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13.0 statistical considerations

13.1 **Primary Endpoint**
Progression-free survival (PFS), defined as the interval from randomization to progression or death due to any cause, whichever occurs first.  
13.2 **Secondary Endpoints**
Overall survival (OS), defined as the interval from randomization to death due to any cause Time to local-regional progression (LRP), defined as the interval from randomization to local or regional progression; Time to distant metastasis (DM), defined as the interval from randomization to distant metastasis; Adverse events definitely, probably, or possibly related to treatment (CTCAE, v. 4) within 1 year of completion of all treatment.  
13.3 **Stratification**
Patients will be stratified by Zubrod Performance Score (0 vs. 1); histology (squamous vs. nonsquamous); and clinical stage (IIIA vs. IIIB).  
13.4 Sample Size **and Power Justification**
In LA-NSCLC, the expected 1-year progression-free survival (PFS) with the concurrent chemoradiotherapy is assumed to be 50% based on RTOG 0617 (Bradley 2011). The target improvement for the added MET is to increase the 1-year PFS to 65%. Assuming the PFS are exponentially distributed (at least approximately) with constant hazards in the both treatment arms, the primary hypothesis is equivalent to reduce the monthly hazard λ from 0.0578 to 0.0359, i.e., a hazard ratio of 0.622 or 37.8% relative hazard reduction. This study will be a randomized phase II screening trial as proposed by Rubinstein et al (2005). The randomization of experimental and standard arms is set to be 1:1. With 152 analyzable patients (76 analyzable patients per arm) and 102 PFS events, there will be 85% power to detect a 37.8% relative reduction in hazard at a significance level of 0.1 (one-sided). Adjusting for a 10% rate of ineligibility, **a maximum of 168** patients is required for this trial.  
For the most recently completed RTOG trial in the same patient population, the randomized phase III study RTOG 0617, the average monthly accrual rate was more than 10 cases/month. Therefore, a monthly accrual rate of 7 patients is assumed in this study, and the total accrual is expected to be completed in 2 years. The study is expected to reach the required number of PFS events 1 year after accrual completion. During the first 6 months following activation, little accrual is anticipated while the trial is being approved by institutional review boards (IRBs). A total of 3.5 years is projected to observe the required number of PFS events and conduct the final analysis (if applicable).
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13.5 statistical analysis plan

