extend to complications of disease not attributable to disease progression or protocol therapy. These delays will not be considered protocol deviation.  
2 During Phase I all patents will receive Temozolomide in a dose according to escalation schedule from 30 to 50 mg/m2 PO daily. During Phase II patents, randomized to Arm B will receive Temozolomide according to dose estimated during Phase I
3Weight, vital signs, ECOG performance status and complete neurological exam 4 As indicated in section **11.3**, all subjects will be offered the opportunity to complete an NIH
advanced directives form. This should be done preferably at baseline but can be done at any time during the study as long as the capacity to do so is retained. The completion of the form is strongly recommended, but is not required 5 Cardiology assessments: 2-D Echocardiogram or MUGA-scan; to be repeated every 3 months
(+/- 2 weeks to adjust to participant visits)
6 During Year 1, CT of chest/abdomen/pelvis and contrast brain MRI will be repeated every 6 weeks (- 3/ +14 days). Beginning with Year 2, imaging may be completed every 12 weeks (- 3/ +14 days) at the discretion of the PI.  
7 Sodium, potassium, chloride, carbon dioxide, BUN, creatinine or measured creatinine clearance, glucose, AST, ALT, bilirubin total and direct, calcium, total protein, albumin, alkaline phosphatase, magnesium 8 Urine or serum HCG 9 On baseline and every Odd Numbered Cycle, to be completed within +/- 1week of the MRI
assessment date. Optional for people who do not speak English.  
10 Primary tumor paraffin block or unstained slides (minimum of 20 slides) will be requested at enrollment to perform comparative genomic analysis as part of correlative studies planned for this clinical trial.  
11 Only applies to participants that have undergone tumor resection at recurrence or prior to study enrollment.  
12 LP guided by fluoroscopy at Baseline and C3D1 are mandatory, three LP guided by fluoroscopy after C3 are optional.  
13 Tests done at Baseline do not need to be repeated on Day 1 of Cycle 1 if performed during 72 hours before C1D1. Tests can be done 72 hours before Day 1 of every cycle.  
14 End of treatment visit will occur approximately 30 days after the last dose of study drug. If the participant cannot return to the Clinical Center for this visit, a request will be made to collect required clinical labs from a local physician or laboratory. If this is not possible, participants may be assessed by telephone for symptoms.  
15 Screening and Day 1 cycle visits for Cycle 3 and beyond may be completed by remote visit with a member of the study team (e.g., if the participant is not able to return to the NIH CC). Remote visits will be conducted in compliance with NIH guidelines and FDA regulations. A participant may be referred to their local provider for pre-treatment assessment, T-DM1 IV treatment, and lab tests, or asked to come to the NIH CC for an in-person assessment, if clinically indicated, and at the discretion of the PI. In the case of any visits with participants' local providers, records will be obtained. There is no variation among labs for tests being performed; they are all standardized tests. In the event that the participant is unable to return to the NIH Clinical Center during a follow up visit, imaging may be delayed, but will be completed as soon as feasible. Collection of research blood, CSF or urine will be omitted or collected in a delayed fashion.  
16 Pharmacokinetics samples will be collected one time only, Phase I only. See Section **5.1**.  
17 Every effort will be made to collect PT/ INR and aPTT baseline and as clinically indicated.  
Routine testing may resume when tubes become available.
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3.6 cost and compensation 3.6.1

NIH does not bill health insurance companies or participants for any research or related clinical care that participants receive at the NIH Clinical Center. If some tests and procedures performed outside the NIH Clinical Center, participants may have to pay for these costs if they are not covered by insurance company. Medicines that are not part of the study treatment will not be provided or paid for by the NIH Clinical Center. 3.6.2 Compensation Participants will not be compensated on this study.
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3.6.3 reimbursement

The NCI will cover the costs of some expenses associated with protocol participation. Some of these costs may be paid directly by the NIH and some may be reimbursed to the participant/guardian as appropriate. The amount and form of these payments are determined by the NCI Travel and Lodging Reimbursement Policy.
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3.7 criteria for removal from

Prior to documenting removal from study, effort must be made to have all subjects complete a safety visit approximately 30 days following the last dose of study therapy. 3.7.1 Criteria for removal from protocol therapy  
Progressive disease (distant brain metastases or systemic disease progression)
Necessity to increase dose of corticosteroid Participant requests to be withdrawn from active therapy Unacceptable Toxicity as defined in sections 3.1 and 3.3 Investigator discretion Positive pregnancy test 3.7.2 Off-Study Criteria
Participant requests to be withdrawn from study Death Investigator discretion PI decision to end the study Screen failure Lost to follow-up
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3.7.3 lost to follow-up

