rtog foundation rtog 3505
late phase 2 and phase

NOTE: Investigators **MUST** immediately report to the sponsor **ANY** Serious Adverse Events, whether or not they are considered related to the investigational agent(s)/intervention (21 CFR 312.64)
An adverse event is considered serious if it results in ANY of the following outcomes:  
1) Death 2) A life-threatening adverse event 3) An adverse event that results in inpatient hospitalization or prolongation of existing hospitalization for ≥ 24 hours 4) A persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions 5) A congenital anomaly/birth defect.  
6) Important Medical Events (IME) that may not result in death, be life threatening, or require hospitalization may be considered serious when, based upon medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. (FDA, 21 CFR 312.32; ICH E2A and ICH E6).  
| ALL SERIOUS adverse events that meet the above criteria MUST be immediately reported to RTOG  via the SAE Report Form within the timeframes detailed in the table below. Grade 1  Grade 2  Grade 3  Grade 4 & 5  Hospitalization Timeframes Timeframes Timeframes Timeframes Resulting in  Hospitalization  10 Calendar Days ≥ 24 hrs 24-Hour 5 Calendar  Days Not resulting  in Hospitalization  ≥ 24 hrs Not required 10 Calendar Days   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
rtog foundation rtog 3505
expedited ae reporting timelines are

o "24-Hour; 5 Calendar Days" - The AE must initially be reported via the SAE Report Form within 24 hours of learning of the AE, followed by a complete expedited report within 5 calendar days of the initial 24-hour report.  
o "10 Calendar Days" - A complete expedited report on the AE must be submitted within 10 calendar days of learning of the AE.  
1Serious adverse events that occur more than 100 days after the last administration of investigational agent/intervention and are considered related to protocol treatment require reporting as follows:
Expedited 24-hour notification followed by complete report within 5 calendar days for:
All Grade 4, and Grade 5 AEs Expedited 10 calendar day reports for:
Grade 2 adverse events resulting in hospitalization or prolongation of hospitalization Grade 3 adverse events
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specific protocol inclusions and exceptions

Hemophagocytic lymphohistiocytosis is an *inclusion* to expedited reporting. Hemophagocytic lymphohistiocytosis is a rare but potentially fatal disease of normal but overactive histiocytes and lymphocytes. It is a manifestation of cytokine release syndrome. Occurring at any grade, hemophagocytic lymphohistiocytosis must be reported in an expedited manner as a serious adverse event.
rtog foundation rtog 3505
8. registration and study entry

8.1 Regulatory Requirements Please refer to the study-specific guide for investigators and research staff for detailed procedures regarding the following: requirements for regulatory collection and RT credentialing. The RTOG Foundation 3505 Study Guide is posted on the RTOG website https://www.rtog.org/clinicaltrials/rtogfoundationstudies/rtogfoundationstudy3505.aspx .  
8.2 RT **Registration Requirements**
For detailed information on the specific technology requirement required for this study, please refer to the study specific guide on the RTOG Foundation 3505 protocol page of the RTOG website, www.rtog.org.  
8.2.1 Pre-Registration Credentialing Requirements for IMRT/3DCRT
See the study-specific guide on the RTOG Foundation 3505 protocol page of the RTOG website, www.rtog.org, for details of RT pre-registration credentialing requirements for IMRT/3DCRT.  
8.2.2 Pre-Registration Requirements for TRIAD Installation See the study-specific guide on the RTOG Foundation 3505 protocol page of the RTOG website, www.rtog.org, for details on TRIAD installation requirements needed prior to patient enrollment.  
8.3 **Patient Enrollment**
See the study-specific guide on the RTOG Foundation 3505 protocol page of the RTOG
website, www.rtog.org.
rtog foundation rtog 3505
9.0 drug information 9.1 investigational