13.5.1 Statistical Methods PFS is defined as the time from randomization to disease progression or death from any cause, whichever occurs first. A rigorous definition of disease progression is provided in Section 11.4 (a second primary cancer is not treated as a PFS event). PFS rates will be estimated using the KaplanMeier method (1958), and the differences between arms will be tested using a log-rank test (Mantel 1966). A multivariate analysis with the Cox proportional hazard model (1972) for PFS will be performed with the stratification variables as fixed variables to assess the treatment effect adjusting for patient-specific risk factors.  
Overall survival (OS) is similarly analyzed as time-to-event data, where the OS rates will be estimated using the Kaplan-Meier method, and the differences between treatment arms will be tested using the log-rank test. A competing risks analysis approach also will be used when analyzing time to local-regional progression (TTLRP) and time to distant metastasis (TTDM). For TTLRP, only local-regional progression will be counted as events (failures), and patients who die or have a distant metastasis without a local-regional progression will be treated as competing risk.  
Analogously, for TTDM, only distant metastasis will be counted as events, and deaths or localregional progression without distant metastasis will be treated as competing risks. A cumulative incidence approach will be used to estimate the respective rates, and the corresponding differences in LRP and DM will be evaluated using Gray's test (Gray 1988).  
Due to the concerns of potential subjective bias introduced in investigator-determined progression
(Dodd 2008), all PET/CT scans will be collected and archived for a planned retrospective central radiologic review. These PET/CT scans will be blindly reviewed by the Radiologic Co-Chair. The centrally-reviewed progression results will be properly incorporated in the sensitivity analyses (FDA
2007) to assure the robustness of analysis results.  
The rate of treatment-related adverse events using NCI Common Terminology Criteria for Adverse Events (CTCAE, v. 4) will be reported with the frequency and severity (e.g., type, grade, and attribution) by arm, and analyzed using Chi-square test or Fisher's exact test wherever applicable.  
The analysis will be performed at the time of primary endpoint analysis. Logistic regression (Agresti 1990) will be used to model the distribution of adverse events with and without adjustment for covariates. Both unadjusted and adjusted odds ratios and the respective 95% confidence intervals will be computed and tested at a significance level of 0.05 (2-sided).  
13.5.2 Significance Testing for Early Termination and Reporting A group sequential test with one planned interim analysis and a final analysis will be performed. The interim analysis will be based on the primary endpoint as defined in Section 13.1.1, and will be carried out when half of the number of expected PFS events (51 events) is reached. At the planned interim analysis, the p-value from the log-rank test will be compared to 0.01 for rejecting the null hypothesis, and the observed hazard ratio (experimental/standard) will be compared with 1.0 (Wieand 1994) for futility. If the p-value associated with the resulting test statistics is less than or equal to 0.01, then we will stop accrual to the trial (if applicable) and conclude MET improves PFS of these patients; if the observed hazard ratio is greater than or equal to 1.0, a decision about whether to terminate accrual and release the results will be made after consultation with the NRG Oncology Data Monitoring Committee (DMC) and NCI/CTEP. If the efficacy boundary is not crossed and the observed hazard ratio is less than 1.0, the final analysis will occur when there are at least 102 PFS events observed. A significance level of 0.09 for the final analysis was derived in order to preserve a 0.1 significance level (1-sided) for the entire study.  
At the protocol-planned interim analysis, the results from the test assessing the treatment efficacy and futility will be reported to the NRG Oncology Data Monitoring Committee (DMC). The protocol statistician may recommend early reporting of the results and/or stopping accrual (if applicable) of the trial if either efficacy or futility boundary is crossed. The accrual rate, treatment compliance, safety of the treatments, and the importance of the study also are considered in making such a recommendation. The results will be reported to the NRG Oncology DMC. The DMC will then make a recommendation about the trial to the NRG Oncology Group Chairs.  
13.5.3 Significance Testing for Final Analysis The final analysis will be performed on an intent-to-treat basis, such that all eligible cases will be included in the arm to which the patient was randomized regardless of what treatment the patient actually received. The major analysis will occur after at least 102 PFS events have been observed, unless an early stopping rule is satisfied. It will include:
Tabulation of all cases entered and those excluded from the analyses with the reasons for exclusion given; Distributions of important prognostic baseline variables; The frequencies and severity of AEs by treatment arms; Compliance rate of treatment delivery; Observed results with respect to the primary and secondary endpoints.  
All eligible patients randomized will be included in the comparison and will be grouped by assigned treatment in the analysis. The primary hypothesis of treatment benefit will be tested using the stratified log-rank statistic with a significance level of 0.1 (1-sided), given that the interim analysis will have been carried out. Additional analyses of treatment effect will be performed using the Cox proportional hazards model with the stratification factors included as fixed covariates, as well as any factors that show an imbalance between the arms and any other factor known to be associated with outcome.  
13.5.4 Interim Analysis to Monitor Study Progress The NRG Oncology Data Monitoring Committee (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. In general, the interim reports will contain information about the patient accrual rate, a projected completion date for the accrual phase, patient exclusion rates and reasons following registration, compliance rate of treatment delivery, distributions of pretreatment characteristics and important prognostic baseline variables, and the frequencies and severity of treatment-related adverse events. The interim reports will not contain the results from the treatment comparisons with respect to the efficacy endpoints. Interim reports with statistical analyses are prepared every 6 months until the initial publication reporting the treatment results has been submitted. The reports contain:
Patient accrual rate with a projected completion date (while the study is still accruing)
Total patients accrued Distributions of important pretreatment and prognostic baseline variables The frequencies and severity of adverse events by treatment arm Compliance rates of treatment delivery 13.5.5 CDUS Reporting 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 **Statistical Considerations for Translation Research**
The translational research objectives are considered in exploratory nature. The predictive and prognostic potential for the proposed biomarkers may become scientifically obsolete or the assay technology may evolve over time, making the technology outlined in the current protocol obsolete when the study is finished. As such, no marker assays will be conducted on the collected specimens. When sufficient information is available from the parent study, a full correlative study protocol for the marker studies detailing the scientific hypothesis, research plan, clinical outcome, assay methods for each biomarker, and a more complete statistical section (with adequate power justification and analysis plan) will be submitted and subjected to CTEP review in accordance with National Clinical Trials Network (NCTN) policies.  
To determine if the study may offer exploratory yet still meaningful findings whether a mutation of interest may predict PFS changes in patients treated with metformin, we summarize the following table as a preliminary evaluation for the interaction effects between marker status (such as TP53, STK11 (LKB1) or KRAS) and treatment assignments. Based on the design parameters and the method proposed by Peterson and George [Peterson 1993], we summarize the statistical power to detect interaction effects (ratio of hazard ratios) of 0.33, at 2-sided significance level of 0.05.  
We denote monthly hazard rates as λ, expected number of events of respective subgroups as E[N], and the hazard ratio between marker + and - as Δ. The subscripts indicate the corresponding treatment arms. The assumed monthly hazard rates and associated expected number of events are listed in the following Table accordingly.  
| Prevalence  of Marker  +/-   | Arm 1    | Arm 2   | Interaction   |          |        |             |       |       |       |
|------------------------------|----------|---------|---------------|----------|--------|-------------|-------|-------|-------|
| Marker -                     | Marker + | Δ1      | Marker -      | Marker + | Δ2     | Δ21= Δ2/ Δ1 | Power |       |       |
| 0.33                         | λ        | 0.0578  | 0.0578        | 1.0      | 0.0487 | 0.0161      | 0.33  | 0.33  | 63.2% |
| E[N]                         | 38       | 19      | 35            | 8        |        |             |       |       |       |
| λ                            | 0.0505   | 0.0758  | 1.5           | 0.0442   | 0.0219 | 0.5         | 0.33  | 70.4% |       |
| E[N]                         | 35       | 21      | 33            | 11       |        |             |       |       |       |
| λ                            | 0.0459   | 0.0918  | 2.0           | 0.0408   | 0.0269 | 0.67        | 0.33  | 72.1% |       |
| E[N]                         | 34       | 22      | 32            | 12       |        |             |       |       |       |
| 0.5                          | λ        | 0.0578  | 0.0578        | 1.0      | 0.0586 | 0.0193      | 0.33  | 0.33  | 74.7% |
| E[N]                         | 28       | 28      | 28            | 14       |        |             |       |       |       |
| λ                            | 0.0471   | 0.0707  | 1.5           | 0.0497   | 0.0246 | 0.5         | 0.33  | 77.2% |       |
| E[N]                         | 26       | 31      | 26            | 17       |        |             |       |       |       |
| λ                            | 0.0407   | 0.0813  | 2.0           | 0.0438   | 0.0289 | 0.67        | 0.33  | 77.8% |       |  
E[N] 24 32 25 19 Based on the table above, the proposed study will have a reasonably good power to detect a strong predictive (interaction) effect (HR=0.33).
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13.7 gender and minorities

