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5 biospecimen collection 5.1 correlative

Correlative studies rationale is discussed in detail in section 1.3 of this protocol. Studies will address aspects of TMZ resistance, molecular alterations in brain metastatic tumor cells, the neuroinflammatory response, and markers of cognition. Biospecimen collection and storage protocols are summarized below.  
| Version Date: 06/06/2022 Test/assay   | Volume                                                                                                                                         | Type of                                                                                                                                        | Collection point                                 | Location of                                      |
|---------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------|--------------------------------------------------|
| (approx.)                             | tube***                                                                                                                                        | specimen analysis                                                                                                                              |                                                  |                                                  |
| Whole genome                          | Tumor                                                                                                                                          | if participant                                                                                                                                 |                                                  |                                                  |
| sequence                              | sample                                                                                                                                         | undergoes surgery  immediately before  enrollment or at the  time of disease  progression.  Primary tumor  paraffin block or  unstained slides | Brastianos Lab at  MGH or Carter  Lab at Harvard |                                                  |
| MGMT Analysis                         | Tumor                                                                                                                                          | if participant                                                                                                                                 |                                                  |                                                  |
| sample                                | undergoes surgery  immediately before  enrollment or at the  time of disease  progression.  Primary tumor  paraffin block or  unstained slides | Steeg Lab at NCI                                                                                                                               |                                                  |                                                  |
| MGMT Analysis                         | CSF, 2 ml                                                                                                                                      | 2 ml in  plypropylene  tubes                                                                                                                   | C1D1, C3D1, every  Odd Numbered  Cycle after C3* | Steeg Lab at NCI                                 |
| Cell free DNA for                     | CSF, 10 ml                                                                                                                                     | Streck CellFree DNA                                                                                                                                                |                                                  |                                                  |
| sequencing                            | Blood  Collection  Tube (BCT                                                                                                                   | C1D1, C3D1, every  Odd Numbered  Cycle after C3*                                                                                               | Brastianos Lab at  MGH or Carter  Lab at Harvard |                                                  |
| Neuroinflammatory                     | CSF, 2 ml                                                                                                                                      | 2 ml in                                                                                                                                        |                                                  |                                                  |
| marker analysis                       | polypropylene  tubes                                                                                                                           | C1D1, C3D1, every  Odd Numbered  Cycle after C3*                                                                                               | Steeg Lab at NCI                                 |                                                  |
| Cell free DNA for                     | Plasma, 10                                                                                                                                     | Two Streck                                                                                                                                     |                                                  |                                                  |
| sequencing                            | ml                                                                                                                                             | Cell-Free  DNA BCTs  or  BD  Vacutainer  EDTA tubes                                                                                            | D1 of each cycle                                 | Brastianos Lab at  MGH or Carter  Lab at Harvard |
| MGMT analysis                         | Plasma, 10                                                                                                                                     | Two Streck                                                                                                                                     |                                                  |                                                  |
| ml                                    | Cell-Free  DNA BCTs  or  BD  Vacutainer  EDTA tubes                                                                                            | D1 of each cycle                                                                                                                               | Steeg Lab at NCI                                 |                                                  |  
| Test/assay                                                                             | Volume                                  | Type of                                                | Collection point   | Location of      |
|----------------------------------------------------------------------------------------|-----------------------------------------|--------------------------------------------------------|--------------------|------------------|
| (approx.)                                                                              | tube***                                 | specimen analysis                                      |                    |                  |
| Exosome analysis                                                                       | Plasma, 5-                              | EDTA (purple                                           | D1 of each cycle   | Lyden Lab at     |
| 10 ml                                                                                  | top) tubes                              | Weill Medical  College                                 |                    |                  |
| Neuroinflammatory                                                                      | Blood, 10                               | Serum                                                  |                    |                  |
| marker analysis                                                                        | ml                                      | separation  tube without  separator gel                | D1 of each cycle   | Steeg Lab at NCI |
| MicroRNA-34c                                                                           | Blood, 5                                | Serum                                                  |                    |                  |
| (miR-34c)                                                                              | ml                                      | separation  tube without  separator gel                | D1 of each cycle   | Steeg Lab at NCI |
| PK samples                                                                             | Blood, 3                                | Sodium                                                 |                    |                  |
| ml                                                                                     | heparin  collection  tubes (green  top) | Pre dose,1, 2, 3, 4  and 8 hours post  Temozolomide ** | Figg Lab at NCI    |                  |
| *CSF at Baseline and C3D1 are mandatory, every Odd Numbered Cycle after C3 is optional |                                         |                                                        |                    |                  |  
*CSF at Baseline and C3D1 are mandatory, every Odd Numbered Cycle after C3 is optional
**PK blood will be collected in a sample of the phase I study subjects in order to determine exposure to temozolomide, in one day, at schedule determined in the table. In this instance, PK data will be collected in the C1 PK CRF regardless of the time point of collection. *** Please note that tubes and media may be substituted based on availability with the permission of the PI or laboratory investigator.
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5.2 sample collection

Please e-mail NCIBloodcore@mail.nih.gov at least 24 hours before transporting samples (the Friday before is preferred). For sample pickup, page 102-11964. For immediate help, call 240-760-6180 (main blood processing core number) or, if no answer, 240-760-6190 (main clinical pharmacology lab number). For questions regarding sample processing, contact NCIBloodcore@mail.nih.gov The samples will be processed, barcoded, and stored in Dr. Figg's lab until requested by the investigator. All samples sent to the Blood Processing Core (BPC) will be barcoded, with data entered and stored in Labmatrix utilized by the BPC. This is a secure program, with access to Labmatrix limited to defined Figg lab personnel, who are issued individual user accounts. Installation of Labmatrix is limited to computers specified by Dr. Figg. These computers all have a password restricted login screen. Labmatrix creates a unique barcode ID for every sample and sample box, which cannot be traced back to participants without Labmatrix access. The data recorded for each sample includes the participant ID, name, trial name/protocol number, time drawn, cycle time point, dose, material type, as well as box and freezer location. Participant demographics associated with the clinical center participant number are provided in the system. For each sample, there are notes associated with the processing method (delay in sample processing, storage conditions on the ward, etc.). 5.2.1 Samples will be coded, linked in the BPC and sent for final analysis to:
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brastianos lab

Priscilla K. Brastianos, MD Central Nervous System Metastasis Program Division of Neuro-Oncology, Massachusetts General Hospital 55 Fruit Street, Yawkey 9E Boston, MA 02115 Phone: 617-643-1938 Fax: 617-643-2591 Email: pbrastianos@mgh.harvard.edu
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lyden lab

David C. Lyden, MD, PhD Stavros S. Niarchos Professor Department of Pediatrics, and Cell and Developmental Biology Weill Medical College of Cornell University 413 E. 69th Street, Box 284 New York, NY 10021 Phone: 646-962-6238 Fax: 646-962-0574 e-mail: dcl2001@med.cornell.edu Steeg Lab 1. Patricia S. Steeg, Ph.D.  
a. Email: steegp@mail.nih.gov b. Phone: 301-402-2732 2. Brunilde Gril, Ph.D.  
a. Email: grilbrun@mail.nih.gov b. Phone: 301-451-6445 Building 37, Room 1126 NIH, Bethesda, MD 20892 5.2.2 Tumor, Blood Samples and CSF samples: Fresh frozen brain tumor tissue will be requested from participants undergoing surgery immediately before enrollment or at the time of disease progression. The slides and tissue blocks are stored indefinitely. All specimens are catalogued and retrieved utilizing the laboratory information systems. The use of any stored specimens for research purposes is only allowed when the appropriate IRB approval has been obtained. In some cases, this approval has been obtained via the original protocol on which the participant was enrolled. Blood samples will be collected at Day 1 of each cycle. CSF, guided by fluoroscopy done at Baseline and C3D1 are mandatory, every Odd Numbered Cycle after C3 is optional. Effective with Amendment (version date 07/12/2021): PK blood samples will be collected per designated time points (pre dose, 1, 2, 3, 4, and 8 hours post Temozolomide) in one day only, in a sample of the participants remaining in the Phase I portion of the study. The collection will follow as: collect 3mL of blood into 4ml (green top) sodium heparin collection tubes. Mix by gently inverting 6 times. Place the tubes immediately on wet ice-water bath after collection. Centrifuge samples within 1 hour after collection at 1300 x g for 10 minutes at 4°C. Place the centrifuged tubes on wet ice. Separate the plasma from the blood into a labeled polpropylene freezer vial. The samples should be processed to plasma within 30 minutes from centrifugation and the pH adjusted to <4 with the use of 8.5% phosphoric acid (15µL of 8.5% phosphoric acid per 0.5mL of plasma). Plasma should then be stored frozen at -70°C until subsequent batch analysis. The collection and analysis will be perfomed by the Figg lab at NCI. 5.2.2.1 MGMT methylation in tumor and blood samples.  
MGMT methylation will be tested at protein level by IHC and at mRNA level by methylationdependent PCR in the Steeg laboratory. For DNA methylation analysis, a procedure previously published will be utilized 92. For IHC, the previously published procedure will be utilized 57.  
5.2.2.2 Neuroinflammatory response and neuronal damage metrics in tumor and blood samples. Analysis of GFAP, UCH-L1 and MicroRNA-34c (miR-34c) levels in serum and CSF will be performed by the Steeg Lab.
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5.3 sample storage tracking and

Each participant sample set will be coded with a unique participant identifier. No participant specific information is encoded in this identifier. The protocol scientific investigators handling samples will be blinded as to as to the participant identification, participant data and outcome. The amount of blood that may be drawn from adult participants (i.e., those persons 18 years of age or older) for research purposes shall not exceed 10.5 mL/kg or 550 mL, whichever is smaller, over any eight-week period. 5.3.2 Sample Storage, Tracking and Disposition Samples will be ordered in CRIS and tracked through a Clinical Trial Data Management system. Should a CRIS screen not be available, the CRIS downtime procedures will be followed. Samples will not be sent outside NIH without appropriate approvals and/or agreements, if required. Barcoded samples are stored in barcoded boxes in a locked freezer at either -20° or -80° C according to stability requirements. These freezers are located onsite in the BPC and offsite at NCI Frederick Central Repository Services in Frederick, MD. Visitors to the laboratory are required to be accompanied by laboratory staff at all times. Access to stored clinical samples is restricted. Samples will be stored until requested by a researcher named on the protocol. All requests are monitored and tracked in Labmatrix. All researchers are required to sign a form stating that the samples are only to be used for research purposes associated with this trial (as per the IRB approved protocol) and that any unused samples must be returned to the BPC. It is the responsibility of the NCI Principal Investigator to ensure that the samples requested are being used in a manner consistent with IRB approval. Following completion of this study, samples will remain in storage as detailed above. Access to these samples will only be granted following IRB approval of an additional protocol, granting the rights to use the material. If, at any time, a participant withdraws from the study and does not wish for their existing samples to be utilized, the individual must provide a written request. Following receipt of this request, the samples will be destroyed (or returned to the participant, if so requested). The PI will record any loss or unanticipated destruction of samples as a deviation. Reporting will be per the requirements of section 7.2. Sample barcodes are linked to participant demographics and limited clinical information. This information will only be provided to investigators listed on this protocol, via registered use of the Labmatrix. It is critical that the sample remains linked to participant information such as race, age, dates of diagnosis and death, and histological information about the tumor, in order to correlate genotype with these variables. All specimens obtained in the protocol are used as defined in the protocol. Any specimens that are remaining at the completion of the protocol will be stored in the conditions described below. The study will remain open so long as sample or data analysis continues. Samples from consenting subjects will be stored until they are no longer of scientific value or if a subject withdraws consent for their continued use, at which time they will be destroyed. If the participant withdraws consent the participants data will be excluded from future distributions, but data that have already been distributed for approved research use will not be able to be retrieved.
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5.4 samples for genetic/genomic analysis

