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9.0 other therapy

9.1 Permitted Supportive Therapy All supportive therapy for optimal medical care will be given during the study period at the discretion of the attending physician(s) within the parameters of the protocol and documented on each site's source documents as concomitant medication.  
9.1.1 Anticonvulsants: No limitations in type of anti-convulsants used.  
9.1.2 Antiemetics: Prophylactic medications are not needed. For nausea, anti-emetics may be used based on the treating physician's preference.  
9.1.3 Anticoagulants: These may be used for the treatment of DVT's or PE's if they occur. Preference should be given for LMW Heparins, but Coumadin is allowed.  
9.1.4 Antidiarrheals: Used as needed per treating physician's discretion.  
9.1.5 Analgesics: Used as needed per treating physician's discretion.  
9.1.6 Hematopoietic Growth Factors: Used as needed per treating physician's discretion.  
9.1.7 Herbal products: Are allowed but patients should inform treating physician and these should be recorded.  
9.1.8 Nutritional supplementation: Are allowed but patients should inform treating physician and these should be recorded.  
10.2.1 Tissue (For collection, processing and shipping information please see Specimen Collection Summary below and Appendix III.)
One H&E stained slide (slide can be a duplicate cut stained H&E of the diagnostic slide
(block); it does not have to be the diagnostic slide itself)
A corresponding paraffin-embedded tissue block of the tumor (the block must match the H&E
being submitted) or a 2 mm diameter core of tumor tissue, punched from the tissue block containing the tumor with a punch tool and submitted in a plastic tube labeled with the surgical pathology number. **Note:** A kit with the punch, tube, and instructions can be obtained free of charge from the NRG Oncology Biospecimen Bank. Block or core must be clearly labeled with the pathology identification number and block number that corresponds to the Pathology Report (see below).  
o The submitted material must be from malignant tumor, not necrotic or fibrotic tissue.  
If the submitted material is reviewed and is not tumor, the site may be assessed a protocol violation.  
A Pathology Report documenting that the submitted block or core contains tumor. The report must include the NRG Oncology protocol number and patient's case number. The patient's name and/or other identifying information should be removed from the report. The surgical pathology numbers and information must NOT be removed from the report.  
A Specimen Transmittal (ST) Form clearly stating that tissue is being submitted for NRG
Oncology Biospecimen Bank; if for translational research, this should be stated on the form. The form must include the NRG Oncology protocol number and patient's case number.  
10.2.2 Blood and Urine (For collection, processing and shipping information please see Specimen Collection Summary below and Appendix III.)
A Specimen Transmittal Form documenting the type of Biospecimen, the date of collection of the biospecimen; the NRG Oncology protocol number, the patient's case number, time point of study, and method of storage, for example, stored at -80 C, must be included.  
10.2.3 Storage Conditions for All Specimens Store frozen specimens at -80 C (-70C to -90C) until ready to ship. If a -80C Freezer is not available:
Samples can be stored short term in a -20 C freezer (non-frost free preferred) for up to one week (please ship out Monday-Wednesday only; Canada: Monday-Tuesday).  
OR:
Samples can be stored in plenty of dry ice for up to one week, replenishing daily (ship out Monday-Wednesday only; Canada: Monday-Tuesday).  
OR:
Samples can be stored in liquid nitrogen vapor phase (ship out Monday-Wednesday only; Canada: Monday-Tuesday).  
Please indicate on the ST Form the storage conditions used and time stored.  
| Specimens for Banking/Translational Research                                                                                                                                        |                                                                                                                                                                       |                                                                                                                 |                                                                                                                         |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------|
| Specimens taken from                                                                                                                                                                | Collected when:                                                                                                                                                       | Submitted as:                                                                                                   | Shipped:                                                                                                                |
| patient:                                                                                                                                                                            |                                                                                                                                                                       |                                                                                                                 |                                                                                                                         |
| Representative H&E  stained slides of the  primary tumor                                                                                                                            | Pre-treatment                                                                                                                                                         | H&E stained slide                                                                                               | Slide shipped ambient                                                                                                   |
| A paraffin-embedded tissue  block of the primary tumor  taken before initiation of  treatment or a 2 mm  diameter core of tissue,  punched from the tissue  block with a punch tool | Pre-treatment                                                                                                                                                         | Paraffin-embedded  tissue block or punch  biopsy (must match the  H&E slide being  submitted)                   | Block or punch shipped  ambient. Cold packs are  recommended during  warm weather to help  prevent blocks from  melting |
| SERUM: 5-10 mL of whole  blood in 1 red-top tube and  centrifuge                                                                                                                    | (1) Pre-treatment                                                                                                                                                     | Frozen serum samples  containing 0.5 mL per  aliquot in 1 mL cryovials  (five to eight)                         | Serum sent frozen on dry  ice via overnight carrier                                                                     |
| (2) 8 weeks after  treatment initiation                                                                                                                                             |                                                                                                                                                                       |                                                                                                                 |                                                                                                                         |
| PLASMA: 5-10 mL of  anticoagulated whole blood  in EDTA tube #1 (purple/  lavender top) and  centrifuge                                                                             | (1) Pre-treatment                                                                                                                                                     | Frozen plasma samples  containing 0.5 mL per  aliquot in 1 mL cryovials  (five to eight)                        | Plasma sent frozen on dry  ice via overnight carrier                                                                    |
| (2) 8 weeks after  treatment initiation                                                                                                                                             |                                                                                                                                                                       |                                                                                                                 |                                                                                                                         |
| Whole blood for DNA: 5-10  mL of anticoagulated whole  blood in EDTA tube #2  (purple/ lavender top) and  mix                                                                       | (1) Pre-treatment  (Note: if site missed this  timepoint they may collect  whole blood at any  timepoint or follow up visit,  but this must be noted on  the ST Form) | Frozen whole blood  samples containing 1.0  mL per aliquot in 1 mL  cryovials (three-five)                      | Whole blood sent frozen  on dry ice via overnight  carrier                                                              |
| 10-20 mL clean-catch urine                                                                                                                                                          | (1) Pre-treatment                                                                                                                                                     | Two 5-10 mL urine  aliquots in 2 sterile 15 ml  polypropylene centrifuge  tubes. Store frozen at  -20 or 80 C | Urine sent frozen on dry  ice via overnight carrier                                                                     |
| (2) 8 weeks after  treatment initiation                                                                                                                                             |                                                                                                                                                                       |                                                                                                                 |                                                                                                                         |  
10.2.5 Submit materials for Specimen Banking, Translational Research as follows:
U. S. Postal Service Mailing Address: For Non-frozen Specimens Only NRG Oncology Biospecimen Bank University of California San Francisco UCSF Box 1800 2340 Sutter Street, Room S341 San Francisco, CA 94143-1800 Courier Address (FedEx, UPS, etc.): For Trackable FFPE and ALL Frozen Specimens NRG Oncology Biospecimen Bank University of California San Francisco 2340 Sutter Street, Room S341 San Francisco, CA 94115 Questions: 415-476-7864/FAX 415-476-5271; RTOG@ucsf.edu  
10.3 Reimbursement (4/15/14)
Please note that with the start of the new NCI National Clinical Trials Network (NCTN) Program, NCI funds for reimbursement for protocol-specified biospecimen materials will be distributed per the requirements/methods specified by the new NCTN Program. This information will be made available with the other registration materials in the Oncology Patient Enrollment Network (OPEN)
portal system. OPEN will serve as the registration system for all patient enrollments onto NCIsponsored NCTN trials, including this study, which will be transitioned into the new Program from the NCI-sponsored Cooperative Group Clinical Trials Program.  
10.4 Confidentiality/Storage
(See the Patient Tissue Consent Frequently Asked Questions, http://www.rtog.org/Researchers/BiospecimenResource/BiospecimenResourceFAQs.aspx for further details.)
10.4.1 Upon receipt, the specimen is labeled with the NRG Oncology protocol number and the patient's case number only. The NRG Oncology Biospecimen Bank database only includes the following information: the number of specimens received, the date the specimens were received, documentation of material sent to a qualified investigator, type of material sent, and the date the specimens were sent to the investigator. No clinical information is kept in the database.  
10.4.2 Specimens for tissue banking will be stored for an indefinite period of time. Specimens for the translational research component of this protocol will be retained until the study is terminated, unless the patient has consented to storage for future studies. If at any time the patient withdraws consent to store and use specimens, the material will be returned to the institution that submitted it.
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11.0 patient assessments 11.1 study