To supplement the toxicity information contained in this document, investigators must obtain the current version of the BMS-936558 (nivolumab) Investigator Brochure (IB) for comprehensive pharmacologic and safety information. Also see Appendix I. The IB can be accessed on the RTOG Foundation 3505 protocol page of the RTOG website, www.rtog.org. Sites must use their username and password to access the protocol page and the IB.  
| Table                                                                     | Product Description                                      |                                                              |                                         |                     |                                |
|---------------------------------------------------------------------------|----------------------------------------------------------|--------------------------------------------------------------|-----------------------------------------|---------------------|--------------------------------|
| Product  Description and  Dosage Form                                     | Potency                                                  | Primary  Packagin g (Volume)/  Label  Type                   | Secondary  Packaging (Qty) /Label  Type | Appearance          | Storage Conditions (per label) |
| Nivolumab BMS936558-01  Solution for  Injection*                                                                           | 100 mg/vial (10  mg/mL)                                  | Carton of  5 or 10  vials                                    | 10cc Type 1 flint                       | Clear to opalescent |                                |
| glass vial                                                                | colorless to pale  yellow liquid. May  contain particles | 2 to 8 degreesC.  Protect from light,  freezing, and shaking |                                         |                     |                                |
| *Nivolumab/placebo may be labeled as BMS-936558-01 Solution for Injection |                                                          |                                                              |                                         |                     |                                |  
0.9% Sodium Chloride Injection solution is to be used as nivolumab/placebo. Placebo (0.9% Sodium Chloride Injection) and diluent solutions (0.9% sodium chloride injection, 5% dextrose injection) should also be sourced by investigative sites if available and permitted by local regulations. If stored in a glass front refrigerator, vials should be stored in the carton. Recommended safety measures for preparation and handling of nivolumab include laboratory coats and gloves. For additional details on prepared drug storage and use time of nivolumab under room temperature/light and refrigeration, please refer to Appendix I and the BMS-936558 (nivolumab) Investigator Brochure section for "Recommended Storage and Use Conditions"
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9.1.1 drug ordering and accountability

Bristol-Myers Squibb will supply Nivolumab free of charge to patients on study in the U.S. and Canada. An on-site unblinded pharmacist will be responsible for mixing placebo per instructions provided in Appendix I. The drug will be distributed by a third party vendor under contract to RTOG Foundation. Drug accountability records must be maintained at all sites according to good clinical practices. See the study-specific guide on the RTOG Foundation 3505 **protocol page of the RTOG** website, www.rtog.org **, for details of drug shipment.**
9.2 **Commercial Agent**
Cisplatin and Etoposide Sites must refer to the package insert for detailed pharmacologic and safety information.  
9.2.1 Availability/Supply Please see Section 5 for administration instructions. Please refer to the current FDAapproved package insert provided with each drug and the site-specific pharmacy for toxicity information and instructions for drug preparation, handling, and storage.
rtog foundation rtog 3505
10. pathology/biospecimens 10.1 tissue/specimen submission

In this study, it is **required** that tissue be submitted to the Biospecimen Bank at UCSF for PD-L1 expression analysis for all patients. It is highly recommended (but optional for the patient) that if any tissue remains after analysis, it will be stored at the Biospecimen Bank at UCSF for future research. In addition, it is highly recommended (but optional for the patient) that serum and peripheral blood specimens be submitted for the two tests described in Section 10.3, Peripheral blood for phenotyping the lymphocytes and serum for banking at UCSF for future translational research. Tissue from a biopsy or surgical specimen will be obtained from formalin fixed and paraffin embedded (FFPE) in tissue blocks. Sites must verify that an archived tumor block is available prior to Step 1 registration to the trial although the actual submission can be immediately after Step 1 registration but at least 5 weeks prior to Registration Step 2. Institutions must ship FFPE tissue as outlined in Section 10.2 to the Biospecimen Bank at UCSF by overnight courier. As noted in section 3.2.15, we recognize the difficulty in submitting tissue from stage 3 lung cancer patients in which the total available tissue may be small. We have designed three possibilities: Preferred: H&E slide and archived tissue tumor block. If sites wish the block to be returned they may submit the block and the Biospecimen Bank will cut the unstained for the required study and then punch and return the block. Alternative: If an archived tumor block sample cannot be shipped for this study, then two 3mm punches from the core needle biopsy blocks may alternatively be provided for analysis in addition to the H&E slide. Minimum: One (1) H&E stained slide (required) and five (5) positively charged unstained slides cut at 3-4 microns from the same block as the H&E. Unstained slides must be stored at 4C same day as being cut and shipped with a cold pack at 4C within 5 business days after being cut. The date that the unstained slides were cut must be noted on the ST form. Do NOT store or ship unstained slides at room temperature as they will not be able to be used for the mandatory testing. Please pack slides well to avoid breakage. If any slides are received broken the site must resubmit additional slides before the biobank can send for mandatory PDL1 testing. Ship FFPE biospecimens for mandatory central review and whole blood and serum samples **for banking** to: (do NOT ship fresh peripheral blood to this address) Biospecimen Bank at UCSF 2340 Sutter Street, Room S341 San Francisco, CA 94115
rtog foundation rtog 3505
ship all fresh peripheral blood

Hooper Laboratory Attn.: Rhonda Kean (laboratory manager) or Craig Hooper, PhD JAH 454 1020 Locust Street Philadelphia, PA 19107 Phone: 215-503-1559 See the tables below for mandatory and optional specimen collection. See further details of specimen collection/processing/shipping on the RTOG web site, http://www.rtog.org/LinkClick.aspx?fileticket=1SFlEVxSui4%3d&tabid=281.
rtog foundation rtog 3505
10.2 mandatory studies