5.4.1 Description of the scope of genetic/genomic analysis Tumor whole-exome sequencing will be performed in collaboration with the Brastianos and Carter' laboratories at Harvard.  
Tumor cfDNA and primary tumor DNA sequencing in collaboration with Brastianos and Carter Laboratories We plan to characterize cfDNA from CSF samples and blood at enrollment and during treatment. 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.  
Exosomes analysis in collaboration with Dr. Lyden's laboratory at Weill Cornell Medical College.  
5.4.2 Privacy and Confidentiality of medical information/biological specimens
Fresh tumor and blood samples will be stored in a minus 80-degree freezer. The samples of each participant will be barcoded. At no time will participant's names be used on the blood and tissue samples. Sometimes, because a group collaboration or journal policy requires it, a subject's genetic data may be deposited in a database such as dbGaP. Although there is controlled access to such a database, such a submission carries theoretical risks of revealing the identity of the subject. This is discussed in the consent. 5.4.3 A Certificate of Confidentiality will be obtained for the study as described in section 11.5.12.  
5.4.4 Management of Results
Subjects will be contacted if a clinically actionable gene variant is discovered. Clinically actionable findings for the purpose of this study are defined as disorders appearing in the American College of Medical Genetics and Genomics recommendations for the return of incidental findings that is current at the time of primary analysis. (A list of current guidelines is maintained on the CCR intranet: https://ccrod.cancer.gov/confluence/display/CCRCRO/Incidental+Findings+Lists). Subjects will be contacted at this time with a request to provide a blood sample to be sent to a CLIA certified laboratory. 5.4.5 Genetic counseling If the research findings are verified in the CLIA certified lab, the subject will be offered the opportunity to come to NIH (at our expense) to have genetic education and counseling with the NCI Genetics Branch to explain this result. If the subject does not want to come to NIH, a referral to a local genetic healthcare provider will be provided (at their expense). This is the only time during the course of the study that incidental findings will be returned. No interrogations regarding clinically actionable findings will be made after the primary analysis.
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6 data collection and evaluation

The PI will be responsible for overseeing entry of data into a 21 CFR Part 11-compliant data capture system provided by the NCI CCR and ensuring data accuracy, consistency and timeliness. The principal investigator, associate investigators/research nurses and/or a contracted data manager will assist with the data management efforts. Primary and final analyzed data will have identifiers so that research data can be attributed to an individual human subject participant. All adverse events, including clinically significant abnormal findings on laboratory evaluations, regardless of severity, will be followed until return to baseline or stabilization of event. Document AEs from the first study treatment, Study Day 1, through 30 days after the subject received the last administration of the study agent. An abnormal laboratory value will be recorded in the database as an AE **only** if the laboratory abnormality is characterized by any of the following:
Results in discontinuation from the study  
Is associated with clinical signs or symptoms Requires treatment or any other therapeutic intervention Is associated with death or another serious adverse event, including hospitalization. Is judged by the Investigator to be of significant clinical impact If any abnormal laboratory result is considered clinically significant, the investigator will provide details about the action taken with respect to the test drug and about the participant's outcome.
End of study procedures: Data will be stored according to HHS, FDA regulations and NIH Intramural Records Retention Schedule as applicable. Loss or destruction of data: Should we become aware that a major breach in our plan to protect subject confidentiality and trial data has occurred, this will be reported expeditiously per requirements in section **7.2.1**.
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6.1.1 source documents

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

Data may be entered from the source documents directly into eCRFs for each participant enrolled in this study. The principal investigator or research nurse will review the eCRFs for completeness and accuracy. Independent audits may also be conducted by NCI personnel to ensure completeness and accuracy of data in study database.
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6.1.3 data quality assurance

The research team will monitor each participant's dataset throughout the study. Source document review will be made against entries on the eCRF and a quality assurance check will be performed to ensure that the investigator is complying with the protocol and regulations. In addition, after the research team (data managers) completes the CRFs a research nurse or physician at the NCI will review and verify the data.
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what data will be shared?

I will share human data generated in this research for future research as follows:
Coded, linked data in an NIH-funded or approved public repository.  
Coded, linked data in BTRIS (automatic for activities in the Clinical Center)
Coded, linked or identified data with approved outside collaborators under appropriate agreements.
Data will be shared through:
An NIH-funded or approved public repository: clinicaltrials.gov.  
BTRIS (automatic for activities in the Clinical Center)
Approved outside collaborators under appropriate individual agreements.  
Publication and/or public presentations.  
Data will be shared:
Before publication. At the time of publication or shortly thereafter.  
6.2.2 Genomic Data Sharing Plan
Unlinked genomic data will be deposited in public genomic databases such as dbGaP in compliance with the NIH Genomic Data Sharing Policy.
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6.3 response criteria

For the purposes of this study, participants should be re-evaluated for response every 6 weeks. In addition to baseline images, confirmatory images should also be obtained 3 weeks following initial documentation of objective response.
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6.3.1 rano-bm criteria for evaluation

The primary endpoint of this study, however, is 12-month freedom from distant brain metastases after initial SRS or surgery. A combination of the neurological examination and MRI brain scan will be used to define progression. Due to improvements in neuroimaging and the fact that tumor growth in certain regions of the CNS is without immediate neurologic signs and symptoms, greater reliance is placed on neuroimaging to define progression. Brain MRI assessment for CNS disease progression will be done every 6 weeks. Time to distant brain CNS failure will be scored from the date of randomization to the date of diagnosis of a new focus of contrast enhancement outside the prior SRS region. Any lesions that cannot be undoubtedly classified as new or increasing tumor will have to be confirmed to be present on two consecutive MRI scans and growing, following recommendations of The Response Assessment in Neuro-Oncology Brain Metastases (RANOBM) 116. (**Appendix D)**
Participants with questionable progression or recurrence of tumor in areas of brain previously treated by SRS, that cannot be differentiated from necrosis post SRS without biopsy, will be continued in protocol treatment and follow-up, as long as no local treatment is recommended by their primary physicians (oncology or radiation therapy). In the instance wherein a lesion is found retrospectively and pre-dated study therapy, that lesion or lesions may receive local treatment if deemed in the best interest of the partcipant. Participants indicated to receive WBRT or new SRS because of local recurrence in brain will be off protocol and censored for analysis. Participants with systemic progression with no CNS new lesions will also be off protocol and censored for analysis. Participants with new brain tumor lesions away from previously treated (by SRS or surgery) are the ones considered progression of disease, independently of the systemic status of disease and/or concomitant recurrence in brain areas previously treated by SRS/surgery. They will be off protocol for progression with distant brain metastases and will be accounted for in the statistical analysis. 6.3.1.1 Definitions of Response Measurable Disease: Bi-dimensionally measurable lesions with clearly defined margins by MRI scan. Objective Status, To Be Recorded at Each Evaluation: If there are too many measurable lesions to measure at each evaluation, choose the largest two to be followed before a patient is entered on study. The remaining lesions will be considered evaluable for the purpose of objective status determination. Unless progression is observed, objective status can only be determined when ALL measurable and evaluable sites and lesions are assessed.
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response criteria