| Gender                                    |         |       |       |
|-------------------------------------------|---------|-------|-------|
| Ethnic Category                           | Females | Males | Total |
| Hispanic or Latino                        | 4       | 6     | 10    |
| Not Hispanic or Latino                    | 66      | 92    | 158   |
| Ethnic Category: Total of all subjects    | 70      | 98    | 168   |
| Gender                                    |         |       |       |
| Racial Category                           | Females | Males | Total |
| American Indian or Alaskan Native         | 0       | 1     | 1     |
| Asian                                     | 2       | 2     | 4     |
| Black or African American                 | 7       | 10    | 17    |
| Native Hawaiian or other Pacific Islander | 0       | 1     | 1     |
| White                                     | 61      | 84    | 145   |
| Racial Category: Total of all subjects    | 70      | 98    | 168   |
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Wu R-R, Jin H, Gao K, Twamley EW, Ou J-J, Shao P, et al. Metformin for treatment of antipsychoticinduced amenorrhea and weight gain in women with first-episode schizophrenia: a double-blind, randomized, placebo-controlled study. *Am J Psychiatry*. 169(8):813–21, 2012. PMID: 22711171 APPENDIX I STUDY PARAMETER TABLE: **PRE-TREATMENT ASSESSMENTS** (8/10/16)
*See Section 11.2 for details and exceptions  
| Assessments                                                                                    | Prior to Registration         | Prior to Treatment   |
|------------------------------------------------------------------------------------------------|-------------------------------|----------------------|
| (calendar days)                                                                                | (calendar days)               |                      |
| History/physical exam, including  height, weight, BSA, and vital  signs                        | 30                            |                      |
| Performance status                                                                             | X                             |                      |
| CT scan with contrast or MRI of  lung and upper ab through  adrenals (see. 3.1.4)              | 45                            |                      |
| MRI or CT of brain with contrast                                                               | 45                            |                      |
| Whole-body FDG-PET/CT                                                                          | 45                            |                      |
| CBC/differential; ANC, platelets;                                                              | 14                            |                      |
| hemoglobin Serum creatinine or creatinine                                                      | 14                            |                      |
| clearance and eGFR Total bilirubin, ALT, AST, Alk                                              | 14                            |                      |
| Phos, Blood glucose Serum albumin                                                              | 14                            |                      |
| Serum pregnancy (if applicable)                                                                | 72 hrs. prior to registration |                      |
| Examination by Rad Onc & Med                                                                   | 42                            |                      |
| Onc †Tissue submission for banking                                                             | X                             |                      |
| †Whole blood and plasma                                                                        | X                             |                      |
| submission for banking †For patients who consent to participate in this component of the study |                               |                      |
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appendix i study parameter table