11.1.1 Urine should be assessed via dipstick or random urine creatinine and protein before each dose of bevacizumab. If dipstick is > 2 + then a UPC ratio should be performed.  
11.1.2 Vital signs should be assessed before each dose of bevacizumab (on treatment day) to make sure there is no increase in blood pressure.  
11.1.3 MR diffusion and perfusion imaging at 8 and 24 weeks post-treatment start is optional but strongly recommended. Repeat MR diffusion and perfusion imaging at progression is optional but strongly recommended as further confirmation of true tumor progression and not radiation necrosis.  
11.2 Measurement of Response The primary measure of response will be by serial measures of the product of the two largest cross-sectional diameters using MacDonald Criteria. Response will also be evaluated in this study using the international criteria proposed by the Response Evaluation Criteria in Solid Tumors (RECIST) Committee [JNCI 92(3): 205-216, 2000], see Section 11.4.  
11.2.1 Complete Response (CR)
Requires all of the following: complete disappearance of the measurable enhancing lesion sustained for at least 4 weeks; no new lesions; and no corticosteroids.  
11.2.2 Partial Response (PR)
Requires all of the following: ≥ 50% decrease compared with baseline in the sum of products of perpendicular diameters of the measurable enhancing lesion sustained for at least 4 weeks; no new lesions; and stable or reduced corticosteroid dose.  
11.2.3 Stable Disease (SD)
Requires all of the following: does not qualify for complete response, partial response, or progression and is receiving stable or decreasing doses of steroids. This will not require a confirmatory scan.  
11.2.4 Progression (P)
Defined by any of the following: ≥ 25% increase in sum of the products of perpendicular diameters of enhancing lesions provided that the patient has not had his/her dose of steroids decreased since the last evaluation period; and any new lesions. This will not require a confirmatory scan. A concomitant decrease in steroid dose will rule out a progression designation during the initial 12 weeks after completion of RT.  
11.2.5 Pseudo-progression Due to possible radiation effects using large dose per fraction, the initial scan 6 weeks following RT should NOT be used to declare progression. In the absence of neurologic worsening OR a new distant area of tumor, the initial post-radiation scan should not be used to declare progression. Progressive worsening on subsequent imaging studies usually distinguishes true progression from pseudo-progression.  
11.2.6 If true progression is determined by subsequent imaging, then the date of progression returns to the earlier date with increasing mass.  
11.3 Criteria for Evaluation of Therapy Effectiveness 11.3.1 Tumor response and regrowth can frequently be difficult to measure directly. Serial neurological exams and CT/MRI scans may provide a guide to the actual course. Time interval to progression will be measured from registration until deterioration is documented by the individual investigator using these guides. The patient should consistently be followed with the same diagnostic imaging study (CT or MRI). (**NOTE**: CT option ONLY for patients unable to undergo MR imaging because of non-compatible devices.)
11.3.2 Overall survival will be measured from registration until death. Progression-free survival will be measured from registration until the first occurrence of progression or death.  
11.3.3 The quality of survival will be measured by neurological functional classification and performance status.  
11.3.4 Toxicities will be measured using the CTCAE criteria, version 4.0.
(page 5 of 5)
11.4 rano response criteria

Efficacy determinations using RANO response criteria (Wen 2010) also will be obtained.  
11.4.1 Response Criteria Radiographic response should be determined in comparison to the tumor measurement obtained at pretreatment baseline for determination of response, and the smallest tumor measurement at either pretreatment baseline or following initiation of therapy for determination of progression. The tables below outline the criteria for radiographic changes following therapy and summarize RANO response criteria. In the event that the radiographic changes are equivocal and it is unclear whether the patient is stable or has developed progressive disease, it is permissible to continue treatment and observe the patient closely, for example at 4 weekly intervals. If subsequent imaging studies demonstrate that progression has occurred, the date of progression should be the scan at which this issue was first raised.  
All measurable and non-measurable lesions should be assessed using the same techniques as baseline. Ideally patients should be imaged on the same MRI, or least with the same magnet strength, for the duration of the study to reduce difficulties in interpreting changes.  
All measurable and non-measurable lesions must be assessed using the same techniques as baseline.
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criteria for response assessment incorporating