Stable/No Progression: No evidence of new measurable lesions on MR imaging. The designation of Stable/No Progression requires a minimum of 12 weeks duration. All measurable and evaluable sites must be assessed using the same techniques as baseline. Partial response: ≥ 50% reduction in the sum of products of all measurable lesions over baseline sum observed using the same techniques as baseline. The patient must be on a stable or decreased dose of corticosteroids to be evaluable for response. Complete response: Complete resolution of all lesions. The patient cannot be on any corticosteroids with the exception of adrenal replacement doses. Progression: 25% increase in the sum of products of all measurable lesions over smallest sum observed (over baseline if no decrease) using the same techniques as baseline, OR clear worsening of any evaluable disease, OR appearance of any new lesion/site, OR failure to return for evaluation due to death or deteriorating condition (unless clearly unrelated to this cancer). Unknown: Progression has not been documented and one or more measurable or evaluable sites have not been assessed. 6.3.2 RECIST for Systemic Evaluation Systemic response and progression will be evaluated in this study using the new international criteria proposed by the revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version 1.1) [*Eur J Ca* 45:228-247, 2009]. Changes in the largest diameter (unidimensional measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph nodes are used in the RECIST criteria. If participant develop additional systemic lesions without CNS progression and require new therapy, they will be taken off the trial. This group will be followed separately to determine their RFS. Additional participant will be accrued to meet the 49 goal with one year of follow up. 6.3.2.1 Definitions Evaluable for toxicity: All participant will be evaluable for toxicity from the time of their first treatment with Temozolomide and/or T-DM1. Evaluable for objective response: Only those participant who have measurable disease present at baseline, have received at least one cycle of therapy, and have had their disease re-evaluated will be considered evaluable for response. These participant will have their response classified according to the definitions stated below. (Note: Participant who exhibit objective disease progression prior to the end of cycle 1 will also be considered evaluable.) Evaluable Non-Target Disease Response: Participant who have lesions present at baseline that are evaluable but do not meet the definitions of measurable disease, have received at least one cycle of therapy, and have had their disease re-evaluated will be considered evaluable for non-target disease. The response assessment is based on the presence, absence, or unequivocal progression of the lesions. 6.3.2.2 Disease Parameters Measurable disease: Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as:
By chest x-ray: >20 mm; By CT scan:  
o Scan slice thickness 5 mm or under: as >10 mm o Scan slice thickness >5 mm: double the slice thickness
With calipers on clinical exam: >10 mm.  
All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters). Malignant lymph nodes. To be considered pathologically enlarged and measurable, a lymph node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). At baseline and in follow-up, only the short axis will be measured and followed. Non-measurable disease. All other lesions (or sites of disease), including small lesions (longest diameter <10 mm or pathological lymph nodes with ≥10 to <15 mm short axis), are considered non-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by CT or MRI), are considered as non-measurable. Note: Cystic lesions that meet the criteria for radiographically defined simple cysts should not be considered as malignant lesions (neither measurable nor non-measurable) since they are, by definition, simple cysts. 'Cystic lesions' thought to represent cystic metastases can be considered as measurable lesions, if they meet the definition of measurability described above. However, if non-cystic lesions are present in the same patient, these are preferred for selection as target lesions. Target lesions. All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, should be identified as **target lesions** and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. It may be the case that, on occasion, the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected. A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the sum, then only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease. Non-target lesions. All other lesions (or sites of disease) including any measurable lesions over and above the 5 target lesions should be identified as **non-target lesions** and should also be recorded at baseline. Measurements of these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each should be noted throughout follow-up. 6.3.2.3 Methods for Evaluation of Measurable Disease All measurements should be taken and recorded in metric notation using a ruler or calipers. All baseline evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 weeks before the beginning of the treatment. The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be imaged but are assessable by clinical exam. Clinical lesions: Clinical lesions will only be considered measurable when they are superficial
(e.g., skin nodules and palpable lymph nodes) and 10 mm diameter as assessed using calipers
(e.g., skin nodules). In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended. Chest x-ray: Lesions on chest x-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung. However, CT is preferable. Conventional CT and MRI: This guideline has defined measurability of lesions on CT scan based on the assumption that CT slice thickness is 5 mm or less. If CT scans have slice thickness greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness. MRI is also acceptable in certain situations (e.g. for body scans). Use of MRI remains a complex issue. MRI has excellent contrast, spatial, and temporal resolution; however, there are many image acquisition variables involved in MRI, which greatly impact image quality, lesion conspicuity, and measurement. Furthermore, the availability of MRI is variable globally. As with CT, if an MRI is performed, the technical specifications of the scanning sequences used should be optimized for the evaluation of the type and site of disease. Furthermore, as with CT, the modality used at follow-up should be the same as was used at baseline and the lesions should be measured/assessed on the same pulse sequence. It is beyond the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body parts, and diseases. Ideally, the same type of scanner should be used and the image acquisition protocol should be followed as closely as possible to prior scans. Body scans should be performed with breath-hold scanning techniques, if possible. PET-CT: At present, the low dose or attenuation correction CT portion of a combined PET-CT is not always of optimal diagnostic CT quality for use with RECIST measurements. However, if the site can document that the CT performed as part of a PET-CT is of identical diagnostic quality to a diagnostic CT (with IV and oral contrast), then the CT portion of the PET-CT can be used for RECIST measurements and can be used interchangeably with conventional CT in accurately measuring cancer lesions over time. Note, however, that the PET portion of the CT introduces additional data which may bias an investigator if it is not routinely or serially performed. Ultrasound: Ultrasound is not useful in assessment of lesion size and should not be used as a method of measurement. Ultrasound examinations cannot be reproduced in their entirety for independent review at a later date and, because they are operator dependent, it cannot be guaranteed that the same technique and measurements will be taken from one assessment to the next. If new lesions are identified by ultrasound in the course of the study, confirmation by CT or MRI is advised. If there is concern about radiation exposure at CT, MRI may be used instead of CT in selected instances. Endoscopy, Laparoscopy: The utilization of these techniques for objective tumor evaluation is not advised. However, such techniques may be useful to confirm complete pathological response when biopsies are obtained or to determine relapse in trials where recurrence following complete response (CR) or surgical resection is an endpoint. Tumor markers: Tumor markers alone cannot be used to assess response. If markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response. Specific guidelines for both CA-125 response (in recurrent ovarian cancer) and PSA response (in recurrent prostate cancer) have been published [*JNCI* 96:487-488, 2004; J Clin Oncol 17, 3461-3467, 1999; *J Clin Oncol* 26:1148-1159, 2008]. In addition, the Gynecologic Cancer Intergroup has developed CA-125 progression criteria which are to be integrated with objective tumor assessment for use in first-line trials in ovarian cancer [*JNCI* 92:1534-1535, 2000]. Cytology, Histology: These techniques can be used to differentiate between partial responses (PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as germ cell tumors, where known residual benign tumors can remain). The cytological confirmation of the neoplastic origin of any effusion that appears or worsens during treatment when the measurable tumor has met criteria for response or stable disease is mandatory to differentiate between response or stable disease (an effusion may be a side effect of the treatment) and progressive disease. FDG-PET: While FDG-PET response assessments need additional study, it is sometimes reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in assessment of progression (particularly possible 'new' disease). New lesions on the basis of FDG-PET imaging can be identified according to the following algorithm:  
a. Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of PD
based on a new lesion.  
b. No FDG-PET at baseline and a positive FDG-PET at follow-up: If the positive FDGPET at follow-up corresponds to a new site of disease confirmed by CT, this is PD. If the positive FDG-PET at follow-up is not confirmed as a new site of disease on CT, additional follow-up CT scans are needed to determine if there is truly progression occurring at that site (if so, the date of PD will be the date of the initial abnormal FDGPET scan). If the positive FDG-PET at follow-up corresponds to a pre-existing site of disease on CT that is not progressing on the basis of the anatomic images, this is not PD.
c. FDG-PET may be used to upgrade a response to a CR in a manner similar to a biopsy in cases where a residual radiographic abnormality is thought to represent fibrosis or scarring. The use of FDG-PET in this circumstance should be prospectively described in the protocol and supported by disease-specific medical literature for the indication. However, it must be acknowledged that both approaches may lead to false positive CR due to limitations of FDG-PET and biopsy resolution/sensitivity.  
Note: A 'positive' FDG-PET scan lesion means one which is FDG avid with an uptake greater than twice that of the surrounding tissue on the attenuation corrected image. 6.3.2.4 Response Criteria 6.3.2.4.1 Evaluation of Target Lesions Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. Partial Response (PR): At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum of diameters. Progressive Disease (PD): At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions). Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum of diameters while on study. 6.3.2.4.2 Evaluation of Non-Target Lesions Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (<10 mm short axis). Note: If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered in complete clinical response. Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits. Progressive Disease (PD): Appearance of one or more new lesions and/or *unequivocal progression* of existing non-target lesions. *Unequivocal progression* should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase. Although a clear progression of "non-target" lesions only is exceptional, the opinion of the treating physician should prevail in such circumstances, and the progression status should be confirmed at a later time by the review panel (or Principal Investigator). 6.3.2.4.3 Evaluation of Best Overall Response The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria.  
| Target    | Non-Target                                                                                                                                                                                                                                                                                                                      | New     | Overall                                           | Best Overall Response when   |
|-----------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------|---------------------------------------------------|------------------------------|
| Lesions   | Lesions                                                                                                                                                                                                                                                                                                                         | Lesions | Response                                          | Confirmation is Required*    |
| CR        | CR                                                                                                                                                                                                                                                                                                                              | No      | CR                                                | >4 wks. Confirmation**       |
| CR        | Non                                                                                                                                                                                                                                                                                                                                 |         |                                                   |                              |
| CR/Non-PD | No                                                                                                                                                                                                                                                                                                                              | PR      |                                                   |                              |
| CR        | Not                                                                                                                                                                                                                                                                                                                             | No      | PR                                                |                              |
| evaluated | No                                                                                                                                                                                                                                                                                                                              | PR      | >4 wks. Confirmation**                            |                              |
| PR        | Non                                                                                                                                                                                                                                                                                                                                 |         |                                                   |                              |
| CR/NonPD/not  evaluated           |                                                                                                                                                                                                                                                                                                                                 |         |                                                   |                              |
| SD        | Non                                                                                                                                                                                                                                                                                                                                 |         |                                                   |                              |
| CR/NonPD/not  evaluated           | No                                                                                                                                                                                                                                                                                                                              | SD      | Documented at least once >4  wks. from baseline** |                              |
| PD        | Any                                                                                                                                                                                                                                                                                                                             | Yes or  | PD                                                |                              |
| No        |                                                                                                                                                                                                                                                                                                                                 |         |                                                   |                              |
| Any       | PD***                                                                                                                                                                                                                                                                                                                           | Yes or  | PD                                                |                              |
| No        | no prior SD, PR or CR                                                                                                                                                                                                                                                                                                           |         |                                                   |                              |
| Any       | Any                                                                                                                                                                                                                                                                                                                             | Yes     | PD                                                |                              |
| *         | See RECIST 1.1 manuscript for further details on what is evidence of a new lesion.                                                                                                                                                                                                                                              |         |                                                   |                              |
| **        | Only for non-randomized trials with response as primary endpoint.                                                                                                                                                                                                                                                               |         |                                                   |                              |
| ***       | In exceptional circumstances, unequivocal progression in non-target lesions may be accepted as  disease progression.                                                                                                                                                                                                            |         |                                                   |                              |
| Note:     | Participant with a global deterioration of health status requiring discontinuation of treatment  without objective evidence of disease progression at that time should be reported as "symptomatic  deterioration." Every effort should be made to document the objective progression even after  discontinuation of treatment. |         |                                                   |                              |  
For Participant with Measurable Disease (i.e., Target Disease)  
| Non-Target Lesions                                                                                                                                                                                                                             | New Lesions   | Overall Response   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------|--------------------|
| CR                                                                                                                                                                                                                                             | No            | CR                 |
| Non-CR/non-PD                                                                                                                                                                                                                                  | No            | Non-CR/non-PD*     |
| Not all evaluated                                                                                                                                                                                                                              | No            | not evaluated      |
| Unequivocal PD                                                                                                                                                                                                                                 | Yes or No     | PD                 |
| Any                                                                                                                                                                                                                                            | Yes           | PD                 |
| * 'Non-CR/non-PD' is preferred over 'stable disease' for non-target disease since SD is increasingly used  as an endpoint for assessment of efficacy in some trials so to assign this category when no lesions can be  measured is not advised |               |                    |  
6.3.3 Duration of Response Duration of overall response: The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented. Duration of stable disease: Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements. 6.3.4 Relapse -Free Survival RFS is defined as the duration of time from start of treatment to time of progression or death, whichever occurs first. 6.3.5 Validation of Disease Assessment (Safety Mechanisms) The following practices will ensure accuracy in disease assessment: Dr. Ritu Shah, Neuroradiolgist, or her designee will be provided with a list of patients scheduled for imaging by a member of the WMB team approximately 4-5 days in advance of clinic except in cases where urgent imaging is warranted. In the case of urgent scans, a member of the WMB team will make every effort to contact Dr. Shah with as much notice as the clinical situation allows prior to and/or after the urgent imaging as been performed. The electronic medical record (EMR) protocol order set for Brain MRIs has been modified to include the following language provided by the Neuroradiology Leadership, "Comment on any new lesions relative to baseline. Any enhancing lesion 5 x 5mm or greater described for the FIRST time is to be called as a NEW lesion, even if it can be seen in retrospect on older studies. For comparison, use Baseline scan of _________." Should additional clarity or requests be made by Neuroradiology regarding order language, we will continue to amend the orders in a timely manner. Every effort will be made that each brain MRI will be initially read by neuroradiologist on call the day scans are performed or on the day patient(s) present to clinic; this review is to rule out any major finding requiring immediate attention. To review specific imaging findings related to the study, Dr. Shah will meet with Dr Zimmer to review the images within 7 days, optimally within 24-48 hours. Dr. Zimmer will document the patient respective findings in the EMR then route for Dr. Shah's co-signature. In the absence of Dr. Shah, she may delegate her responsibilities to a Neuroradiology colleague.
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6.4 toxicity criteria

The following adverse event management guidelines are intended to ensure the safety of each patient while on the study. The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting. All appropriate treatment areas should have access to a copy of the CTCAE version 4.0. A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm\#ctc_40).
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7 nih reporting requirements/data and

Please refer to definitions provided in Policy 801: Reporting Research Events found at:
https://irbo.nih.gov/confluence/pages/viewpage.action?pageId=36241835\#Policies&Guidance800Series-ComplianceandResearchEventReportingRequirements.
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7.2 ohsrp office of compliance

Please refer to the reporting requirements in Policy 801: Reporting Research Events and Policy 802 Non-Compliance Human Subjects Research found at:
https://irbo.nih.gov/confluence/pages/viewpage.action?pageId=36241835\#Policies&Guidance800Series-ComplianceandResearchEventReportingRequirements.  
7.2.2 IRB Requirements for PI Reporting at Continuing Review Please refer to the reporting requirements in Policy 801: Reporting Research Events found at:
https://irbo.nih.gov/confluence/pages/viewpage.action?pageId=36241835\#Policies&Guidance800Series-ComplianceandResearchEventReportingRequirements.
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7.3 nci clinical director reporting

Problems expeditiously reported to the OHSRP in iRIS will also be reported to the NCI Clinical Director. A separate submission is not necessary as reports in iRIS will be available to the Clinical Director. In addition to those reports, all deaths that occur within 30 days after receiving a research intervention should be reported via email to the Clinical Director unless they are due to progressive disease. To report these deaths, please send an email describing the circumstances of the death to the Clinical Director/designee at NCICCRQA@mail.nih.gov within one business day of learning of the death.
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7.4 nih required data and

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

This protocol requires monitoring by the NCI CCR Data Safety Monitoring Board (DSMB) as described in Section **9.4.6**. Interim outcome results will not be revealed to the investigators of the trial; results will be presented to the investigators prior to final accrual to the trial only if the DSMB recommends early termination of the trial. 7.4.3 Corrective Action Plan The study has implemented a corrective action plan to assure timely and accurate disease assessment. Please reference Section **6.3.5** Validation of Disease Assessment (Safety Mechanisms). Additionally, the study will undergo oversight by an Independent Monitoring Committee (**7.4.4**). 7.4.4 Independent Monitoring Committee (IMC) Additional oversight for this study will be provided by an Independent Monitoring Committee consisting of the Principal Investigator and two qualified investigators who are independent of the study team. The committee meetings will be conducted annually for the duration of time that participants are having disease response evaluations per protocol. Ad hoc meetings may be called at more frequent intervals for cause at the request of the PI or the independent reviewers. During these meetings, the committee will review the results of the findings from the procedures described in Section **6.3.5** Validation of Disease Assessment (Safety Mechanisms) to ensure disease assessment is managed accurately in accordance with protocol requirements. These results will be provided to the IMC by the PI or designee. The PI will be responsible for promptly alerting the independent reviewers of any missed disease assessments identified during the validation process. Event reporting and event management will be submitted for appropriate adjudication and reported promptly to the IRB.
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8 safety reporting criteria to

Principal Investigator shall report any SAE, SUSAR, Medical Device Event, Device Deficiency or Incident information, including, but not limited to, all initial and follow-up information involving any Study subject in the Study. Serious Adverse Event and Suspected Unexpected Serious Adverse Reaction, Medical Device Event, Potential Incident, Device Deficiency or Incident Reporting: Principal Investigator shall report any SAE, SUSAR, Medical Device Event, Device Deficiency or Incident information, including, but not limited to, all initial and follow-up information involving any Study subject in the Study. Notification shall be in the form of a completed CIOMS I/MedWatch (or other mutually agreed upon format) within two (2) business days of learning of the information. This information shall be transmitted to Merck GS using the contact information provided below or such other modified contact information as provided by Merck in writing. All information shall be transmitted in the English language and contain the reporter's name and the Study subject identifier code. SUSAR information will be reported unblinded if the Study Drug has been blinded in the Study. Randomization codes for all other SAEs will be provided to Merck GS at end of Study if the Study Drug has been blinded in the Study.  
Merck may define certain Non-Serious Events of Interest. If any Non-Serious Events of Interest are defined, Merck will provide such information in writing to Principal Investigator at the time of Protocol approval, execution of the CTA or anytime thereafter. Reporting of any defined NonSerious Events of Interest will be handled in the same manner as SAEs unless mutually agreed otherwise in writing by the parties. All reports of Study Drug exposure during pregnancy or lactation (including a female partner of a male Study subject using the Study Drug), whether associated with an AE or not, must be reported to Merck GS in accordance with the timelines and contact information for an SAE. Principal Investigator shall follow pregnancies to term to obtain the outcome of the pregnancy. The outcome of the pregnancy shall be forwarded to Merck GS. The Principal Investigator shall fully comply with all of their respective reporting obligations to the applicable regulatory authorities with respect to any AE, SAE or SUSAR that arises from the Study.  
SAE reports and any other relevant safety information are to be forwarded to Merck GS facsimile number: 215-993-1220. A MedWatch form available at http://www.fda.gov/medwatch/
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9 statistical considerations 9.1 statistical