| Assessments                                                                                                               | During Treatment                                                                           |                          |    |
|---------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------|--------------------------|----|
| Weekly during MET  Dose Escalation  (Arm 2 Only)                                                                          | Weekly during                                                                              | Within 48 hours Prior to |    |
| Concurrent Treatment                                                                                                      | Each Cycle of                                                                              |                          |    |
| Consolidation Treatment                                                                                                   |                                                                                            |                          |    |
| History & Physical                                                                                                        | X                                                                                          | X                        |    |
| exam BSA Recalculation                                                                                                    | If there is a change of more than 10% of body  weight, compared to the registration weight |                          |    |
| CBC/differential and                                                                                                      | X                                                                                          | X                        |    |
| serum creatinine Bilirubin, AST/ALT, alk                                                                                  | X                                                                                          |                          |    |
| phos Blood glucose for Arm 2                                                                                              | X                                                                                          | X                        | X  |
| patients                                                                                                                  | See Section 11.2 for  details.                                                             |                          |    |
| Adverse events                                                                                                            | X                                                                                          | X                        | X  |
| evaluation †Plasma submission for                                                                                         | At week 1 and 6 of                                                                         |                          |    |
| banking                                                                                                                   | chemo/RT                                                                                   |                          |    |
| (corresponding to  weeks 3 and 8 of  metformin treatment  period for patients  randomized to Arm 2)  (see Section 10.2.5) |                                                                                            |                          |    |
| †For patients who consent to participate in this component of the study                                                   |                                                                                            |                          |    |
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appendix i study parameter table

| Assessments                                                             | Follow Up                                                                                                                  |                                                                 |                                                       |
|-------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------|-------------------------------------------------------|
| At 4-6 weeks from                                                       | At 3 months from                                                                                                           | At 6, 9, and 12                                                 |                                                       |
| end of RT                                                               | end of RT                                                                                                                  | mos. from end  of RT or until  progression,  whichever is first | q3 mos. x 2 years;  q6 mos. x 3 years;  then annually |
| History & Physical                                                      | X                                                                                                                          | X                                                               | X                                                     |
| exam Performance status                                                 | X                                                                                                                          | X                                                               | X                                                     |
| Follow-up CT scan  of chest and upper  abdomen with  contrast or MRI    | X                                                                                                                          | X                                                               | See                                                   |
| recommended  timeframes in  Section 11.2                                |                                                                                                                            |                                                                 |                                                       |
| Imaging of other  sites (e.g.  extremities, head)                       | These sites should be imaged per institutional routine if there is suspicion of  new metastatic disease; see Section 11.2. |                                                                 |                                                       |
| Biopsy                                                                  | Recommended for any recurrence,  whether based on CT scan or  PET/CT                                                       |                                                                 |                                                       |
| Adverse event                                                           | X                                                                                                                          | X                                                               | X                                                     |
| evaluation †Plasma submission for  banking                              | At 6 month  follow up visit  (see Section  10.2.5)                                                                         |                                                                 |                                                       |
| †For patients who consent to participate in this component of the study |                                                                                                                            |                                                                 |                                                       |
eligibility checklist (8/4/14) (page 4
appendix ii zubrod performance scale