Complete Response:
Requires all of the following:
a) Complete disappearance of all enhancing measurable and non-measurable disease sustained for at least 4 weeks.  
b) No new lesions. c) Stable or improved non-enhancing (T2/FLAIR) lesions.  
d) Patients must be off corticosteroids (or on physiologic replacement doses only).  
e) Stable or improved clinically.  
Note: Patients with non-measurable disease only cannot have a complete response. The best response possible is stable disease. Partial Response: Requires all of the following:
a) Greater than or equal to 50% decrease compared to baseline in the sum of products of perpendicular diameters of all measurable enhancing lesions sustained for at least 4 weeks.  
b) No progression of non-measurable disease. c) No new lesions. d) Stable or improved non-enhancing (T2/FLAIR) lesions on same or lower dose of corticosteroids compared to baseline scan.  
e) The corticosteroid dose at the time of the scan evaluation should be no greater than the dose at time of baseline scan.  
f) Stable or improved clinically.  
Note: Patients with non-measurable disease only cannot have a partial response. The best response possible is stable disease. Stable Disease: Requires all of the following:
a) Does not qualify for complete response, partial response, or progression. b) Stable non-enhancing (T2/FLAIR) lesions on same or lower dose of corticosteroids compared to baseline scan. In the event that the corticosteroid dose was increased for new symptoms and signs without confirmation of disease progression on neuroimaging, and subsequent follow-up imaging shows that this increase in corticosteroids was required because of disease progression, the last scan considered to show stable disease will be the scan obtained when the corticosteroid dose was equivalent to the baseline dose.  
c) Stable clinically.  
Progression: Defined by any of the following:
a) Greater than > 25% increase in sum of the products of perpendicular diameters of enhancing lesions compared to the smallest tumor measurement obtained either at baseline (if no decrease) or best response, on stable or increasing doses of corticosteroids.*
b) Significant increase in T2/FLAIR non-enhancing lesion on stable or increasing doses of corticosteroids compared to baseline scan or best response following initiation of therapy,* not due to co-morbid events (e.g. radiation therapy, demyelination, ischemic injury, infection, seizures, post-operative changes, or other treatment effects).  
c) Any new lesion. d) Clear clinical deterioration not attributable to other causes apart from the tumor (e.g. seizures, medication side effects, complications of therapy, cerebrovascular events, infection, etc.) or changes in corticosteroid dose.  
e) Failure to return for evaluation due to death or deteriorating condition. f) Clear progression of non-measurable disease.  
*Stable doses of corticosteroids include patients not on corticosteroids  
| Summary of RANO Response Criteria   |             |             |                   |          |
|-------------------------------------|-------------|-------------|-------------------|----------|
| CR                                  | PR          | SD          | PD#               |          |
| Criterion                           |             |             |                   |          |
| T1-Gd +                             | None        | ≥ 50%      | < 50%  - < 25%  | ≥ 25% * |
| T2/FLAIR                            | Stable or  | Stable or  | Stable or        | *       |
| New Lesion                          | None        | None        | None              | Present* |
| Corticosteroids                     | None        | Stable or  | Stable or        | NA**     |
| Clinical Status                     | Stable or  | Stable or  | Stable or        | *       |
| Requirement for  response           | All         | all         | All               | Any*     |  
Abbreviations: RANO, Response Assessment in Neuro-Oncology; CR=complete response; PR=partial response; SD=stable disease; PD=progressive disease; FLAIR, fluid-attenuated inversion recovery;  =
decrease;  = increase
\# Progression occurs when any of the criteria with * is present.  
NA**: Increase in corticosteroids alone will not be taken into account in determining progression in the absence of persistent clinical deterioration.  
11.4.2 Efficacy Assessments
*Progression free survival (PFS)* will be defined from the time the patient enters the study until there is clinical or radiographic evidence of progressive disease (see definition of PD above).  
*Overall survival (OS)* will be defined from the time the patient enters the study to the date of death. Patients not known to have died will be censored for survival as of the last date known alive.  
*PFS and overall survival* will be estimated by using the Kaplan-Meier method.  
The primary endpoint for this study is overall survival.  
11.5 Criteria for Discontinuation of Protocol Treatment Progression of disease (See Section 11.2)
Unacceptable toxicity to the patient (at the discretion of the treating physician) - Reasons for removal must be clearly documented on the appropriate case report form/flow sheet, and NRG Oncology data management must be notified.
If protocol treatment is discontinued, follow up and data collection will continue as specified in the protocol.
12.0 data collection data should
if a data form is

Patients will be identified by initials only (first middle last); if there is no middle initial, a hyphen will be used (first-last). Last names with apostrophes will be identified by the first letter of the last name.  
12.1 Summary of Data Submission (10/9/14)  
Item Due  
Demographic Form (A5) **Within 2 weeks after registration**  
Initial Evaluation Form **(I1)** Pathology Report **(P1) to mail a copy of the**  
report to NRG Oncology  
Treatment Form **(TF)** At completion of each 8-week period (4 doses of bevacizumab per TF form if no protocol modification)  
Follow-up Form **(F1)** At the time of progression and at death. AFTER  
progression or treatment discontinuation: every 2 months for 1 year, then every 6 months for 1 year, then annually.  
MRI scans and Reports (MR, ME) Pre-study (complete series) (see Section 12.2)  
12.2 Summary of Dosimetry Digital Data Submission (Submit to TRIAD; see Section 5.5 for account access and installation instructions) (4/15/14)  
Preliminary Dosimetry Information Digital Data Submission - Treatment Within 1 week of start of RT
Digital data submission includes the following, all in DICOM format:
CT data, critical normal structures, all GTV, CTV, and PTV contours Digital beam geometry beam sets Single dose file for concurrently treated beams Digital DVH data for all required critical normal structures, GTV, CTV, and PTVs for total dose plan All required structures **MUST** be labeled per Table in Section 6.5.1.  
| Progression to TRIAD (see Section 12.2)   |
|-------------------------------------------|  
The "RTOG 1205 Datasheet" is available in the Forms section of the of the NRG Oncology/RTOG web site, http://www.rtog.org/ClinicalTrials/ProtocolTable/StudyDetails.aspx?study=1 205. Submit via TRIAD with the digital data listed above.  
Upon submission of any digital data via TRIAD, complete an online digital data submission form (DDSI) located on the NRG Oncology/RTOG web site at http://www.rtog.org/ClinicalTrials/ProtocolTable/StudyDetails.aspx?study=1205 Item Due
12.0 data collection data should
mri submission

Pre-study MRI (the Scan used to delineate the target volumes for planning. Submit the entire series via TRIAD
Final Dosimetry Information Within 1 week of RT end Radiotherapy Form **(T1) via the web** Daily Treatment Record (T5) on paper to HQ
NOTE: ALL SIMULATION AND PORTAL FILMS AND/OR DIGITAL FILM IMAGES WILL
BE KEPT BY THE INSTITUTION AND ONLY SUBMITTED IF REQUESTED.
12.0 data collection data should
13.0 statistical considerations

13.1 Study Endpoints (11/20/13) 13.1.1 Primary Endpoint Overall Survival, defined as the interval from randomization to death due to any cause.  
13.1.2 Secondary Endpoints Objective response 6-month progression-free survival Progression-free survival, defined as the interval from randomization to progression or death, whichever occurs first Treatment adverse events Grade 3+ acute or delayed CNS toxicity 13.2 Sample Size and Power Justification The primary objective of this study is to determine whether re-irradiation plus bevacizumab
(experimental arm) will improve the overall survival compared to bevacizumab alone (control arm). The design will be a randomized phase II screening trial as proposed by Rubinstein et al (2005). The randomization of experimental and control arms is 1:1. The null hypothesis is that the overall survival for both arms is 9 months, based on data from RTOG 0625/ACRIN 6677 and the BRAIN study. The alternative hypothesis is that patients receiving IMRT plus bevacizumab will have an improvement in overall survival to 13 months, based on the Gutin data. With 160 eligible subjects, there will be 80% power to detect a 31% reduction in the hazard ratio to 0.69 at the significance level of 0.10 (one-sided). Analysis will be performed when 135 events (deaths) are reported, expected to occur 16 to 21 months after trial closure. Guarding against up to a 10%
ineligibility rate, **the final target accrual for this study will be 178 cases**.  
13.3 Patient Accrual Based on the monthly accrual for a prior RTOG recurrent glioblastoma phase II study (RTOG
0625), this study is projected to accrue 10 cases/month. Therefore, the target accrual should be completed within 22 months of study activation, allowing slow accrual in the first 6 months. If the average monthly accrual (6 months after trial activation) is less than 5 patients, the study will be re-evaluated with respect to feasibility.
12.0 data collection data should
13.4 patient stratification and randomization