Safety: Determination of the MTD assessed at the completion of cycle 1 (21 days) Efficacy: Freedom from distant new brain metastases following stereotactic radiosurgery or surgical resection at 1 year, measured by rate of brain relapse free survival (RFS) for temozolomide and TDM-1 vs. TDM-1 alone assessed at approximately 12-24 months after the last participant starts treatment.
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secondary efficacy endpoints

Number of participants with DLTs at each dose level assessed at the completion of cycle 1 (21 days) Tabulated counts of adverse events assessed through 30 days after the completion of study therapy Time to whole brain irradiation compared between the two arms assessed at time of whole brain irradiation Overall survival compared between the two arms assessed at death
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9.2 sample size determination phase

The initial safety evaluation will be done using a standard 3+3 design. Thus, with 3 dose levels, up to 18 participants may be included in the initial safety evaluation. Phase II: Following a safety evaluation, the primary objective of the trial is to determine if the addition of temozolomide to TDM-1 will be associated with an increase in new brain metastases Relapse Free Survival (brain RFS) among participants with breast cancer and resection of brain metastases. While there are no published reports on participants with HER2+ disease and SRS without WBRT, one paper indicated that the 12 month brain RFS is approximately 51% in participants with breast cancer and resected brain metastases that are treated with SRS alone 117. This will be used to help estimate the sample size. The goal would be to determine if this can be increased by a modest amount with the addition of temozolomide. T-DM1 will be used as the backbone systemic treatment. Initially, a small (3 dose level) phase I-type run-in for safety will be needed for evaluation of T-DM1 and temozolomide combination. In the main, randomized cohort of the trial, Kaplan-Meier curves and a one-tailed log-rank test will be the primary analysis methods. Assuming exponential brain RFS curves, the hazard rate for TDM-1 associated with a 50% 12 month brain RFS is 0.0578: approximately a 5.8% probability of relapsing each month when the 12 month brain RFS probability is 50%. If we assume that the addition of temozolomide will increase brain RFS to 65% at 12 months, this corresponds to a hazard rate of 0.0359 and the resulting hazard ratio for the comparison of the two overall actuarial curves would be 1.61. Following the principles of a phase 2.5 trial, to compare these curves and detect a difference with a 0.10 significance level one-tailed log-rank test, 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 participant, with occurrence of 79 total relapses in both arms combined, to have 80% power to compare the curves. It should also be noted that if the true 12 month brain RFS probability without temozolomide were closer to 55%, that 49 participants per arm would provide 83% power for a comparison with 70%, using otherwise identical parameters. Participants will be randomized to receive TDM-1 or TDM-1 + temozolomide after being registered onto the study, and will be stratified prior to randomization for characteristics potentially associated with relapse: 1 brain lesion in the absence of extracranial disease or presence of stable extracranial disease, 2-5 brain lesions in the absence of extracranial disease or stable extracranial disease, >5 brain lesions in the absence of extracranial disease or stable extracranial disease, 1 brain lesion and progressive extracranial disease, 2-5 brain lesions and progressive extracranial disease, >5 brain lesions and progressive extracranial disease. If participants develop additional systemic lesions and require new therapy, they will be taken off treatment. This group will be followed separately to determine their brain RFS. Additional participants will be accrued to meet the 49 goal with one year of follow up. It is expected that approximately 30 to 40 participants per year can be accrued onto this trial, and thus accrual to both phases of the trial will be completed in approximately 3-4 years. Allowing for a very small number of inevaluable participants, the accrual ceiling will be set at 125 participants to permit up to 18 participants in the initial dose escalation phase and 98 randomized evaluable participants.
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9.3 populations for analysis

Phase I: all participants who received at least one dose of the agents Phase II: Modified intention to treat analysis (all participants who received at least one dose of the agents)
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9.4 statistical analyses

9.4.1 General approach Safety data will be tabulated and reported per arm.  
Kaplan-Meier curves will be created for each of the randomized arms and compared using a twotailed log-rank test, with a one-sided 0.10 significance level of interest to be detected.  
9.4.2 Analysis of the primary safety and efficacy endpoints The MTD will be identified based on being the dose level at which 0 or 1 participant in 6 has a DLT. Brain RFS curves will be created using the Kaplan-Meier method based on all participants considered to be evaluable based on having received protocol treatment, and compared using a one-tailed log-rank test, with a one-sided 0.10 significance level of interest to be detected. 9.4.3 Analysis of the secondary efficacy endpoints Number of participants with DLTs at each dose level: tabulated counts Toxicity data: tabulated counts Time to whole brain irradiation compared between the two arms: Kaplan-Meier curves constructed with a two-tailed log-rank test used to compare the arms.  
Overall survival compared between the two arms Kaplan-Meier curves constructed with a twotailed log-rank test used to compare the arms.  
9.4.4 Safety Analyses Toxicity data: tabulated counts and adverse events will be evaluated descriptively. 9.4.5 Baseline Descriptive Statistics Demographic and clinical characteristics of participants on both arms will be tabulated. 9.4.6 Planned interim analyses This study will be monitored by the NCI/CCR Data Safety and Monitoring Board, both for toxicity as well as futility and efficacy with respect to the primary brain RFS endpoint.  
Beginning in the year after twenty total participants have been randomized and treated on the trial, the study will be monitored by a DSMB on an annual basis to evaluate the safety of the two arms. The SAEs (typically grade 3 toxicities, or greater) will be reported according to type of toxicity, and maximal grade noted per participant, for toxicities with at least a possible attribution to the therapy provided on that arm. Comparisons will be made between the two arms using a CochranArmitage test for trend, or other appropriate methods, to determine if there is increased toxicity associated with either arm. In addition, at the first DSMB meeting held following the point at which half of the required total subjects have been enrolled and followed for a minimum of 12 months (that is, after 49 participants have been enrolled, and the most recent of those 49 participants were randomized at least 12 months prior to the date of the DSMB evaluation), a single evaluation for futility will be undertaken. The futility evaluation will be performed as follows: based on the full data available at the time, if the hazard ratio for brain RFS for the two arms is not favoring the temozolomide arm (that is, if the HR=1.0 or greater favoring TDM-1 alone), or at the discretion of the DSMB if results are not clearly defined for other reasons, such as the appearance of the interim KaplanMeier curves, then the trial will end accrual at that time if this is directed by the DSMB.  
Monitoring for efficacy will take place on an annual basis, also beginning after 49 participants have been randomized and followed potentially for a minimum of 6 months. This will be performed using a Lan-DeMets alpha spending function approach based on an O'Brien-Fleming boundary associated with the results of the log-rank test related to the comparison of OS curves. 9.4.7 Exploratory analyses As described in the correlative studies section of the protocol, biomarkers in the CSF and serum will be measured, as well as their changes with treatment. The biomarkers will be evaluated in an exploratory fashion to predict relapse. MGMT methylation status will be analyzed in primary tumor, CSF and serum, as described in the correlative studies section in the protocol. Findings at baseline and changes at different points during the treatment will be compared between participants that benefit and those participants that don't benefit from temozolomide treatment, applying non-parametric tests. In the same manner, questionnaires regarding cognitive and quality of life aspects will be applied and analyzed in an exploratory fashion. The analysis should include: 1. Neurotoxicity and neurocognitive effects between the two arms 2. MDASI-BT subscale (cognitive dysfunction, focal neurologic deficits, and interference) differences between the two arms 3. Baseline MDASI-BT subscales (overall symptom burden, overall interference, cognitive dysfunction) as well as fatigue and change of appetite as prognostic for time to distant metastases and for OS 4. Changes in score over time in select MDASI-BT subscales as prognostic for OS, time to distant metastases, and neurocognitive decline
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10 collaborative agreements 10.1 clinical

The study agent, Temozolomide is provided by MERCK & Co., Inc. under a Clinical Trials Agreement (CTA). Results or study data may be communicated to CTA partner, according to the terms of the NIH- Advanced Accelerator Applications SA CTA.
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11 human subjects protections 11.1

Subjects from all racial and ethnic groups are eligible for this trial if they meet the eligibility criteria. Efforts will be made to extend the accrual to a representative population. If differences in outcome that correlate to racial or ethnic identity are noted, accrual may be expanded or additional studies may be performed to investigate those differences more fully. We are excluding non-English speakers because part of our study objectives is to evaluate neurocognitive effects of disease and treatment. The evaluation of neurocognitive effects relies in tests and questionnaires that require clear comprehension of the language used to apply them. Failures in comprehension of the language could jeopardize the tests results and interpretation.
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11.2 participation of children

The age group for enrollment on this trial is 18 or more years of age. Because no dosing or adverse event data are currently available on the use of T-DM1 or temozolomide in participants < 18 years of age, children are excluded from this study.
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11.3 participation of subjects unable

Adults unable to give consent are excluded from enrolling in the protocol. However, re-consent may be necessary and there is a possibility, though unlikely, that subjects could become decisionally impaired. For this reason and because there is a prospect of direct benefit from research participation (section **11.5**), all subjects will be offered the opportunity to fill in their wishes for research and care, and assign a substitute decision maker on the "NIH Advance Directive for Health Care and Medical Research Participation" form so that another person can make decisions about their medical care in the event that they become incapacitated or cognitively impaired during the course of the study. Note: The PI or AI will contact the NIH Ability to Consent Assessment Team (ACAT) for evaluation to assess ongoing capacity of the subjects and to identify an LAR, as needed. Please see section **11.6.1** for consent procedure.
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11.4 evaluation of benefits and

The primary risk to participants participating in this research study is from the toxicity of Temozolomide and T-DM1, or both drugs. Temozolomide is an investigational agent in the prevention of breast cancer brain metastases. T-DM1 is an approved agent for treatment of metastatic HER2-positive breast cancer. The protocol provides for detailed and careful monitoring of all participants to assess for toxicity. Toxicity data from the phases I and II dose levels will be collected and reviewed to ensure that there were no severe toxicities that would preclude further participant enrollment. Participants will be treated with therapeutic intent and response to the therapy will be closely monitored.
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11.5 risks/benefits analysis 11.5.1 risks

11.5.2 Research Blood Collection Risks Risks of blood draws include pain and bruising in the area where the needle is placed, lightheadedness, and rarely, fainting. When large amounts of blood are collected, low red blood cell count (anemia) can develop. 11.5.3 Risks of Echocardiogram Other than the possibility of some mild discomfort during the test, there are no known risks to an echocardiogram.
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11.5.4 risks of muga scans

In addition to the risks of radiation exposure, the primary risk associated with MUGA scans is an allergic reaction to the contrast material. 11.5.5 Risks of Questionnaires These contain questions that may be sensitive in nature. Subjects are instructed that they may refuse to answer any question that makes them feel uncomfortable, and theat if they have concerns after completing the questionnaire, they are encouraged to contact the study team. 11.5.6 Lumbar Puncture for Research Purposes Risks of lumbar puncture include headache, tenderness or pain in the lower back and bleeding near the puncture site; and pain and numbness that shoots down in the legs. In rare cases, people experience brainstem herniation, which is the movement of brain tissue from its normal position in the skull. 11.5.7 Risks of exposure to ionizing radiation Study participants will be exposed to radiation from the following sources within a one-year period:
Up to 5 lumbar punctures guided by fluoroscopy  
Up to 8 CT scans 1 technetium bone scan Up to 4 MUGA scans
The amount of radiation exposure from these procedures is equal to approximately 11.19 rem. This is equal to roughly the same amount of radiation as 37.3 years' worth of background radiation. About 40 out of 100 people (40%) will get cancer during their lifetime, and 20 out of 100 (20%) will die from cancer. The risk of getting cancer from the radiation exposure in this study is 1.1 out of 100 (1.1%) and of getting a fatal cancer is 0.6 out of 100 (0.6%). Note: MRIs will be performed for participants who are unable to tolerate the CT contrast. 11.5.8 Risks of CT Scans In addition to the radiation risks discussed above, risks associated with CT scans include allergic reaction to and kidney damage from the contrast dye, nausea, vomiting, and anxiety.
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11.5.9 risks of mris

The risks of MRI include anxiety as well as the risks associated with the gadolinium contrast material, which are metallic taste, headache, nausea, and allergic reaction; and in participants with kidney disease, nephrogenic systemic fibrosis (NSF).
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11.5.10 other risks

Risks include the possible occurrence of any of a range of side effects which are listed in the Consent Document or this protocol document. Frequent monitoring for adverse effects will help to minimize the risks associated with administration of the study agents.
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11.5.11 non-physical risks of genetic

Anxiety and stress may arise as a result of the anticipation that unwanted information regarding disease related DNA sequencing or disease tendencies, or misattributed paternity. Participants will be clearly informed that the data related to DNA sequencing and genetic analysis is coded, investigational and will not be shared with participants, family members or health care providers. 11.5.11.2 Risk related to possibility that information may be released This includes the risk that data related to genotype, DNA sequencing or risk for disease tendency or trait can be released to members of the public, insurers, employers, or law enforcement agencies. Although there are no plans to release results to the participants, family members or health care providers, this risk will be included in the informed consent document.
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11.5.11.3 risk to family or