0 Fully active, able to carry on all predisease activities without restriction  
5 Death  
| 1   | Restricted in physically strenuous activity but ambulatory and able to  carry work of a light or sedentary nature. For example, light  housework, office work   |
|-----|-----------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| 2   | Ambulatory and capable of all self-care but unable to carry out any  work activities. Up and about more than 50% of waking hours   |
|-----|------------------------------------------------------------------------------------------------------------------------------------|  
| 3   | Capable of only limited self-care, confined to bed or chair 50% or  more of waking hours   |
|-----|--------------------------------------------------------------------------------------------|  
| 4   | Completely disabled. Cannot carry on self-care. Totally confined to  bed   |
|-----|----------------------------------------------------------------------------|
appendix iii ajcc staging system
appendix ii zubrod performance scale

| TX                                                                                                         | Primary tumor cannot be assessed, or tumor proven by the presence of malignant cells in  sputum or bronchial washings but not visualized by imaging or bronchoscopy                                                                                                                                                                                                                                                                            |
|------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| T0                                                                                                         | No evidence of primary tumor.                                                                                                                                                                                                                                                                                                                                                                                                                  |
| Tis                                                                                                        | Carcinoma in situ                                                                                                                                                                                                                                                                                                                                                                                                                              |
| T1                                                                                                         | Tumor 3 cm or less in greatest dimension, surrounded by lung or visceral pleura, without  bronchoscopic evidence of invasion more proximal than the lobar bronchus (i.e., not in the  main bronchus)*                                                                                                                                                                                                                                          |
| T1a                                                                                                        | Tumor 2 cm or less in greatest dimension                                                                                                                                                                                                                                                                                                                                                                                                       |
| T1b                                                                                                        | Tumor more than 2 cm but 3 cm or less in greatest dimension                                                                                                                                                                                                                                                                                                                                                                                    |
| T2                                                                                                         | Tumor more than 3 cm but 7 cm or less with any of the following features (T2 tumors with  these features are classified T2a if 5 cm or less): Involves main bronchus, 2 cm or more  distal to the carina; Invades the visceral pleura PL1 or PL2); Associated with atelectasis or  obstructive pneumonitis that extends to the hilar region but does not involve the entire lung                                                               |
| T2a                                                                                                        | Tumor more than 3 cm but 5 cm or less in greatest dimension                                                                                                                                                                                                                                                                                                                                                                                    |
| T2b                                                                                                        | Tumor more than 5 but 7 cm or less in greatest dimension                                                                                                                                                                                                                                                                                                                                                                                       |
| T3                                                                                                         | Tumor more than 7 cm or one that directly invades any of the following: parietal (PL3), chest  wall (including superior sulcus tumors), diaphragm, phrenic nerve, mediastinal pleura,  parietal pericardium; or tumor in the main bronchus (less than 2 cm distal to the carina* but  without involvement of the carina; or associated atelectasis or obstructive pneumonitis of  the entire lung or separate tumor nodule(s) in the same lobe |
| T4                                                                                                         | Tumor of any size that invades any of the following: mediastinum, heart, great vessels,  trachea, recurrent laryngeal nerve, esophagus, vertebral body, carina, separate tumor  nodules in a different ipsilateral lobe                                                                                                                                                                                                                        |
| *The uncommon superficial spreading tumor of any size with its invasive component limited to the bronchial |                                                                                                                                                                                                                                                                                                                                                                                                                                                |
appendix iii ajcc staging system
regional lymph nodes (n)