A randomized phase II trial will be conducted for bevacizumab-naïve recurrent GBM patients. Age and Karnofsky performance status (KPS) are demonstrated to be prognostic of survival for recurrent GBM (Carson 2007). In addition, previous RTOG recurrent GBM trials (RTOG 0625, 0627) show that age, KPS, and resection type have prognostic value in overall survival.  
Therefore, for this NRG Oncology recurrent GBM study, patients will be stratified by age (<50 vs. ≥50), KPS (60 vs. 70-80 vs.90-100), and resection type (yes vs. no/biopsy only), and then randomized to either the control arm or experimental arm in a permuted block design using the method described by Zelen (1974).
12.0 data collection data should
13.5 analyses plans

13.5.1 Statistical Methods Overall and progression-free survival rates will be estimated using the Kaplan-Meier method (Kaplan 1958), and differences between treatment arms will be tested in the log-rank test (Mantel 1966). Overall survival will be measured from the date of randomization to the date of death or, otherwise, the last follow-up date on which the patient was reported alive. Progression-free survival will be measured from the date of randomization to the date of first progression or death or, otherwise, the last follow-up date on which the patient was reported alive. Objective response rate, grade 3+ toxicities rate, acute or delayed CNS toxicity rate, and 6-month progression-free survival rate, will be estimated using an exact binomial distribution together with 95% confidence interval. The difference between the 2 groups will be tested using a chi square test. In terms of grade 3+ toxicity and acute/delayed toxicity, all patients receiving any protocol treatment (bevacizumab or combination of RT with bevacizumab) should be included. For the statistical comparison of the primary endpoint (overall survival), the type I error is set as 0.1 (1sided), while for all the secondary endpoints, the statistical comparison between groups will serve as an exploratory purpose without multiple comparison adjustment, with type I error of 0.05 (2sided) for each comparison. Multivariate analyses with the Cox proportional hazard model (Cox 1972) for overall and progression-free survival will be performed with the stratification variables as fixed variables to assess the treatment effect adjusting patient-specific risk factors. The covariates evaluated for the multivariate models are assigned protocol treatment, tumor volume at the time of treatment, time since original diagnosis, stratification factors, and other prognostic factors. Proportional hazard assumptions will be checked using different graphical or time-varying coefficients testing methods. If the data clearly do not follow proportional hazards, other statistical models will be used to fit the data instead. Possible alternatives are to use the stratified Cox proportional hazard model, accelerated failure model, or partition the time axis into sections where proportional hazard assumption holds.  
13.5.2 Interim Toxicity and Futility Analysis Due to a possible increased incidence of grade 3+ CNS toxicity (possibly, probably, or definitely related to treatment) occurring during the first 6 months post RT in the experimental arm, a special interim analysis will be performed after the first 20 patients enrolled to the experimental arm have a minimum 6-month follow-up. If the incidence of grade 3+ CNS toxicity (based on RTOG 0625) is 20% higher, or 30%, in the experimental arm, the trial will be halted due to lack of safety. The interim toxicity analysis results will be reported to the NRG Oncology DMC. The DMC will then make a recommendation about the trial to the NRG Oncology Group Co-Chairs. The interim futility analysis will be performed when 50% of the required events (68 deaths) are reported. The analysis will be performed on an intent-to-treat basis, with all eligible cases included in the treatment arm to which they were randomized regardless of what treatment the patients actually received. The primary endpoint, overall survival, will be tested. The futility will be tested using the conditional probability under the alterative hypothesis of detecting the hypothesized treatment benefit favoring the experimental arm at the final analysis given the observed data. The results from testing the treatment futility will be reported to the NRG Oncology DMC. The responsible statistician may recommend early reporting of the results and/or stopping accrual (if applicable) of the trial if the conditional power is less than 0.1. The accrual rate, treatment compliance, safety of the treatments, and the importance of the study are also considered in making such a recommendation. The results will be reported to the RTOG DMC with the treatment blinded. The DMC will then make a recommendation about the trial to the NRG
Oncology Group Co-Chair.  
13.5.3 Interim Analysis to Monitor Study Progress Interim reports with statistical analyses are prepared every 6 months until the initial manuscript reporting the treatment results has been submitted. The reports contain:
The patient accrual rate with a projected accrual completion date Accrual by institution The pretreatment characteristics of accrued patients The frequency and severity of toxicities The results of any completed study chair modality reviews The interim reports will not contain the results from the treatment comparisons with respect to the efficacy endpoints (overall survival, progression-free survival, treatment response). The NRG Oncology DMC will review the accrual to the study and the rate of adverse events on the study at least twice per year until the initial results of the study have been presented to the scientific community.  
13.5.4 Significance Testing for Final Analysis The final analysis will be performed on an intent-to-treat basis, such that all eligible cases on the study will be included in the arm to which they were randomized regardless of what treatment the patients actually received. The analysis to report the final results of treatment comparison between the experimental arm and the control arm will be undertaken when 135 events (deaths)
have been reported. A one-sided log-rank test will be performed to test the difference in overall survival between the two treatment arms. If the p value is less than protocol-specified 0.10 (one sided), the study statistician will reject the null hypothesis and conclude that the experimental arm
(re-radiation plus bevacizumab) is promising in prolonging overall survival, therefore supporting the development of a confirmatory phase III trial comparing this regimen to the current standard treatment. All information reported in the interim analyses to monitor the study progress (above)
and the treatment compliance with respect to re-radiation and chemotherapy will also be included in the final report.  
13.5.5 This study will be monitored by the Clinical Data Update System (CDUS) version 3.0. Cumulative CDUS data will be submitted quarterly by electronic means. Reports are due January 31, April 30, July 31, and October 31.  
13.6 Gender and Minorities In conformance with the National Institutes of Health (NIH) Revitalization Act of 1993 with regard to inclusion of women and minorities in clinical research, both men and women of all races and ethnic groups are eligible for this study. We will also analyze treatment differences by gender, race, and ethnicity. The following table lists the projected accrual for each racial and ethnic group based upon previous RTOG recurrent GBM trials.  
| Projected Distribution of Gender and Minorities  Gender   |         |       |       |
|-----------------------------------------------------------|---------|-------|-------|
| Ethnic Category                                           | Females | Males | Total |
| Hispanic or Latino                                        | 10      | 13    | 23    |
| Not Hispanic or Latino                                    | 67      | 88    | 155   |
| Ethnic Category: Total of all subjects                    | 77      | 101   | 178   |
| Gender                                                    |         |       |       |
| Racial Category                                           | Females | Males | Total |
| American Indian or Alaskan Native                         | 1       | 1     | 2     |
| Asian                                                     | 3       | 3     | 6     |
| Black or African American                                 | 6       | 6     | 12    |  
| Gender                                    |         |       |       |
|-------------------------------------------|---------|-------|-------|
| Ethnic Category                           | Females | Males | Total |
| Native Hawaiian or other Pacific Islander | 1       | 1     | 2     |
| White                                     | 66      | 90    | 156   |
| Racial Category: Total of all subjects    | 77      | 101   | 178   |
12.0 data collection data should
references