Mandatory Study: Tumor PD-L1 Expression for All Patients Specimens are being collected to determine if patients with PD-L1 positive tumors will have an improved PFS when treated with nivolumab in comparison to patients with PD-L1 negative tumors (see Section 2.7 for further details).  
Required Form: ST form and Pathology report- must have all PHI redacted except date of procedure and pathology accession number.  
Shipping costs: Paid for by sites. Return labels are provided for frozen biospecimens only. International sites are encouraged to have a Fed Ex Customs Broker to help with any custom issues.  
At least 5 weeks prior to Registration Step 2.  
H&E slide and residual FFPE material will remain at the bank for all patients who consent to banking. Blocks can be returned upon request but will be punched prior to returning.
rtog foundation rtog 3505
ship specimens to

Biospecimen Bank at UCSF 2340 Sutter Street, Room S341 San Francisco, CA 94115  
| For questions, contact: Biospecimen Bank at UCSF; RTOG@ucsf.edu; 415-476-7864/FAX 415-476-5271 Specimen Type Collection Time  Collection  Points Information and  Requirements Shipping Representative  H&E stained slides  of the primary  tumor Pre-treatment H&E stained slide (must match the  block being  submitted). Can be  a duplicate cut  Slide shipped  ambient to the  Biospecimen Bank   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| slide, does not have  to be the diagnostic  slide.                                                                                                           |               |                                                                                                                                                                                                                                                                                                                                                                                                                             |                                                                                                                                                                                                                                                                                                                                       |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| A corresponding  paraffin embedded  tissue block, OR  two 3mm  embedded punches  of the primary  tumor OR 5  unstained slides as  specified in section  10.1 | Pre-treatment | Paraffin-embedded  tissue block or  punch biopsies  from the block. If  embedded punches  are submitted the  H&E from the  punch block must  also be submitted.  If 5 unstained slides are being provided  instead of the block  they must be stored  at 4⁰C and shipped  at 4⁰C within 5  business days after  being cut. The date that the  slides were cut must be noted on the ST  form. See Section  10.1 for details. | Block or punches shipped ambient (ship with a cold  pack during warm  weather) to the  Biospecimen Bank.  NOTE: Unstained  slides must be  shipped with cold  pack by overnight  courier. Pack slides  well to avoid  breakage, If slides  are received broken  this will delay  testing and site will  have to resubmit  the slides. |  
10.3 Optional Specimen Collection for RTOG 3505 Research and Banking  
Optional Study #1: SNP analyses and Serum for immune phenotype  
Patients must be offered the opportunity to consent to optional specimen collection. If the  
patient consents to participate, the site is required to submit the patient's specimens as specified in the protocol. Sites are not permitted to delete the specimen component from the protocol or from the sample consent.  
| patient consents to participate, the site is required to submit the patient's specimens as  specified in the protocol. Sites are not permitted to delete the specimen component from  the protocol or from the sample consent. Specimens are being collected to determine immune phenotype, and co-stimulatory/coinhibitory receptors with clinical response.  Required Form: ST form  Biospecimen Kits: Available from the Biospecimen Bank at UCSF.  Shipping days for frozen specimens: Monday-Wednesday (U.S. sites); MondayTuesday (Canada and Non-North American).  Shipping costs: Return labels are provided for peripheral blood or frozen  biospecimens only. Batch shipping is encouraged for all sites with access to a - 70⁰C to -90⁰ Freezer. Canada and International sites will be required to have  access to a -70⁰C to -90⁰Freezer to batch ship samples and may have to provide   |
|---|  
| their own outer shipping containers. International sites are encouraged to have a  Fed Ex Customs Broker to help with any custom issues.   |
|--------------------------------------------------------------------------------------------------------------------------------------------|
| Ship specimens to: Biospecimen Bank at UCSF 2340 Sutter Street, Room S341 San Francisco, CA 94115 For questions, contact: Biospecimen Bank at UCSF; RTOG@ucsf.edu; 415-476-7864/FAX 415-476-5271 Specimen Type Collection Time  Collection  Points Information and  Requirements Shipping Whole blood for  DNA- collected in  EDTA tube Pre-treatment only Invert tubes several  times, then aliquot  1ml per aliquot into  1 ml cryovials (3-5  per tube drawn).  Store at -70⁰C to - 90⁰ until shipped. Sent frozen on dry  ice via overnight  carrier to the  Biospecimen Bank Batch shipping of  multiple cases in  one shipment is  encouraged. Serum: One 5 mL   Pre-treatment; red top (clot) tube  Postchemoradiation/ before nivolumab/ placebo  60days after  nivolumab/placebo  at study  discontinuation for  either toxicity or  progression Serum centrifuged  and aliquotted. Frozen serum  samples containing minimum of 0.5mL  per aliquot in 1 mL  cryovials (5 per  tube drawn).  Samples should be  frozen and stored at  a -70⁰C to -90⁰; see  Appendix III for  processing  instructions. Serum sent frozen  on dry ice via  overnight carrier to  the Biospecimen  Bank Batch shipping of all time points or multiple cases in  one shipment is  encouraged.                                                                                                                                            |
rtog foundation rtog 3505
optional study #2 peripheral blood