Family members or relatives may or may not want to be aware of familial tendencies or genetic risks of disease which may cause anxiety about possible future health problems. As previously noted, participants will be given the option in the consent document to be notified of any medically significant and actionable incidental findings.
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11.5.12 certificate of confidentiality

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

The potential benefit to a participant on this study is a reduction or stability in the volume of their tumor systemically or outside of the brain, as well as potentially the prevention or delay in development of new metastatic lesions in the brain, which may or may not have favorable impact on symptoms and/or survival.
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11.5.14 risks/benefits analysis

The potential benefits from this therapy are stabilization or shrinkage of disease outside the brain and a reduction in chances of developing new tumor lesions in the brain, with decrease of symptoms caused by the brain tumor such as neurological deficits and headache. Given the efforts to minimize risk with the administration of this combination, this protocol involves greater than minimal risk, but presents the potential for direct benefit to individual subjects.
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11.6 consent process and documentation

The informed consent document will be provided as a physical or electronic document to the participant or consent designee(s) as applicable for review prior to consenting. A designated study investigator will carefully explain the procedures and tests involved in this study, and the associated risks, discomforts and benefits. In order to minimize potential coercion, as much time as is needed to review the document will be given, including an opportunity to discuss it with friends, family members and/or other advisors, and to ask questions of any designated study investigator. A signed informed consent document will be obtained prior to entry onto the study. The initial consent process as well as re-consent, when required, may take place in person or remotely (e.g., via telephone or other NIH approved remote platforms used in compliance with policy, including HRPP Policy 303) per discretion of the designated study investigator and with the agreement of the participant/consent designee(s). Whether in person or remote, the privacy of the subject will be maintained. Consenting investigators (and participant/consent designee, when in person) will be located in a private area (e.g., clinic consult room). When consent is conducted remotely, the participant/consent designee will be informed of the private nature of the discussion and will be encouraged to relocate to a more private setting if needed. Consent will be documented with required signatures on the physical document (which includes the printout of an electronic document sent to participant) or as described below, with a manual
(non-electronic) signature on the electronic document. When required, witness signature will be obtained similarly as described for the investigator and participant.
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manual (non-electronic) signature on electronic

When a manual signature on an electronic document is used for the documentation of consent at the NIH Clinical Center, this study will use the following to obtain the required signatures:  
Adobe platform (which is not 21 CFR Part 11 compliant); or, iMedConsent platform (which is 21 CFR Part 11 compliant)
During the consent process, participants and investigators will view individual copies of the approved consent document on screens at their respective locations (if remote consent); the same screen may be used when in the same location but is not required. Both the investigator and the subject will sign the document using a finger, stylus or mouse.  
Note: Refer to the CCR SOP PM-2, Obtaining and Documenting the Informed Consent Process for additional information (e.g., verification of participant identity when obtaining consent remotely) found at https://ccrod.cancer.gov/confluence/pages/viewpage.action?pageId=73203825.  
11.6.1 Consent Process for Adults Who Lack Capacity to Consent to Research Participation For participants addressed in section **11.3**, an LAR will be identified consistent with Policy 403 and informed consent obtained from the LAR, as described in Section **11.6**.
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11.7 request for waiver of

Prior to the subject signing the consent for this study pre-screening activities listed in section **2.2.1** may be performed. We request a waiver of consent for these activities as they involve only minimal risk to the subjects. A waiver will not adversely affect the rights and welfare of the subjects given that the activities are only intended to determine suitability for screening for participation in research protocols. These activities could not practicably be carried out without the wavier as central recruiting services, utilized in the NIH Clinical Center, perform pre-screening activities for multiple studies and obtaining consent for each one is beyond their resources. The subjects will be provided with additional pertinent information after participation as they will be informed whether or not they are eligible to sign a consent for additional screening.
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12 regulatory and operational considerations

This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable cause. Written notification, documenting the reason for study suspension or termination, will be provided by the suspending or terminating party to study participants, investigator, funding agency, and regulatory authorities. If the study is prematurely terminated or suspended, the Principal Investigator (PI) will promptly inform study participants, the Institutional Review Board (IRB), and sponsor and will provide the reason(s) for the termination or suspension. Study participants will be contacted, as applicable, and be informed of changes to study visit schedule. Circumstances that may warrant termination or suspension include, but are not limited to:
Determination of unexpected, significant, or unacceptable risk to participants  
Demonstration of efficacy that would warrant stopping Insufficient compliance to protocol requirements Data that are not sufficiently complete and/or evaluable Determination that the primary endpoint has been met Determination of futility
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, and satisfy the sponsor, IRB and as applicable, Food and Drug Administration (FDA).
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12.2 quality assurance and quality

The clinical site will perform internal quality management of study conduct, data and biological specimen collection, documentation and completion. An individualized quality management plan will be developed to describe a site's quality management. Quality control (QC) procedures will be implemented beginning with the data entry system and data QC checks that will be run on the database will be generated. Any missing data or data anomalies will be communicated to the site(s) for clarification/resolution. Following written Standard Operating Procedures (SOPs), the monitors will verify that the clinical trial is conducted and data are generated and biological specimens are collected, documented (recorded), and reported in compliance with the protocol, International Conference on Harmonisation Good Clinical Practice (ICH GCP), and applicable regulatory requirements (e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)). The investigational site will provide direct access to all trial related sites, source data/documents, and reports for the purpose of monitoring and auditing by the sponsor, and inspection by local and regulatory authorities.
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12.3 conflict of interest policy

The independence of this study from any actual or perceived influence, such as by the pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who have a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, persons who have a perceived conflict of interest will be required to have such conflicts managed in a way that is appropriate to their participation in the design and conduct of this trial. The study leadership in conjunction with the National Cancer Institute has established policies and procedures for all study group members to disclose all conflicts of interest and will establish a mechanism for the management of all reported dualities of interest.
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12.4 confidentiality and privacy

Participant confidentiality and privacy is strictly held in trust by the participating investigators, their staff, and the sponsor(s). This confidentiality is extended to cover testing of biological samples and genetic tests in addition to the clinical information relating to participants. Therefore, the study protocol, documentation, data, and all other information generated will be held in strict confidence. No information concerning the study or the data will be released to any unauthorized third party without prior written approval of the sponsor. All research activities will be conducted in as private a setting as possible. The study monitor, other authorized representatives of the sponsor, representatives of the Institutional Review Board (IRB), and/or regulatory agencies may inspect all documents and records required to be maintained by the investigator, including but not limited to, medical records (office, clinic, or hospital) and pharmacy records for the participants in this study. The clinical study site will permit access to such records. The study participant's contact information will be securely stored at the clinical site for internal use during the study. At the end of the study, all records will continue to be kept in a secure location for as long a period as dictated by the reviewing IRB, Institutional policies, or sponsor requirements. Study participant research data, which is for purposes of statistical analysis and scientific reporting, will be transmitted to and stored at the NCI CCR. This will not include the participant's contact or identifying information. Rather, individual participants and their research data will be identified by a unique study identification number. The study data entry and study management systems used by the clinical site and by NCI CCR research staff will be secured and password protected. At the end of the study, all study databases will be archived at the NIH. To further protect the privacy of study participants, a Certificate of Confidentiality has been issued by the National Institutes of Health (NIH). This certificate protects identifiable research information from forced disclosure. It allows the investigator and others who have access to research records to refuse to disclose identifying information on research participation in any civil, criminal, administrative, legislative, or other proceeding, whether at the federal, state, or local level. By protecting researchers and institutions from being compelled to disclose information that would identify research participants, Certificates of Confidentiality help achieve the research objectives and promote participation in studies by helping assure confidentiality and privacy to participants.
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13 pharmaceutical information

There will be no IND obtained for the use of any of the commercial agents used in this study. This study meets the criteria for exemption for an IND as this investigation is not intended to support a new indication for use or any other significant change to the labeling; the drugs are already approved and marketed and the investigation is not intended to support a significant change in advertising; and the investigation does not involve a route of administration or dosage level in use in a patient population or other factor that significantly increases the risks (or decreases the acceptability of the risks) associated with the use of the drug product.
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13.1 temozolomide 13.1.1 source

Temozolomide is commercially available. It will be provided by MERCK & Co., Inc. for this study under the agreement described in section **10.1**. NOTE: Effective with Amendment F (version date 06/08/2020): Merck discontinuned production of the Temozolomide 5mg and 20mg capsules, so Merck will purchase those doses from Myonex for delivery to the NIH pharmacy. Merck will continue to provide the 100mg capsules per the original agreement. 13.1.2 Formulation and preparation Temozolomide is an imidazotetrazine derivative. The chemical name of temozolomide is 3,4-  
 dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide. The structural formula is: The material is a white to light tan/light pink powder with a molecular formula of C6H6N6O2 and a molecular weight of 194.15. The molecule is stable at acidic pH (<5) and labile at pH >7; hence Temozolomide can be administered orally and intravenously. The prodrug, temozolomide, is rapidly hydrolyzed to the active 5-(3-methyltriazen-1-yl) imidazole-4-carboxamide (MTIC) at neutral andalkaline pH values, with hydrolysis taking place even faster at alkaline pH. Other Names: methazolastone, Temodar Temozolomide is supplied in white opaque, preservative-free, two-piece, hard gelatin capsules of the following p.o. dosage strengths: 5 mg, 20 mg, 100 mg, 180 mg and 250 mg. Each capsule contains drug substance in combination with lactose, anhydrous NF, colloidal silicon dioxide NF, sodium starch glycolate NF, tartaric acid NF, and stearic acid NF. The capsule shells contain gelatin NF, titanium dioxide USP, and sodium lautyl sulfate NF.
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mode of action

Alkylating agent of imidazotetrazinone class. 13.1.3 Storage and Stability The 5-mg, 20-mg, 100-mg, 140-mg, and 180-mg capsule strengths are available in 5- count and 14-count packages. The 250-mg capsule strength is available in a 5-count package. Each strength of Temozolomide must be dispensed in a separate vial or in its original package (one strength per one container).
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follow the instructions below

Based on the dose prescribed, determine the number of each strength of Temozolomide capsules that are needed. Label each container with the appropriate number of capsules to be taken each day. Dispense to the participant, making sure each container lists the strength (mg) per capsule and that he or she understands to take the appropriate number of capsules of Temozolomide from each package or vial to equal the total daily dose prescribed by the physician. Temozolomide is store at 25°C, excursions permitted to 15-30°C. Repackaging of Temozolomide for dispensing may occur per guidance provided in manufacturer's product labeling.
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13.1.4 administration procedures

Participants should take each day's dose with a full glass of water at the same time each day. Taking the medication on an empty stomach or at bedtime may help ease nausea. If participants are also taking anti-nausea or other medications to relieve the side effects associated with Temozolomide, they should be advised to take these medications 30 minutes before they take Temozolomide. Temozolomide causes the rapid appearance of malignant tumors in rats. Participants SHOULD NOT open or split the capsules. If capsules are accidentally opened or damaged, rigorous precautions should be taken with the capsule contents to avoid inhalation or contact with the skin or mucous membranes. The medication should be kept away from children and pets. The Temozolomide capsules should be swallowed whole and NEVER CHEWED.
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13.1.5 pharmacokinetics

Temozolomide is rapidly and completely absorbed after oral administration; peak plasma concentrations occur in 1 hour. Food reduces the rate and extent of temozolomide absorption. Mean peak plasma concentration and AUC decreased by 32% and 9%, respectively, and Tmax increased 2-fold (from 1.1 to 2.25 hours) when temozolomide was administered after a modified high-fat breakfast. Temozolomide is rapidly eliminated with a mean elimination half-life of 1.8 hours and exhibits linear kinetics over the therapeutic dosing range. Temozolomide has a mean apparent volume of distribution of 0.4 L/kg (%CV=13%). It is weakly bound to human plasma proteins; the mean percent bound of drug-related total radioactivity is 15%.
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13.1.6 metabolism and elimination

Temozolomide is spontaneously hydrolyzed at physiologic pH to the active species, 3-methyl- (triazen-1-yl)imidazole-4-carboxamide (MTIC) and to temozolomide acid metabolite. MTIC is further hydrolyzed to 5-amino-imidazole-4-carboxamide (AIC), which is known to be an intermediate in purine and nucleic acid biosynthesis and to methylhydrazine, which is believed to be the active alkylating species. Cytochrome P450 enzymes play only a minor role in the metabolism of temozolomide and MTIC. Relative to the AUC of temozolomide, the exposure to MTIC and ACI is 2.4% and 23%, respectively. About 38% of the administered temozolomide total radioactive dose is recovered over 7 days; 37.7% in urine and 0.8% in feces. The majority of the recovery of radioactivity in urine is as unchanged temozolomide (5.6%), AIC (12%), temozolomide acid metabolite (2.3%), and unidentified polar metabolites(s) (17%). Overall clearance of temozolomide is about 5.5 L/hr/m2.
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13.1.7 special populations