Brandes AA. State-of-the-art treatment of high-grade brain tumors. *Semin Oncol*. 2003;30:4-9.  
Brenner DJ, Martel MK, Hall EJ. Fractionated regimens for stereotactic radiotherapy of recurrent tumors in the brain. *Int J Radiat Oncol Biol Phys*. 1997;21:819-824.  
Carson KA, Grossman SA, Fisher JD, et al. Prognostic factors for survival in adult patients with recurrent glioma enrolled onto the new approaches to brain tumor therapy CNS consortium phase I and II clinical trials. *J Clin Oncol.* 2007;25:2601-2606.  
Cho KH, Hall WA, Gerbi BJ, et al. Single dose versus fractionated stereotactic radiotherapy for recurrent high-grade gliomas. *Int J Radiat Oncol Biol Phys*. 1999;45:1133-1141.  
Combs SE, Thilmann C, Edler L et al. Efficacy of fractionated stereotactic reirradiation in recurrent gliomas: Long-term results in 172 patients treated in a single institution*. J Clin Oncol*. 2005;23:8863-8869.  
Cox DR. Regression models and life-tables. *J Royal Statistical Society*. 1972 (Series B);34:187-202.  
Cuneo K, Vredenburgh J, Sampson JH et al, Safety and efficacy of stereotactic radiosurgery and adjuvant bevacizumab in patients with recurrent malignant gliomas, *Int J Radiat Oncol Biol Phys.* epub April 12, 2011.  
Ellis LM, Hicklin DJ. VEGF-targeted therapy: mechanisms of anti-tumour activity. *Nat Rev Cancer*.  
2008;8:579-591. Fogh SE, Andrews DW, Glass J, et al. Hypofractionated stereotactic radiation therapy: an effective therapy for recurrent high-grade gliomas. *J Clin Oncol*. 2010;28:3048-3053.  
Friedman HS, Prados M, Wen P, et al. Bevacizumab alone and in combination with irinotecan in recurrent glioblastoma. *J Clin Oncol.* 2009;27:4733-3740.  
Gilbert MR, Wang M, Aldape K, et al. RTOG 0625: a phase II study of bevacizumab with irinotecan in recurrent glioblastoma. *J Clin Oncol*. 2009:27(15S):2011. Gorski DH, Beckett MA, Jaskowiak NT, et al. Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation. *Cancer Res* 1999;59:3374-3378.  
Grosu AL, Weber WA, Franz M, et al: Reirradiation of recurrent high-grade gliomas using amino acid PET (SPECT)/CT/MRI image fusion to determine gross tumor volume for stereotactic fractionated radiotherapy. *Int J Radiat Oncol Biol Phy*s. 2005;63:511-519.  
Gutin PH, Iwamoto FM, Beal K, et al. Safety and efficacy of bevacizumab with hypofractionated stereotactic irradiation for recurrent malignant gliomas. *Int J Radiat Oncol Biol Phys*. 2009;75:156-163.  
Hasselbalch B, Lassen U, Hansen S, et al. Cetuximab, bevacizumab, and irinotecan for patients with primary glioblastoma and progression after radiation therapy and temozolomide: a phase II trial. *Neuro* Oncol. 2010;12:508-516.  
Hovinga KE, Shimizu F, Wang R et al. Inhibition of notch signaling in glioblastoma targets cancer stem cells via an endothelial cell intermediate. *Stem Cells*. 2010;28:1019-1029.  
Hudes RS, Corn BW, Werner-Wasik M, et al. A phase I dose escalation study of hypofractionated stereotactic radiotherapy as salvage therapy for persistent or recurrent malignant glioma. Int J Radiat Oncol Biol Phys.1999;43:293-298.  
Jain RK, di Tomaso E, Duda DG, et al. Angiogenesis in brain tumours. *Nat Rev Neurosci*. 2007;8:610622.  
Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. *J Am Stat Assoc*.  
1958;53:457-481.  
Kim HK, Thornton AF, Greenberg HS, et al. Results of re-irradiation of primary intracranial neoplasms with three-dimensional conformal therapy. *Am J Clin Oncol*.1997;20:358-363.  
Kreisl TN, Kim L, Moore K, et al. Phase II trial of single-agent bevacizumab followed by bevacizumab plus irinotecan at tumor progression in recurrent glioblastoma. *J Clin Oncol.* 2009;27:4733-4740.  
Laing RW, Warrington AP, Graham J, et al. Efficacy and toxicity of fractionated stereotactic radiotherapy in the treatment of recurrent gliomas (phase I/II study). *Radiother Oncol*. 1993;27:22-29.  
Lai A, Tran A, Nghiemphu P, et al. Phase II study of bevacizumab plus temozolomide during and after radiation therapy for patients with newly diagnosed glioblastoma multiforme. *J Clin Oncol*. 2010;29:142148. Lederman G, Wronski M, Arbit E, et al. Treatment of recurrent glioblastoma multiforme using fractionated stereotactic radiosurgery and concurrent paclitaxel. *Am J Clin Oncol*. 2000;23:155-159.  
Lee C-G, Heijn M, di Tomaso E, et al. Anti-vascular endothelial growth factor treatment augments tumor radiation response under normoxic or hypoxic conditions. *Cancer Res*. 2000;60:5565-5570 Levin VA, Bidaut L, Hou P, et al. Randomized double-blind placebo-controlled trial of bevacizumab therapy for radiation necrosis of the central nervous system. *Int J Radiat Oncol Biol Phys*. 2011;79:14871495.  
Mantel N. Evaluation of survival data and two new rank order statistics arising in its consideration. *Cancer* Chem Rep. 1966;5:163-170. Mayer R, Sminia P. Reirradiation tolerance of the human brain. *Int J Radiat Oncol Biol Phys*.  
2008;70:1350-1360. Nieder C, Astner ST, Mehta MP et al. Improvement, clinical course, and quality of life after palliative radiotherapy for recurrent glioblastoma. *Am J Clin Oncol*. 2008;31:300-305.  
Niyazi M, Ganswindt U, Birgit S et al. Irradiation and bevacizumab in high-grade glioma retreatment settings*. Int J Radiat Oncol Biol Phys.* October 27, 2010. Epub ahead of print.  
Provencio M, Sanchez A, Garrido P, et al.New molecular targeted therapies integrated with radiation therapy in lung cancer. *Clin Lung Cancer*. 2010;11:91-97.  
Quant EC, Norden AD, Drappatz J, et al. Role of a second chemotherapy in recurrent malignant glioma patients who progress on bevacizumab. *Neuro Oncol*. 2009;11:550-555.  
Reardon D, Desjardins A, Vredenburgh JJ, et al. Metronomic chemotherapy with daily, oral etoposide plus bevacizumab for recurrent malignant glioma: phase II study. *Br J Cancer*. 2009;101:1986-1994.  
Rubinstein LV, Korn EL, Freidlin B, et al. Design issues of randomized phase II trials and a proposal for phase II screening trials. *J Clin Oncol*. 2005;23:7199-7206.  
Sathornsumetee S, Desjardins A, Vredenburgh JJ, et al. Phase II study of bevacizumab and erlotinib in patients with recurrent malignant glioma. *Neuro Oncol*. 2010;12:1300-1310.  
Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. *New Engl J Med*. 2005;352:987-996.  
VanderSpek L, Fisher B, Bauman G, et al. 3D conformal radiotherapy and cisplatin for recurrent malignant glioma. *Can J Neurol Sci*. 2008;35:57-64.  
Verhoeff JJ, Lavini C, van Linde ME, et al. Bevacizumab and dose-intense temozolomide in recurrent high grade glioma. *Ann Oncol*. 2010;21:1723-1727.  
Vredenburgh JJ, Desjardins A, Herndon JE, 2nd, et al. Phase II trial of bevacizumab and irinotecan in recurrent malignant glioma. *Clin Cancer Res*. 2007;13:1253-1259a.  
Vredenburgh JJ, Desjardins A, Herndon JE, 2nd, et al. Bevacizumab plus irinotecan in recurrent glioblastoma multiforme. *J Clin Oncol*. 2007;25:4722-4729b.  
Vredenburgh JJ, Cloughesy T, Samant M, et al, Corticosteroid use in patients with glioblastoma at first or second relapse treated with bevacizumab in the BRAIN study. *Oncologist.* 2010;15:1329-1334.  
Vredenburgh JJ, Desjardins A, Reardon DA, et al. The addition of bevacizumab to standard radiation therapy and temozolomide followed by bevacizumab, temozolomide, and irinotecan for newly diagnosed glioblastoma. *Clin Cancer Res.* 2011:17:4119-4124.  
Wong ET, Hess KR, Gleason MJ, et al. Outcomes and prognostic factors in recurrent glioma patients enrolled onto phase II clinical trials. *J Clin Oncol.* 1999;17:2572-2578.  
Zelen M. (1974). The randomization and stratification of patients to clinical trials. *J Chronic Dis*. 27:36575.
appendix i study parameter tables
references