NX Regional lymph nodes cannot be assessed N0 No regional lymph nodes metastasis N1 Metastasis to ipsilateral peribronchial and/or ipsilateral hilar lymph nodes, and intrapulmonary nodes including involvement by direct extension N2 Metastasis to ipsilateral mediastinal and/or subcarinal lymph node(s) N3 Metastasis in contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene, or supraclavicular lymph node(s)
appendix iii ajcc staging system
distant metastasis (m) m0 no

M1 Distant metastasis M1a Separate tumor nodule(s) in a contralateral lobe tumor with pleural nodules or malignant pleural (or pericardial) effusion* M1b Distant metastasis
* Most pleural (and pericardial effusions with lung cancer are due to tumor. In a few patients, however, multiple cytopathologic examinations of pleural (pericardial) fluid are negative for tumor, and the fluid is nonbloody and is not an exudate. Where these elements and clinical judgment dictate that the effusion is not related to the tumor, the effusion should be excluded as a staging element, and the patient should be classified as M0.  
| STAGE GROUPING  Occult Carcinoma   | TX, N0, M0                                            |
|------------------------------------|-------------------------------------------------------|
| Stage 0                            | Tis, N0, M0                                           |
| Stage IA                           | T1a-b, N0, M0                                         |
| Stage IB                           | T2a, N0, M0                                           |
| Stage IIA                          | T2b, N0, M0 T1a-b, N1, M0 T2a, N1, M0                 |
| Stage IIB                          | T2b, N1, M0 T3, N0, M0                                |
| Stage IIIA                         | T1a-b, N2, M0 T2a-b, N2, M0 T3, N1-2, M0 T4, N0-1, M0 |
| Stage IIIB                         | T1a-b, N3, M0 T2a-b, N3, M0 T3, N3, M0 T4, N2-3, M0   |
| Stage IV                           | Any T, Any N, M1a-b                                   |  
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 ALL 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).
appendix iii ajcc staging system
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 with paper towel, 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**.  
**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.
appendix iv biospecimen collection) nrg
ffpe specimens

This Kit allows sub-sampling of an FFPE block for submission to the NRG Oncology **Biospecimen**  
  
  
  
  
Bank - **San Francisco. 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.  
Step 2 Label the punch tool with the proper specimen ID 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 - San Francisco 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: NRGBB@ucsf.edu or call 415-476-**
RTOG(7864)/Fax 415-476-5271.  
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 APPENDIX IV: BIOSPECIMEN COLLECTION
Courier Address (FedEx, UPS, etc.): For ALL Frozen Specimens or Trackable shipments NRG Oncology Biospecimen Bank University of California San Francisco 2340 Sutter Street, Room S341 San Francisco, CA 94115
appendix iv biospecimen collection
ffpe specimens

NRG Oncology BLOOD COLLECTION KIT INSTRUCTIONS
This Kit is for collection, processing, storage, and shipping of Plasma, or whole blood *(as* specified by the protocol):
appendix iv biospecimen collection
kit contents

Two Purple Top EDTA tube for plasma (A) One Purple Top EDTA tube for Whole Blood (B)
Thirty-five (15) 1 ml cryovials for pre-tx; 45 1 ml cryovials for other plasma time point Biohazard bags (2) and Absorbent shipping material (2)
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 iv biospecimen collection
preparation and processing of plasma

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 the 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.  
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 iv biospecimen collection
(b) whole blood for dna

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".
appendix iv biospecimen collection
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 the NRG Oncology study and case numbers, collection date/time, time point collected and clearly mark specimen as "blood".  
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.  
PLEASE MAKE SURE THAT EVERY SPECIMEN IS LABELED and include collection time **point on**  
  
the ST Form.  
PLEASE MAKE SURE THAT EVERY SPECIMEN IS LABELED and include collection time point on
ST Form.
appendix iv biospecimen collection
appendix iv biospecimen collection freezing

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 (ST) Form the storage conditions used and time stored.
appendix iv biospecimen collection
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 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 NRGBB@ucsf.edu or call (415)476-7864.
appendix iv biospecimen collection
shipping address

Courier Address (FedEx, UPS, etc.): For ALL **Frozen Specimens**
NRG Oncology Biospecimen Bank University of California San Francisco 2340 Sutter Street, Room S341 San Francisco, CA 94115 For questions, call 415-476-7864 or e-mail: NRGBB@ucsf.edu