Creatinine Clearance: Population pharmacokinetic analysis indicates that creatinine clearance over the range of 36-130 mL/min/m2 has no effect on the clearance of temozolomide after oral administration. The pharmacokinetics of temozolomide have not been studied in participants with severely impaired renal function (CLcr < 36 mL/min/m2). Caution should be exercised when temozolomide is administered to participants with severe renal impairment. Temozolomide has not been studied in participants on dialysis.  
Hepatically Impaired Participants: In a pharmacokinetic study, the pharmacokinetics of temozolomide in participants with mild to moderate hepatic impairment (Child's-Pugh Class I-II) were similar to those observed in participants with normal hepatic function. Caution should be exercised when temozolomide is administered to participants with severe hepatic impairment. Gender: Population pharmacokinetic analysis indicates that women have an approximately 5% lower clearance (adjusted for body surface area) for temozolomide than men. Women have higher incidences of grade 4 neutropenia and thrombocytopenia in the first cycle of therapy than men. Population pharmacokinetic analysis indicates that age (range 19-78 years) has no influence on the pharmacokinetics of temozolomide. In the anaplastic astrocytoma study population, participants 70 years of age or older had a higher incidence of grade 4 neutropenia and grade 4 thrombocytopenia in the first cycle of therapy than participants under 70 years of age. In the entire safety database, however, there did not appear to be a higher incidence in participants 70 years of age or older. 13.1.8 Incompatibilities and Toxicity Drug-Drug Interactions: In a multiple dose study, administration of temozolomide with ranitidine did not change the Cmax or AUC values for temozolomide or MTIC. Population analysis indicates that administration of valproic acid decreases the clearance of temozolomide by about 5%. The clinical implication of this effect is not known. Population analysis failed to demonstrate any influence of co-administered dexamethasone, prochlorperazine, phenytoin, carbamazepine, ondansetron, H2- receptor antagonists, or phenobarbital on the clearance of orally administered temozolomide. Known Potential Adverse Events Hematologic: Thrombocytopenia, leukopenia, myelodysplastic syndrome Gastrointestinal: Nausea, vomiting, anorexia Hepatic: Elevated liver enzymes (reversible) Skin: Rash Neurologic: Convulsions, weakness on one side of the body, abnormal coordination, paralysis Other: Constipation, diarrhea, stomatitis, fatigue, decreased performance status, headache  
| Likely (occurring in more than 20% of participants) Fatigue Alopecia   | Constipation                                                                                      |                           |
|------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------|---------------------------|
| Headache                                                               | Nausea                                                                                            | Anorexia                  |
| Seizures                                                               | Vomiting                                                                                          |                           |
| Common (occurring in 3-20% of participants) Dizziness Depression       | Weight gain                                                                                       |                           |
| Abnormal muscle movements/                                             | Difficulty sleeping                                                                               | Loss of urinary control   |
| coordination Abnormal gait                                             | Drowsiness                                                                                        | Urinary tract infection   |
| Hemiplegia or partial paralysis                                        | Skin rash                                                                                         | Frequent urination        |
| Upper and/or lower extremity                                           | Pruritus                                                                                          | Abnormal vision           |
| edema Weakness (such as weakness on                                    | Dry skin                                                                                          | Blurry vision             |
| one side of the body) Tickling/tingling sensation                      | Mucositis                                                                                         | Diplopia or double vision |
| Pain (such as in the abdomen,                                          | Dysphagia                                                                                         | Fever                     |
| joints, back, and/or muscles) Breast pain in females                   | Dysgeusia or taste changes                                                                        | Head cold                 |
| Confusion                                                              | Diarrhea                                                                                          | Cough                     |
| Memory problems                                                        | Excess steroid in the body  (possible bruising and/or  increase in size of the face  and/or neck) | Sore throat               |
| Anxiety                                                                | Viral infection                                                                                   | Sinusitis                 |
| Dyspnea                                                                | Allergic reaction                                                                                 |                           |  
Temozolomide may commonly cause low blood cell counts (white blood cells, red blood cells, and platelets). This means that while you take the drug, there is more of a chance of getting an infection, including pneumonia. You may become anemic and/or have problems with bleeding, bruising, fatigue, and/or shortness of breath. You may need a blood transfusion.  
| Rare but serious (occurring in fewer than 3% of participants) Bone marrow disease where not  New occurrence of cancer  Steven-Johnson Syndrome enough blood cells are made (including myeloid leukemia) Hallucinations Allergic skin reaction Damage from radiation (such as  skin damage) Nervous system disease  Severe skin damage with loss of  Pneumonitis (lung  (possible pain and/or weakness) a large portion of skin  inflammation) Neuropathies (nerve damagepossible numbness, tingling, and  pain) Weight loss Flu-like symptoms Hyperglycemia (possible  Fever due to low white blood  Injection site reactions (skin  diabetes) cell counts redness, irritation, pain, itching,  swelling, and/or warmth)   |
|---|  
Hypokalemia (possible weakness)  
Bruising Opportunistic infection  
Severe allergic reaction Hemorrhage Herpes infection causing painful  
skin rash (shingles)  
Temozolomide is potentially mutagenic and should be handled with appropriate precautions by both staff and participants. Capsules should not be opened. If capsules are accidentally opened or damaged, rigorous precautions should be taken with the capsule contents to avoid inhalation or contact with the skin or mucous membranes. Procedures for proper handling and disposal of anticancer drugs should be considered. Contraindications: Temozolomide is contraindicated in participants who have a history of a hypersensitivity reaction to any of its components or to DTIC.
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pregnancy category d

| unclear if these side effects were caused by temozolomide, but they may be: BUN blood level increase Death Cholecystitis Creatinine blood elevated Hypoxia Pancreatitis Lymphopenia Weight loss GI perforation Thrombocytopenia Epistaxis Dehydration Neutropenia Sepsis   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Temozolomide may cause fetal harm when administered to a pregnant woman. There are no adequate and well-controlled studies in pregnant women. If this drug is used during pregnancy, or if the participant becomes pregnant while taking this drug, the participant should be apprised of the potential hazard to the fetus. Women of childbearing potential should be advised to avoid becoming pregnant during therapy with temozolomide. Treatment of a man with temozolomide may increase the risk of birth defects if he causes a woman to become pregnant while he is taking temozolomide. Men treated with temozolomide may have difficulty causing a woman to become pregnant after their treatment is completed. Men receiving temozolomide should be directed to use effective contraception while they are being treated. There is insufficient data to know what the risk of subsequent problems with fertility will be. Similarly, women who are treated with temozolomide may have difficulty becoming pregnant in the future and may at be at increased risk of having children with birth defects. There is insufficient evidence to determine what the risk of these complications will be.
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13.2 t-dm1

13.2.1 Source T-DM1 will be used in its standard approved indication by the FDA. T-DM1 will be purchased from commercial sources by the NIH CC Pharmacy. 13.2.2 Formulation and Preparation T-DM1 (ado-trastuzumab emtansine) is a HER2-targeted antibody-drug conjugate (ADC) which contains the humanized anti-HER2 IgG1, trastuzumab, covalently linked to the microtubule inhibitory drug DM1 (a maytansine derivative) via the stable thioether linker MCC (4-[Nmaleimidomethyl] cyclohexane-1-carboxylate). Emtansine refers to the MCC-DM1 complex.  
The antibody trastuzumab, is a well characterized recombinant monoclonal antibody product  
 produced by mammalian (Chinese hamster ovary) cells, and the small molecule components (DM1 and MCC) are produced by chemical synthesis. Ado-trastuzumab emtansine contains an average of 3.5 DM1 molecules per antibody. Ado-trastuzumab emtansine has the following chemical structure:
T-DM1 (ado-trastuzumab emtansine) is a sterile, white to off-white preservative free lyophilized powder in single-use vials. Each vial contains 100 mg or 160 mg ado-trastuzumab emtansine. Following reconstitution, each single-use vial contains ado-trastuzumab emtansine (20 mg/mL), polysorbate 20 [0.02% (w/v)], sodium succinate (10 mM), and sucrose [6% (w/v)] with a pH of 5.0 and density of 1.026 g/mL. The resulting solution containing 20 mg/mL ado-trastuzumab emtansine is administered by intravenous infusion following dilution. 13.2.3 Supply and handling: T-DM1(ado-trastuzumab emtansine) is supplied as: one 100 mg vial, single use vial or one 160 mg vial, single use vial per carton. Store vials in a refrigerator at 2°C to 8°C (36°F to 46°F) until time of reconstitution. *Do not freeze* or shake.
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13.2.4 formulation and preparation

In order to prevent medication errors, it is important to check the vial labels to ensure that the drug being prepared and administered isT-DM1 (ado-trastuzumab emtansine) and not trastuzumab. Lyophilized powder in single-use vials: 100 mg per vial or 160 mg per vial of ado-trastuzumab emtansine.
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13.2.5 administration

Administer T-DM1 as an intravenous infusion only with a 0.2 or 0.22 micron in-line polyethersulfone (PES) filter. Do not administer as an intravenous push or bolus. Do not mix TDM1, or administer as an infusion, with other medicinal products. In order to improve traceability of biological medicinal products, the tradename and the batch number of the administered product should be clearly recorded (or stated) in the patient file.
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13.2.5.1 reconstitution

Use aseptic technique for reconstitution and preparation of dosing solution. Appropriate procedures for the preparation of chemotherapeutic drugs should be used. Using a sterile syringe, slowly inject 5 mL of Sterile Water for Injection into the 100 mg T-DM1 vial, or 8 mL of Sterile Water for Injection into the 160 mg T-DM1 vial to yield a solution containing 20 mg/mL. Swirl the vial gently until completely dissolved. *Do not shake.* Inspect the reconstituted solution for particulates and discoloration. The reconstituted solution should be clear to slightly opalescent and free of visible particulates. The color of the reconstituted solution should be colorless to pale brown. Do not use if the reconstituted solution contains visible particulates or is cloudy or discolored. The reconstituted lyophilized vials should be used immediately following reconstitution with Sterile Water for Injection. If not used immediately, the reconstituted T-DM1 vials can be stored for up to 24 hours in a refrigerator at 2ºC to 8ºC (36°F to 46°F); discard unused T-DM1 after 24 hours. *Do not freeze.* The reconstituted product contains no preservative and is intended for single-use only. 13.2.5.2 Dilution Determine the correct dose (mg) of T-DM1. Calculate the volume of the 20 mg/mL reconstituted T-DM1 solution needed. Withdraw this amount from the vial and add it to an infusion bag containing 250 mL of 0.9% Sodium Chloride Injection. *Do not use Dextrose (5%) solution.* Gently invert the bag to mix the solution in order to avoid foaming. The diluted T-DM1 infusion solution should be used immediately. If not used immediately, the solution may be stored in a refrigerator at 2°C to 8°C (36°F to 46°F) for up to 24 hours prior to use. This storage time is additional to the time allowed for the reconstituted vials. *Do not freeze or* shake. 13.2.6 Pharmacokinetics The pharmacokinetics of T-DM1 was evaluated in a phase 1 study and in a population pharmacokinetic analysis for the ado-trastuzumab emtansine conjugate (ADC) using pooled data from 5 trials in participants with breast cancer. A linear two-compartment model with first-order elimination from the central compartment adequately describes the ADC concentration-time profile. In addition to ADC, the pharmacokinetics of total antibody (conjugated and unconjugated trastuzumab), DM1 were also determined. The pharmacokinetics of T-DM1 are summarized below. 13.2.7 Distribution Maximum concentrations (Cmax) of ADC and DM1 were observed close to the end of infusion. In Study 1, mean (SD) ADC and DM1 Cycle 1 Cmax following KADCYLA administration was 83.4 (16.5) µg/mL and 4.61 (1.61) ng/mL, respectively. 18 of 24 In vitro, the mean binding of DM1 to human plasma proteins was 93%. *In vitro,* DM1 was a substrate of P-glycoprotein (P-gp). Based on population pharmacokinetic analysis, the central volume of distribution of ADC was 3.13 L.
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13.2.8 metabolism and elimination