| Pre-Treatment Assessments                                                                                                                                       |                              |                         |               |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------|-------------------------|---------------|
| Assessment                                                                                                                                                      | Prior to registration        | Within 14 days prior to | Pre-Treatment |
| registration                                                                                                                                                    |                              |                         |               |
| Histopath dx  (P1: pathology  report to be  mailed to HQ per  section 12.0)                                                                                     | X                            |                         |               |
| History                                                                                                                                                         | X                            |                         |               |
| Physical                                                                                                                                                        | X                            |                         |               |
| Neurological                                                                                                                                                    |                              | X                       |               |
| exam  Tumor imaging  MRI/CT scan (pts  without recent  resection; see  Section 3.1.1for  details) (MR  diffusion and  perfusion imaging  strongly  recommended) |                              | Within 21 days          |               |
| Tumor imaging  MRI/CT scan (pts  with recent  resection; see  Section 3.1.4 for  details) (MR  diffusion and  perfusion imaging  strongly  recommended)         | And recommended to be within |                         |               |
| 96 h post-surgery                                                                                                                                               |                              |                         |               |
| Karnofsky status                                                                                                                                                | X                            |                         |               |
| CBC w/                                                                                                                                                          |                              | X                       |               |
| differential  Bilirubin                                                                                                                                         |                              | X                       |               |
| SGOT or AST  Serum creatinine                                                                                                                                   | X                            |                         |               |
| Urine protein:  creatinine or urine  dipstick                                                                                                                   |                              | X                       |               |
| Serum pregnancy                                                                                                                                                 |                              | Within 7 days           |               |
| test (if applicable)  MGMT  methylation status  (see Section 4 for  details)                                                                                    |                              | X                       |               |
| Tissue, blood,  urine for banking  (if patient  consents; see  Section 10 for  details)                                                                         |                              | X                       |               |  
| Assessments During Treatment                                              |                                                   |                                        |
|---------------------------------------------------------------------------|---------------------------------------------------|----------------------------------------|
| Assessment                                                                | q8 weeks (± 3 days)                               | At 8 weeks, 24 weeks, and  progression |
| Physical                                                                  | RT arm: weekly during RT  Non-RT arm: q8 weeks    |                                        |
| Weight                                                                    | X (See 7.1.1; done prior to  each drug dose)      |                                        |
| Neurological exam                                                         | X                                                 |                                        |
| Tumor imaging                                                             | X                                                 |                                        |
| MRI/CT scan  Karnofsky status                                             | X                                                 |                                        |
| CBC w/ differential                                                       | X                                                 |                                        |
| Urine protein:  creatinine or urine  dipstick                             | X (See 11.1; done prior to  each drug dose)       |                                        |
| MR diffusion and  perfusion imaging  (optional but strongly  recommended) |                                                   | X                                      |
| Blood pressure                                                            | X (See 11.1; done prior to                        |                                        |
| monitoring                                                                | each drug dose)                                   |                                        |
| AE evaluation                                                             | X                                                 |                                        |
| Blood, urine for  banking  (if patient consents)                          | At 8 weeks after treatment  initiation (± 3 days) |                                        |  
| Follow-Up Assessments   |                                                                                                                                                             |
|-------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Assessments             | After treatment discontinuation or progression: q8 weeks for 1 year, then q6  months for 1 year, then annually (± 2 weeks).  (See Section 12.1 for details) |
| Physical                | X                                                                                                                                                           |
| Karnofsky status        | X                                                                                                                                                           |
| AE evaluation           | X                                                                                                                                                           |
appendix i study parameter tables
appendix ii karnofsky performance scale

100 Normal; no complaints; no evidence of disease  
| 90   | Able to carry on normal activity; minor signs or symptoms of disease          |
|------|-------------------------------------------------------------------------------|
| 80   | Normal activity with effort; some sign or symptoms of disease                 |
| 70   | Cares for self; unable to carry on normal activity or do active work          |
| 60   | Requires occasional assistance, but is able to care for most personal  needs  |
| 50   | Requires considerable assistance and frequent medical care                    |
| 40   | Disabled; requires special care and assistance                                |
| 30   | Severely disabled; hospitalization is indicated, although death not  imminent |  
| 20   | Very sick; hospitalization necessary; active support treatment is  necessary   |
|------|--------------------------------------------------------------------------------|  
10 Moribund; fatal processes progressing rapidly 0 Dead Appendices for NRG Oncology Biospecimen Collection *(as specified by the protocol)*.  
NRG Oncology FFPE Specimen Plug Kit Collection NRG Oncology Blood Collection Kit Instructions NRG Oncology Urine Collection Kit Instructions Shipping Instructions:
U.S. Postal Service Mailing Address: For FFPE or Non-frozen Specimens Only NRG Oncology Biospecimen Bank University of California San Francisco UCSF Box 1800 2340 Sutter Street, Room S341 San Francisco, CA 94143-1800 Courier Address (FedEx, UPS, etc.): For Frozen or Trackable Specimens NRG Oncology Biospecimen Bank University of California San Francisco 2340 Sutter Street, Room S341 San Francisco, CA 94115 Include all NRG Oncology paperwork in pocket of biohazard bag. Check that the Specimen Transmittal (ST) Form has the consent boxes checked off. Check that all samples are labeled with the NRG Oncology study and case number, and include date of collection as well as collection time point (e.g., pretreatment, post-treatment).  
**FFPE Specimens:**
o Slides should be shipped in a plastic slide holder/slide box. Place a small wad of padding in top of the container. If you can hear the slides shaking it is likely that they will break during shipping.  
o FFPE Blocks can be wrapped in paper or placed in a cardboard box with padding. Do not wrap blocks with bubble wrap or gauze. Place padding in top of container so that if you shake the container the blocks are not shaking If you can hear the block shaking it might break during shipping.  
o Slides, Blocks, or Plugs can be shipped ambient or with a cold pack either by United States Postal Service (USPS) to the USPS address (94143) or by Courier to the Street Address
(94115). **Do NOT ship on Dry Ice**.
appendix i study parameter tables
frozen specimens