A participant will be considered lost to follow-up if he or she fails to return for 4 scheduled visits and is unable to be contacted by the study site staff.  
The following actions must be taken if a participant fails to return to the clinic for a required study visit:
The site will attempt to contact the participant and reschedule the missed visit within 14 days, counsel the participant on the importance of maintaining the assigned visit schedule. and ascertain if the participant wishes to and/or should continue in the study.  
Before a participant is deemed lost to follow-up, the investigator or designee will make every effort to regain contact with the participant (where possible, 3 telephone calls and, if necessary, an IRB approved certified letter to the participant's last known mailing address or local equivalent methods). These contact attempts should be documented in the participant's medical record or study file.  
Should the participant continue to be unreachable, he or she will be considered to have withdrawn from the study with a primary reason of lost to follow-up.
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4 concomitant medications/measures 4.1 t-dm1

No formal drug-drug interaction studies with T-DM1 have been conducted. *In vitro* studies indicate that DM1, the cytotoxic component of T-DM1, is metabolized mainly by CYP3A4 and to a lesser extent by CYP3A5. Concomitant use of strong CYP3A4 inhibitors (e.g., ketoconazole, itraconazole, clarithromycin, atazanavir, indinavir, nefazodone, nelfinavir, ritonavir, saquinavir, telithromycin, and voriconazole) with T_DM1 should be avoided due to the potential for an increase in DM1 exposure and toxicity (**Appendix B**). Consider an alternate medication with no or minimal potential to inhibit CYP3A4. If concomitant use of strong CYP3A4 inhibitors is unavoidable, consider delaying T-DM1 treatment until the strong CYP3A4 inhibitors have cleared from the circulation (approximately 3 elimination half-lives of the inhibitors) when possible. If a strong CYP3A4 inhibitor is coadministered and T-DM1 treatment cannot be delayed, participants should be closely monitored for adverse reactions.
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4.2 temozolomide 4.2.1 drug interactions

Administration of valproic acid decreases oral clearance of temozolomide by about 5% and should be avoided in this study participants.
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4.2.2 general

All supportive therapy for optimal medical care will be given during the study period at the discretion of the attending physician(s) within the parameters of the protocol and documented on each site's source documents as concomitant medication.  
Platelet support: Prophylactic use of platelet is only used when Platelet count < 20 x 109 /L. Platelet transfusion is provided for active bleeding participants with thrombocytopenia to keep platelet count above 50 x 109 /L.  
Corticosteroids: Corticosteroids will be allowed at enrollment and during the first month of treatment with T-DM1 after SRS, up to a dose of no more than 10 mg of dexamethasone daily or equivalent. Participants that need to continue corticosteroids after the initial month will not be allowed to increase the dose after that period. Participants requiring dose increase will be off protocol. G-CSF administration: Routine prophylactic use is not permitted. However, therapeutic use in participants with complications (severe neutropenia with fever), may be considered at the investigator's discretion. Febrile Neutropenia: It may be managed according to the local institution's Infectious Disease guidelines. Measures may include appropriate laboratory testing, including blood and urine cultures and the institution of broad-spectrum antibiotics. If a source for the fever is not identified or the fever resolves when the neutrophil count recovers, antibiotics may be discontinued and the participant observed. Pneumocystis Pneumonia Prophylaxis: recommended to participants in use of temozolomide and CD4 counts <200/mm3.  
Antiemetics: Prophylactic and therapeutic use is allowed. Other concomitant medications and the therapies considered necessary for the well-being of the participant may be given at the discretion of the treating physician. All concomitant medications must be recorded. Bisphosphonates, regardless of indication, provided subjects have been on stable doses for at least 2 weeks prior to randomization. Stable dose should be maintained during the investigational product treatment period. Subjects requiring initiation of bisphosphonate treatment, during the course of this study, should be discontinued due to progressive disease unless disease progression can be completely ruled out and clearly documented in the subject's source documentation. Other anticancer experimental therapies: No other anticancer therapy (including chemotherapy, radiation, hormonal treatment or immunotherapy) of any kind is permitted during the study period. No other antitumor drugs under investigation may be used concomitantly with the study drug.