In vitro studies indicate that DM1, the small molecule component of T-DM1, undergoes metabolism by CYP3A4/5. DM1 did not inhibit or induce major CYP450 enzymes *in vitro.* In human plasma, ado-trastuzumab emtansine catabolites MCC-DM1, Lys-MCC-DM1, and DM1 were detected at low levels. Based on population pharmacokinetic analysis, following intravenous infusion of T-DM1, the clearance of the ADC was 0.68 L/day and the elimination half-life (t1/2) was approximately 4 days. No accumulation of T-DM1 was observed after repeated dosing of intravenous infusion every 3 weeks. Based on population pharmacokinetic analysis (n=671), body weight, sum of longest diameter of target lesions by RECIST, HER2 extracellular domain (ECD) concentrations, AST, albumin, and baseline trastuzumab concentrations were identified as statistically significant covariates for adotrastuzumab emtansine clearance. However, the magnitude of effect of these covariates on adotrastuzumab emtansine exposure suggests that, with the exception of body weight, these covariates are unlikely to have a clinically meaningful effect on T-DM1 exposure. Therefore, the body weight based dose of 3.6 mg/kg every 3 weeks without correction for other covariates is considered appropriate. Drug to drug interaction: 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. 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. 13.2.9 Special populations Pregnancy: There is a pregnancy exposure registry that monitors pregnancy outcomes in women exposed to T-DM1 during pregnancy. Encourage women who receive T-DM1 during pregnancy or within 7 months prior to conception, to enroll in the MotHER Pregnancy Registry by contacting 1-800-6906720 or visiting http://www.motherpregnancyregistry.com/. In addition, there is a pregnancy pharmacovigilance program for T-DM1. If T-DM1 is administered during pregnancy, or if a participant becomes pregnant while receiving T-DM1 or within 7 months following the last dose of T-DM1, health care providers and participants should immediately report T-DM1 exposure to Genentech at 1-888-835-2555. T-DM1 can cause fetal harm when administered to a pregnant woman. There are no available data on the use of T-DM1 in pregnant women. Cases of oligohydramnios and oligohydramnios sequence manifesting as pulmonary hypoplasia, skeletal abnormalities, and neonatal death were observed in the postmarketing setting in participants treated with trastuzumab, the antibody component of T-DM1. Based on its mechanism of action, the DM1 component of T-DM1 can also cause embryo-fetal harm when administered to a pregnant woman. Apprise the participant of the potential risks to a fetus. There are clinical considerations if T-DM1 is used in a pregnant woman, or if a participant becomes pregnant within 7 months following the last dose of T-DM1. The estimated background risk of major birth defects and miscarriage for the indicated population is unknown. In the U.S. general population, the estimated background risk of major birth defects and miscarriage in clinically recognized pregnancies is 2-4% and 15-20%, respectively. Fetal/Neonatal Adverse Reactions Monitor women who received T-DM1 during pregnancy or within 7 months prior to conception for oligohydramnios. If oligohydramnios occurs, perform fetal testing that is appropriate for gestational age and consistent with community standards of care.  
Human Data There are no available data on the use of T-DM1 in pregnant women. In the postmarketing setting, cases of oligohydramnios, and of oligohydramnios sequence, manifesting in the fetus as pulmonary hypoplasia, skeletal abnormalities and neonatal death were observed after treatment with trastuzumab during pregnancy. These case reports described oligohydramnios in pregnant women who received trastuzumab either alone or in combination with chemotherapy. In some case reports, amniotic fluid index increased after trastuzumab was stopped. In one case, trastuzumab therapy resumed after amniotic index improved, and oligohydramnios recurred.  
Animal Data There were no reproductive and developmental toxicology studies conducted with ado-trastuzumab emtansine. DM1, the cytotoxic component of T-DM1, disrupts microtubule function. DM1 is toxic to rapidly dividing cells in animals and is genotoxic, suggesting it has the potential to cause embryotoxicity and teratogenicity. In studies where trastuzumab was administered to pregnant cynomolgus monkeys during the period of organogenesis at doses up to 25 mg/kg given twice weekly (about 7 times the clinical dose), trastuzumab crossed the placental barrier during the early (Gestation Days 20 to 50) and late (Gestation Days 120 to 150) phases of gestation. The resulting concentrations of trastuzumab in fetal serum and amniotic fluid were 15 of 24 approximately 33% and 25%, respectively, of those present in the maternal serum but were not associated with adverse developmental effects. Lactation There is no information regarding the presence of ado-trastuzumab emtansine in human milk, the effects on the breastfed infant, or the effects on milk production. DM1, the cytotoxic component of T-DM1, may cause serious adverse reactions in breastfed infants based on its mechanism of action. Advise women not to breastfeed during treatment and for 7 months following the last dose of T-DM1. There were no animal lactation studies conducted with ado-trastuzumab emtansine or the cytotoxic component of T-DM1 (DM1). In lactating cynomolgus monkeys, trastuzumab was present in breast milk at about 0.3% of maternal serum concentrations after pre- (beginning Gestation Day 120) and post-partum (through Post-partum Day 28) doses of 25 mg/kg administered twice weekly (about 7 times the clinical dose of T-DM1). Infant monkeys with detectable serum levels of trastuzumab did not exhibit any adverse effects on growth or development from birth to 1 month of age. Verify the pregnancy status of females of reproductive potential prior to the initiation of T-DM1. Contraception Females T-DM1 can cause embryo-fetal harm when administered during pregnancy. Advise females of reproductive potential to use effective contraception during treatment and for 7 months following the last dose of T-DM1 Males Because of the potential for genotoxicity, advise male participants with female partners of reproductive potential to use effective contraception during treatment with T-DM1 and for 4 months following the last dose. Infertility Based on results from animal toxicity studies, T-DM1 may impair fertility in females and males of reproductive potential. It is not known if the effects are reversible Geriatric Use Of 495 participants who were randomized to T-DM1 in the randomized trial, 65 participants (13%) were ≥ 65 years of age and 11 participants (2%) were ≥ 75 years of age. In participants ≥ 65 years old (n=138 across both treatment arms) the hazard ratios for progression-free survival (PFS) and Overall Survival (OS) were 1.06 (95% CI: 0.68, 1.66) and 1.05 (95% CI: 0.58, 1.91), respectively. Population pharmacokinetic analysis indicates that age does not have a clinically meaningful effect on the pharmacokinetics of ado-trastuzumab emtansine
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renal impairment

No dedicated renal impairment trial for T-DM1 has been conducted. Based on the population pharmacokinetics, as well as analysis of Grade 3 or greater adverse drug reactions and dose modifications, dose adjustments of T-DM1 are not needed in participants with mild (creatinine clearance [CLcr] 60 to 89 mL/min) or moderate (CLcr 30 to 59 mL/min) renal impairment. No dose adjustment can be recommended for participants with severe renal impairment (CLcr less than 30 mL/min) because of the limited data available.
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hepatic impairment

No adjustment to the starting dose is required for participants with mild or moderate hepatic impairment. T-DM1 was not studied in patients with severe hepatic impairment. Closely monitor participants with hepatic impairment due to known hepatotoxicity observed with T-DM1.  
13.2.10 Incompatibilities and Toxicity Incompatibilities are addressed in Section **4.1.1** Known Potential Adverse Events Hematologic: Thrombocytopenia, neutropenia, anemia Gastrointestinal: Nausea, vomiting, diarrhea Hepatic: Elevated liver enzymes Skin: Rash, pruritus Neurologic: headache, peripheral neuropathy Other: fatigue, decreased performance status  
| Likely (occurring in more than 25% of participants) Fatigue Hemorrhage            | Increased transaminases   |              |
|-----------------------------------------------------------------------------------|---------------------------|--------------|
| Nausea                                                                            | Thrombocytopenia          | Constipation |
| Musculoskeletal pain                                                              | Headache                  | Epistaxis    |
| Common (occurring in 3-25% of participants) Blood alkaline phosphatase  Dysgeusia | Dyspnea                   |              |
| increased Hypokalemia                                                             | Dizziness                 | Cough        |
| Myalgia                                                                           | Insomnia                  | Pruritus     |
| Arthralgia                                                                        | Rash                      | Hypertension |
| Increased bilirubin                                                               |                           |              |  
| Rare but serious / G3-4 (occurring in fewer than 3% of participants) Hypokalemia Musculoskeletal pain Peripheral neuropathy Hemorrhage Hypertension Hepatic failure has been observed in two participants (0.2%) with HER2-positive metastatic breast   |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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89. Hassel JC, Sucker A, Edler L, et al. MGMT gene promoter methylation correlates with tolerance of temozolomide treatment in melanoma but not with clinical outcome. British Journal of Cancer 2010; 103(6): 820-6. 90. Spitzwieser M, Holzweber E, Pfeiler G, Hacker S, Cichna-Markl M. Applicability of HIN-1, MGMT and RASSF1A promoter methylation as biomarkers for detecting field cancerization in breast cancer. *Breast Cancer Research* 2015; 17: 13. 91. Kato K, Hara A, Kuno T, et al. Aberrant promoter hypermethylation of p16 and MGMT genes in oral squamous cell carcinomas and the surrounding normal mucosa. *Journal of Cancer* Research and Clinical Oncology 2006; 132(11): 735-43. 92. Fiano V, Trevisan M, Trevisan E, et al. MGMT promoter methylation in plasma of glioma patients receiving temozolomide. *Journal of Neuro-Oncology* 2014; 117(2): 347-57. 93. Majchrzak-Celinska A, Paluszczak J, Kleszcz R, et al. Detection of MGMT, RASSF1A, p15INK4B, and p14ARF promoter methylation in circulating tumor-derived DNA of central nervous system cancer patients. *Journal of Applied Genetics* 2013; 54(3): 335-44. 94. Balana C, Carrato C, Ramirez JL, et al. Tumour and serum MGMT promoter methylation and protein expression in glioblastoma patients. *Clinical & Translational Oncology* 2011; 13(9): 677-85. 95. Hoffmann AC, Kaifi JT, Vallbohmer D, et al. Lack of Prognostic Significance of Serum DNA Methylation of DAPK, MGMT, and GSTPI in Patients With Non-Small Cell Lung Cancer. Journal of Surgical Oncology 2009; 100(5): 414-7. 96. Yu M, Song S, Nguyen S, Gu HH, Wong E. Cerebrospinal fluid (CSF) O-6methylguanine-methyltransferase (MGMT) from patients with glioblastoma multiforme. *NeuroOncology* 2007; 9(4): 549-.  
97. Palmieri D, Duchnowska R, Woditschka S, et al. Profound Prevention of Experimental Brain Metastases of Breast Cancer by Temozolomide in an MGMT-Dependent Manner. Clinical Cancer Research 2014; 20(10): 2727-39. 98. Fitzgerald D, Palmieri D, Hua E, et al. Reactive glia are recruited by highly proliferative brain metastases of breast cancer and promote tumor cell colonization. *Clin Exp Metast* 2008; 25: 799-810. 99. Steeg PS, Camphausen KA, Smith QR. Brain metastases as preventive and therapeutic targets. *Nature Reviews Cancer* 2011; 11(5): 352-63. 100. Freedman RA, Gelman RS, Wefel JS, et al. Translational Breast Cancer Research Consortium (TBCRC) 022: A Phase II Trial of Neratinib for Patients With Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer and Brain Metastases. *Journal of Clinical* Oncology 2016; 34(9): 945-+.  
101. Habets EJJ, Dirven L, Wiggenraad RG, et al. Neurocognitive functioning and healthrelated quality of life in patients treated with stereotactic radiotherapy for brain metastases: a prospective study. *Neuro-Oncology* 2016; 18(3): 435-44. 102. Caine C, Deshmukh S, Gondi V, et al. CogState computerized memory tests in patients with brain metastases: secondary endpoint results of NRG Oncology RTOG 0933. *Journal of* Neuro-Oncology 2016; 126(2): 327-36. 103. Triebel KL, Gerstenecker A, Meneses K, et al. Capacity of patients with brain metastases to make treatment decisions. *Psycho-Oncology* 2015; 24(11): 1448-55. 104. Fitzgerald DP, Subramanian P, Deshpande M, et al. Opposing Effects of Pigment Epithelium-Derived Factor on Breast Cancer Cell versus Neuronal Survival: Implication for Brain Metastasis and Metastasis-Induced Brain Damage. *Cancer Research* 2012; 72(1): 144-53.  
105. Papa L, Brophy GM, Welch RD, et al. Time Course and Diagnostic Accuracy of Glial and Neuronal Blood Biomarkers GFAP and UCH-L1 in a Large Cohort of Trauma Patients With and Without Mild Traumatic Brain Injury. *Jama Neurology* 2016; 73(5): 551-60. 106. Brophy GM, Mondello S, Papa L, et al. Biokinetic Analysis of Ubiquitin C-Terminal Hydrolase-L1 (UCH-L1) in Severe Traumatic Brain Injury Patient Biofluids. *Journal of* Neurotrauma 2011; 28(6): 861-70. 107. Papa L, Silvestri S, Brophy GM, et al. GFAP Out-Performs S100 beta in Detecting Traumatic Intracranial Lesions on Computed Tomography in Trauma Patients with Mild Traumatic Brain Injury and Those with Extracranial Lesions. *Journal of Neurotrauma* 2014; 31(22): 1815-U11. 108. Papa L, Brophy GM, Hannay HJ, et al. EARLY CSF LEVELS OF UCH-L1 ARE ASSOCIATED WITH MEASURES OF INJURY SEVERITY IN PATIENTS WITH SEVERE TBI. *Journal of Neurotrauma* 2012; 29(10): A143-A. 109. Ren CH, Kobeissy F, Alawieh A, et al. Assessment of Serum UCH-L1 and GFAP in Acute Stroke Patients. *Scientific Reports* 2016; 6: 9. 110. Zovoilis A, Agbemenyah HY, Agis-Balboa RC, et al. microRNA-34c is a novel target to treat dementias. *Embo Journal* 2011; 30(20): 4299-308. 111. Bhatnagar S, Chertkow H, Schipper HM, et al. Increased nnicroRNA-34c abundance in Alzheimer's disease circulating blood plasma. *Frontiers in Molecular Neuroscience* 2014; 7: 11. 112. Pacifici M, Delbue S, Kadri F, Peruzzi F. Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR. *Jove-Journal of Visualized Experiments* 2014; (83): 7. 113. Hoshino A, Costa-Silva B, Shen TL, et al. Tumour exosome integrins determine organotropic metastasis. *Nature* 2015; 527(7578): 329-+. 114. Armstrong TS, Gning I, Mendoza TR, et al. Clinical Utility of the MDASI-BT in Patients with Brain Metastases. *Journal of Pain and Symptom Management* 2009; 37(3): 331-40. 115. Saffer BY, Lanting SC, Koehle MS, Klonsky ED, Iverson GL. Assessing cognitive impairment using PROMIS (R) applied cognition-abilities scales in a medical outpatient sample.  
Psychiatry Research 2015; 226(1): 169-72. 116. Lin NU, Lee EQ, Aoyama H, et al. Response assessment criteria for brain metastases: proposal from the RANO group. *Lancet Oncology* 2015; 16(6): E270-E8. 117. Kased N, Binder DK, McDermott MW, et al. GAMMA KNIFE RADIOSURGERY FOR BRAIN METASTASES FROM PRIMARY BREAST CANCER. *International Journal of* Radiation Oncology Biology Physics 2009; 75(4): 1132-40.
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15 appendices