o Multiple cases may be shipped in the same cooler, but make sure each one is in a separate bag and clearly identified. . If possible keep Serum, Plasma, and Whole Bloods in separate bags.  
o Place specimens and absorbent shipping material in Styrofoam cooler filled with dry ice (at least 7 lbs). There should be plenty of dry ice under and above the specimens. If the volume of specimens is greater than the volume of dry ice then ship in a larger Styrofoam box, or two separate boxes. Any Styrofoam box can be used, as long as it is big enough.  
o Specimens received thawed due to insufficient dry ice or shipping delays will be discarded and the site will be notified.  
o Send frozen specimens on dry ice via overnight courier to the address above. Specimens should only be shipped Monday through Wednesday (Monday-Tuesday for Canada) to prevent thawing due to delivery delays. Saturday or holiday deliveries cannot be accepted. Samples can be stored frozen at -80 C until ready to ship.  
**For Questions regarding collection/shipping please contact the NRG Oncology**
Biospecimen Bank by e-mail: RTOG@ucsf.edu or phone: 415-476-7864 or Fax: 415-4765271.
appendix i study parameter tables
nrg oncology ffpe specimen plug

This Kit allows sub-sampling of an FFPE block for submission to the NRG Oncology Biospecimen  
  
  
  
 Bank. The plug kit contains a shipping tube and a punch tool.  
Step 1 If the block is stored cold, allow it to equilibrate for 30 minutes at room temperature. Place the punch tool on the paraffin block over the selected tumor area. (Ask a pathologist to select area with tumor.) Push the punch into the paraffin block. Twist the punch tool once around to separate the plug from the block. Then pull the punch tool out of the block. The punch should be filled with tissue sample.
appendix i study parameter tables
step 2

Label the punch tool with the proper specimen ID. Include the pathology accession number and block ID. DON'T remove specimen from the punch. Use a separate punch tool for every specimen. Call or e-mail us if you have any questions or need additional specimen plug kits. Step 3 Once punch tool is labeled, place in shipping tube and mail to address below. Please do not mix specimens in the same tube.  
We will remove core specimen from the punch, embed in a paraffin block, and label with specimen ID.  
  
  
*NOTE: If your facility is uncomfortable obtaining the plug but wants to retain the tissue block, please send the entire block to the NRG Oncology Biospecimen Bank and we will sample a plug from the block and return the remaining block to your facility. Please indicate on the submission form the request to perform the plug procedure and return of the block. Ship specimen plug kit, specimen in punch tool, and all paperwork to the address below. For Questions regarding collection/shipping or to order an FFPE Specimen Plug Kit, please contact the NRG Oncology Biospecimen Bank by e-mail: RTOG@ucsf.edu or call 415-476-7864/Fax 415476-5271.  
| U.S. Postal Service Mailing Address:  For Non-frozen Specimens Only                                                                               | Courier Address (FedEx, UPS, etc.):  For Frozen Specimens or Trackable  shipments                                             |
|---------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------|
| NRG Oncology Biospecimen Bank  University of California San Francisco  UCSF Box 1800  2340 Sutter Street, Room S341  San Francisco, CA 94143-1800 | NRG Oncology Biospecimen Bank  University of California San Francisco  2340 Sutter Street, Room S341  San Francisco, CA 94115 |
appendix i study parameter tables
nrg oncology blood collection kit

This Kit is for collection, processing, storage, and shipping of serum, plasma, or whole blood (as
appendix i study parameter tables
specified by the protocol) kit

One Red Top tube for serum (A) One Purple Top EDTA tube for plasma (B)
One Purple Top EDTA tube for Whole Blood (C)
Twenty-one (21) 1 ml cryovials Biohazard bags (3) and Absorbent shipping material (3)
Styrofoam container (inner) and Cardboard shipping (outer) box UN1845 DRY Ice Sticker and UN3373 Biological Substance Category B Stickers Specimen Transmittal (ST) Form and Kit Instructions
appendix i study parameter tables
preparation and processing of serum

Label as many 1ml cryovials (5 to 8) as necessary for the serum collected. Label them with the NRG Oncology study and case number, collection date, time, and time point, and clearly mark cryovials "serum".  
Process:
1. Allow one red top tube to clot for 30 minutes at room temperature.  
2. Spin in a standard clinical centrifuge at ~2500 RPM for 10 minutes at 4C (preferred). If sites are unable to process samples at 4C then spinning at room temperature is acceptable if done within 2 hours of draw but must be noted on the ST Form.  
3. Aliquot 0.5 ml serum into as many cryovials as are necessary for the serum collected (5 to 8)
labeled with NRG Oncology study and case numbers, collection date/time, protocol time-point collected (e.g. pretreatment, post-treatment), and clearly mark specimen as "serum". If an aliquot has less than 0.5 ml in the tube please note the volume on the vial and make a note on the ST Form.  
4. Place cryovials into biohazard bag and immediately freeze at -70 to -90 C, and store frozen until ready to ship. See below for storage conditions.  
5. Store serum at -70 to -90 C until ready to ship on dry ice. See below for storage conditions.
appendix i study parameter tables
please make sure that every

Label as many 1ml cryovials (5 to 8) as necessary for the plasma collected. Label them with the NRG Oncology study and case number, collection date, time, and time point, and clearly mark cryovials "plasma".  
Process:
1. After collection, invert tube(s) multiple times to ensure adequate mixing of EDTA. 2. Centrifuge specimen(s) within one hour of collection in a standard clinical centrifuge at ~2500 RPM for 10 minutes at 4C (preferred). If sites are unable to process samples at 4C then spinning at room temperature is acceptable if done within 2 hours of draw but must be noted on the ST Form.  
3. If the interval between specimen collection and processing is anticipated to be more than one hour, keep specimen on ice until centrifuging is performed.  
4. Carefully pipette and aliquot 0.5 ml plasma into as many cryovials as are necessary for the plasma collected (5 to 8) labeled with NRG Oncology study and case numbers, collection date/time, time point collected and clearly mark specimen as "plasma". Avoid pipetting up the buffy coat layer. If an aliquot has less than 0.5 ml in the tube please note the volume on the vial and make a note on the ST Form.  
5. Place cryovials into biohazard bag and immediately freeze at -70 to -90C.  
6. Store frozen plasma until ready to ship on dry ice.  
7. See below for storage conditions.
appendix i study parameter tables
please make sure that every

The St Form. (C) Whole Blood For Dna (If Requested): Purple Top Edta Tube #2  
Label as many 1ml cryovials (3 to 5) as necessary for the whole blood collected. Label them with the NRG Oncology study and case number, collection date/time, and time point, and clearly mark cryovials "blood".  
Process:
1. After collection, invert tube(s) multiple times to ensure adequate mixing of EDTA. Blood can also be mixed for 5 minutes on a mixer at room temperature.  
2. Carefully pipette and aliquot 1.0 ml blood into as many cryovials as are necessary for the blood collected (3 to 5) labeled with NRG Oncology study and case numbers, collection date/time, time point collected and clearly mark specimen as "blood". **If an aliquot has less**
than 1.0 ml in the tube please note the volume on the vial and make a note on the ST Form.  
3. Place cryovials into biohazard bag and freeze immediately at -70 to -80 Celsius. 4. Store blood samples frozen until ready to ship on dry ice.  
5. See below for storage conditions.
appendix i study parameter tables
please make sure that every

Freezing and Storage:
Freeze Blood samples in a -80C Freezer or on Dry Ice or snap freeze in liquid nitrogen.  
Store at –80C (-70C to -90C) until ready to ship.  
If a -80C Freezer is not available, Samples can be stored short term in a -20C freezer (non-frost free preferred) for up to one week (please ship out Monday-Wednesday only; Canada: MondayTuesday only).  
OR: Samples can be stored in plenty of dry ice for up to one week, replenishing daily
(please ship out on Monday-Wednesday only; Canada: Monday-Tuesday only).  
OR:
Samples can be stored in liquid nitrogen vapor phase (ship out MondayWednesday only; Canada: Monday-Tuesday only).  
Please indicate on Specimen Transmittal Form the storage conditions used and time stored.  
Shipping/Mailing:
Ship specimens on Dry Ice overnight Monday-Wednesday **(Monday-Tuesday from Canada)** to prevent thawing due to delivery delays. Saturday and holiday deliveries cannot be accepted.
Include all NRG Oncology paperwork in a sealed plastic bag and tape to the outside top of the Styrofoam box.  
Wrap frozen specimens of same type (i.e., all serum together, plasma together and whole bloods together) in absorbent shipping material and place each specimen type in a separate biohazard bag. Place specimen bags into the Styrofoam cooler and fill with plenty of dry ice (7-10 lbs/3.5kg minimum). *Add padding to avoid the dry ice from breaking the tubes.*
Place Styrofoam coolers into outer cardboard box, and attach shipping label and UN3373 and UN1895 stickers to outer cardboard box.  
Multiple cases may be shipped in the same cooler, but make sure each one is in a separate bag and that there is enough room for plenty of dry ice. Add padding to avoid the dry ice from breaking the tubes.  
**For questions regarding collection, shipping or to order a Blood Collection Kit, please email RTOG@ucsf.edu or call (415)476-7864.**
appendix i study parameter tables
shipping address

Courier Address (FedEx, UPS, etc.): For all Frozen Specimens NRG Oncology Biospecimen Bank San Francisco University of California San Francisco 2340 Sutter Street, Room S341 San Francisco, CA 94115 For questions, call 415-476-7864 or e-mail: RTOG@ucsf.edu
appendix i study parameter tables
nrg oncology urine collection kit

This Kit is for collection, processing, storage, and shipping of urine specimens. Kit Contents:  
One (1) Sterile Urine collection cup  Two 15 ml polypropylene centrifuge  
tubes  
Two 7 ml disposable pipettes  Biohazard bags  
Absorbent paper towel  Parafilm for sealing outside of tubes
appendix i study parameter tables
preparation and processing of urine

A clean catch urine specimen will be collected. To collect the specimen, use the following instructions:
o Males should wipe clean the head of the penis and females need to wipe between the labia with soapy water/cleansing wipes to remove any contaminants.  
o After urinating a small amount into the toilet bowl to clear the urethra of contaminants, collect a sample of urine in the collection cup.  
o After 10-25 mL urine has been collected, remove the container from the urine stream without stopping the flow of urine.  
o Finish voiding the bladder into the toilet bowl.  
Aliquot 5-10 mls of Urine into each of two 15 ml polypropylene centrifuge tubes (disposable pipets are provided in the kit). Do not fill with more than 10 mls to avoid cracking of tubes due to expansion during freezing. Replace the cap and tighten on the tubes. Make sure the cap is not cross-threaded or placed on incorrectly or leaking will occur.  
Use parafilm to seal the cap around the outside rim of the urine tube to prevent leakage. Discard remaining Urine and collection cup.  
Label the specimen with the NRG Oncology study and case number, collection date and time, time point of collection, and clearly mark specimens as "urine".  
Wrap Urine Tubes with absorbent material (paper towels) and place into biohazard bag and seal the bag. Freeze and store Urine samples in a -20C or -80C freezer until ready to ship.
appendix i study parameter tables
storage and shipping freezing and

Urine specimens may be sent in batches or with other frozen biospecimens, if within 30-60 days of collection. Store at -20C or -80C (-70C to -90C) until ready to ship. If a -80C Freezer is not available:
Samples can be stored short term in a -20 C freezer (non-frost free preferred) for up to one week (please ship out Monday-Wednesday only; Canada: Monday-Tuesday only).  
OR:
Samples can be stored in plenty of Dry Ice for up to one week, replenishing daily (please ship out Monday-Wednesday only; Canada: Monday-Tuesday only).  
Please indicate on Specimen Transmittal Form the storage conditions used and time stored.
appendix i study parameter tables
shipping/mailing

Ship specimens on Dry Ice overnight Monday-Wednesday **(Monday-Tuesday from Canada)** to prevent thawing due to delivery delays. Saturday and holiday deliveries cannot be accepted.  
Include all NRG Oncology paperwork in a sealed plastic bag and tape to the outside top of the Styrofoam box.  
Place sealed specimen bags into the Styrofoam cooler and fill with plenty of dry ice (7-10 lbs/3.5kg minimum). *Add padding to avoid the dry ice from breaking the tubes.*
Place Styrofoam coolers into outer cardboard box, and attach shipping label and UN3373 and UN1895 stickers to outer cardboard box.  
Multiple cases may be shipped in the same cooler, but make sure each one is in a separate bag and that there is enough room for plenty of dry ice. Add padding to avoid the dry ice from breaking the tubes.  
Samples received thawed will be discarded, and a notification will be sent immediately to the Principal Investigator and Clinical Research Assistant of the submitting institution. The institution should send a subsequent sample, collected as close as possible to the original planned collection date.  
For questions regarding ordering, collection, or shipping of a Urine Collection Kit, please e-mail RTOG@ucsf.edu or call (415)476-7864 or fax (415) 476-5271.  
Shipping Address: FedEx/UPS/Courier address (For all frozen samples)
NRG Oncology Biospecimen Bank San Francisco 2340 Sutter Street, Room S341, UCSF
San Francisco, CA 94115 Contact Phone: (415) 476-7864