| 15.1                               | APPENDIX A - PERFORMANCE STATUS CRITERIA ECOG Performance Status Scale Karnofsky Performance Scale                     |                                                            |                                                                     |
|------------------------------------|------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------|---------------------------------------------------------------------|
| Grade                              | Descriptions                                                                                                           | Percent                                                    | Description                                                         |
| 100                                | Normal, no complaints, no                                                                                              |                                                            |                                                                     |
| Normal activity. Fully active,     | evidence of disease.                                                                                                   |                                                            |                                                                     |
| 0                                  | able to carry on all pre-disease                                                                                       | Able to carry on normal activity;                          |                                                                     |
| performance without restriction.   | 90                                                                                                                     | minor signs or symptoms of  disease.                       |                                                                     |
| Symptoms, but ambulatory.          | Normal activity with effort;                                                                                           |                                                            |                                                                     |
| Restricted in physically strenuous | 80                                                                                                                     | some signs or symptoms of  disease.                        |                                                                     |
| 1                                  | activity, but ambulatory and able  to carry out work of a light or                                                     | Cares for self, unable to carry on                         |                                                                     |
| sedentary nature (e.g., light      | 70                                                                                                                     | normal activity or to do active                            |                                                                     |
| housework, office work).           | work.                                                                                                                  |                                                            |                                                                     |
| In bed <50% of the time.           | Requires occasional assistance,                                                                                        |                                                            |                                                                     |
| Ambulatory and capable of all      | 60                                                                                                                     | but is able to care for most of  his/her needs.            |                                                                     |
| 2                                  | self-care, but unable to carry out  any work activities. Up and                                                        | Requires considerable assistance                           |                                                                     |
| about more than 50% of waking      | 50                                                                                                                     | and frequent medical care.                                 |                                                                     |
| hours.                             | 40                                                                                                                     | Disabled, requires special care  and assistance.           |                                                                     |
| 3                                  | In bed >50% of the time.  Capable of only limited self-care,  confined to bed or chair more  than 50% of waking hours. | 30                                                         | Severely disabled,  hospitalization indicated. Death  not imminent. |
| 100% bedridden. Completely         | 20                                                                                                                     | Very sick, hospitalization  indicated. Death not imminent. |                                                                     |
| 4                                  | disabled. Cannot carry on any  self-care. Totally confined to bed  or chair.                                           | 10                                                         | Moribund, fatal processes  progressing rapidly.                     |
| 5                                  | Dead.                                                                                                                  | 0                                                          | Dead.                                                               |
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15.2 appendix b - list

| CYP3A4 INHIBITORS  Strong Inhibitors  (prohibited)                                                                                                                                                                                                                                                                                                                                                                                                                                                                   | Weak Inhibitors  (use with caution, avoid if  possible)                                                                                                                                                                                                                                                                                            |                                                                                                                                                              |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Amprenavir1  Atazanavir1  Clarithromycin  Conivaptan1  Delavirdine1  Fosamprenavir1  Fospropofol1  Imatinib1  Indinavir  Isoniazid1  Itraconazole  Ketoconazole  Miconazole1  Nefazodone  Nelfinavir  Nicardipine1  Posaconazole1  Propofol1  Quinidine1  Ritonavir  Saquinavir2  Telithromycin                                                                                                                                                                                                                      | Moderate Inhibitors  (use with caution, avoid if  possible)  Amiodarone1  Aprepitant  Cimetidine1  Clotrimazole1  Cyclosporine1  Desipramine1  Doxycycline1  Efavirenz1  Erythromycin  Fluconazole  Fosaprepitant1  Grapefruit juice  Haloperidol1  Lidocaine1  Metronidazole1  Norfloxacin1  Sertraline1  Tetracycline1  Verapamil  Voriconazole1 | Chloramphenicol2  Ciprofloxacin2  Diethyldithiocarbamate2  Fluvoxamine2  Gestodene2  Mibefradil2  Mifepristone  Norfluoxetine2  Star fruit2  Troleandomycin2 |
| 1 Cited in Cytochrome P450 Enzymes: Substrates, Inhibitors, and Inducers. In: Lacy CF, Armstrong LL, Goldman MP, Lance LL, eds. Drug Information Handbook 20th ed.  Hudson, OH; LexiComp Inc. 2011-2012: 1810-1818  2 Cited in Flockhart DA. Drug Interactions: Cytochrome P450 Drug Interaction Table. Indiana University School of Medicine (2007).  http://medicine.iupui.edu/clinpharm/ddis/table.asp. Accessed Nov 2011. Note: Drugs without a superscript are cited in both the Lacy and Flockhart references. |                                                                                                                                                                                                                                                                                                                                                    |                                                                                                                                                              |
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15.3 appendix c - oral

Today's Date ________________________________ Cycle \#__________ Patient Name_______________________________ Patient Study ID ___________________ (initials acceptable for patien's name)
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instructions to the patient

1. Complete one form for each cycle (21 days). 2. You will take ___ (number capsule(s)), total _______ mg (dosage) each day. 3. Please, do not eat anything for at least 2 hours before and 1 hour after temozolomide administration.  
4. Record the date, the number of pills you took, and when you took them. 5. If you have any comments or notice any side effects, please record them in the Comments column. 6. Please bring your pill bottle and this form to your physician when you go for your next appointment.  
\# pills and when taken: temozolomide  
| appointment.  # pills and when  taken:  temozolomide  Date Day   | Comments   | Date   | Day   | # pills and when  taken:  temozolomide   | Comments   |
|------------------------------------------------------------------|------------|--------|-------|------------------------------------------|------------|
| Dose                                                             | Time       | Dose   | Time  |                                          |            |
| 1                                                                | 12         |        |       |                                          |            |
| 2                                                                | 13         |        |       |                                          |            |
| 3                                                                | 14         |        |       |                                          |            |
| 4                                                                | 15         |        |       |                                          |            |
| 5                                                                | 16         |        |       |                                          |            |
| 6                                                                | 17         |        |       |                                          |            |
| 7                                                                | 18         |        |       |                                          |            |
| 8                                                                | 19         |        |       |                                          |            |
| 9                                                                | 20         |        |       |                                          |            |
| 10                                                               | 21         |        |       |                                          |            |
| 11                                                               |            |        |       |                                          |            |
| Study Team will complete this section:                           |            |        |       |                                          |            |  
Study Team will complete this section:
1. Date patient started protocol treatment_____________________ 2. Date patient was removed from study _____________________ 3. Patient's planned daily dose______________________________ 4. Total number of pills taken this month ______________________
Physician/Nurse's Signature: ______________________________________________________
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15.4 appendix d- rano criteria

Panel 1: Response assessment of target and non-target lesions Target lesions Complete response Disappearance of all CNS target lesions sustained for at least 4 weeks; with no new lesions, no use of corticosteroids, and patient is stable or improved clinically. Partial response At least a 30% decrease in the sum longest diameter of CNS target lesions, taking as reference the baseline sum longest diameter sustained for at least 4 weeks; no new lesions; stable to decreased corticosteroid dose; stable or improved clinically. Progressive disease At least a 20% increase in the sum longest diameter of CNS target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, at least one lesion must increase by an absolute value of 5 mm or more to be considered progression. Stable disease Neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease, taking as reference the smallest sum longest diameter while on study.  
Non-target lesions Non-target lesions should be assessed qualitatively at each of the timepoints specified in the protocol.  
Complete response Requires all of the following: disappearance of all enhancing CNS non-target lesions, no new CNS lesions. Non-complete response or non-progressive disease Persistence of one or more non-target CNS lesion or lesions. Progressive disease Any of the following: unequivocal progression of existing enhancing non-target CNS lesions, new lesion(s) (except while on immunotherapy-based treatment), or unequivocal progression of existing tumour-related non-enhancing (T2/FLAIR) CNS lesions. In the case of immunotherapy-based treatment, new lesions alone may not constitute progressive disease.  
Lin NU, Lee EQ, Aoyama H, et al. Response assessment criteria for brain metastases: proposal from the RANO group. Lancet Oncology 2015; 16(6): E270-E8
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abbreviated title tmz in breast

| Complete response                                                                                                                                                              |  Partial response                                  | Stable disease                                                                       | Progressive disease                   |                                  |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------|--------------------------------------------------------------------------------------|---------------------------------------|----------------------------------|
| Target lesions                                                                                                                                                                 | None                                               | a30% decrease in sum longest distance    <30% decrease relative to baseline but <20% | =20% increase in sum longest distance |                                  |
| relative to baseline                                                                                                                                                           | increase in sum longest distance relative to nadir | relative to nadir*                                                                   |                                       |                                  |
| Non-target lesions                                                                                                                                                             | None                                               | Stable or improved                                                                   | Stable or improved                    | Unequivocal progressive disease* |
| New lesion(s)†                                                                                                                                                                 | None                                               | None                                                                                 | None                                  | Present"                         |
| Corticosteroids                                                                                                                                                                | Stable or decreased                                | Stable or decreased                                                                  | Not applicable:                       |                                  |
| None                                                                                                                                                                           |                                                    |                                                                                      |                                       |                                  |
| Clinical status                                                                                                                                                                | Stable or improved                                 | Stable or improved                                                                   | Stable or improved                    | Worse'                           |
| Requirement for response    All                                                                                                                                                | All                                                | All                                                                                  | Any ‡                                 |                                  |
| *Progression occus when this criterion is met. I'A new lesion is one that not present on prior scans and is visible in minimum two projections. If a new lesion is equivocal,  |                                                    |                                                                                      |                                       |                                  |
| continued therapy can be corridered, and follow-up assessment will clarify if the new lesion is new disease. If repeat scans confirm there is definitely a new lision, progres |                                                    |                                                                                      |                                       |                                  |
| the initial scanshowing the new lesson. For immunotherapy-based approaches, new lesions alone to do not define progression. Increase In corticosterolds alone will not be take |                                                    |                                                                                      |                                       |                                  |
| progression in the absence of persistent clinical deterioration.                                                                                                               |                                                    |                                                                                      |                                       |                                  |
| Toble 2: Summary of the response criteria for CNS metastases proposed by RANO-BM                                                                                               |                                                    |                                                                                      |                                       |                                  |  
Lin NU, Lee EQ, Aoyama H, et al. Response assessment criteria for brain metastases: proposal from the RANO group. Lancet Oncology 2015; 16 (6): E270-E8