Patients must be offered the opportunity to consent to optional specimen collection. If the patient consents to participate, the site is required to submit the patient's specimens as specified in the protocol. Sites are not permitted to delete the specimen component from the protocol or from the sample consent.  
Specimens are being collected to determine immune phenotype, and co-stimulatory/coinhibitory receptors with clinical response.  
Required Form: Special Study specific ST form Shipping days: Sites should only draw blood samples on Monday-Wednesdays and only ship Monday-Thursdays.  
Shipping labels will be provided with kits for peripheral blood. Canada and International sites may have to provide their own outer shipping containers.  
Sites must notify Rhonda Kean at Rhonda.Kean@jefferson.edu when they are shipping these samples Ship specimens to:
Hooper Laboratory Attn.: Rhonda Kean (laboratory manager) or Craig Hooper, PhD JAH 454 1020 Locust Street Philadelphia, PA 19107  
| Specimen Type                                                              | Collection Time Points                                        | Collection  Information  and  Requirements                 | Shipping      |
|----------------------------------------------------------------------------|---------------------------------------------------------------|------------------------------------------------------------|---------------|
| Peripheral Blood  Mononuclear  Cells (PBMC) - 5 Green top  (heparin) tubes |                                                              | Pre-treatment (day  -14 to day 0)                          |               |
|                                                                           | After  ChemoRT/before  nivolumab/placebo                      |                                                            |               |
|                                                                           | During  nivolumab/placebo:  Day 60                            |                                                            |               |
|                                                                           | at study  discontinuation for either toxicity or  progression | See Appendix  IV for processing and  shipping  information | Ship fresh at |
| room temperature  overnight for  morning delivery  to the Hooper  Laboratory. Ship  MondayThursday                                                                            |                                                               |                                                            |               |  
For questions, contact: 215-503-1559
rtog foundation rtog 3505
optional study #3 tumor tissue

Patients must be offered the opportunity to consent to optional specimen collection. If the patient consents to participate, the site is required to submit the patient's specimens as specified in the protocol. Sites are not permitted to delete the specimen component from the protocol or from the sample consent.  
Required Form: ST form and Pathology report- must have all PHI redacted except date of procedure and pathology accession number Shipping costs: Paid for by sites. Return labels are provided for frozen 68 biospecimens only. International sites are encouraged to have a Fed Ex Customs Broker to help with any custom issues.  
Ship specimens to: Biospecimen Bank at UCSF 2340 Sutter Street, Room S341 San Francisco, CA 94115  
| For questions, contact: Biospecimen Bank at UCSF; RTOG@ucsf.edu; 415-476-7864/FAX 415-476-5271 Specimen Type Collection Time  Collection  Points Information and  Requirements Shipping Representative  H&E stained slides  of the primary  tumor At progression H&E stained slide (must match the  block being  submitted) Slide shipped  ambient to the  Biospecimen Bank A corresponding paraffin-embedded  tissue block or two  3 mm embedded  punches of the  primary tumor taken before  initiation of  treatment At progression Paraffin-embedded  tissue block or  punch biopsies  from the block. If  embedded punches  are submitted the  H&E from the  punch block must  also be submitted Block or punches shipped ambient (ship with a cold  pack during warm  weather) to the  Biospecimen Bank   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
rtog foundation rtog 3505
11. special studies (non-tissue) 11.1

Objectives To characterize immune mediated toxicity from nivolumab following chemoradiation
(CRT) for locally advanced NSCLC;
To evaluate the effect of immune mediated toxicity from nivolumab following CRT
on QOL and patient reported fatigue.
rtog foundation rtog 3505
hypotheses

The primary HRQOL endpoint will be to determine if 15-month QOL (from start of nivolumab/placebo) using the Functional Assessment of Cancer Therapy - Trial Outcome Index for lung cancer (FACT-TOI) is correlated with overall survival (OS). The hypothesis is that long-term QOL using FACT-TOI at 15-month (from start of nivolumab/placebo) will be superior in the CRT followed by nivolumab arm compared to the CRT followed by placebo arm as the addition of nivolumab is expected to improve tumor response to CRT and improve disease-related QOL. Secondary exploratory objectives are:
To compare patient reported fatigue using the PROMIS fatigue short form between the consolidative nivolumab arm compared to placebo. The hypothesis is that acute fatigue at 3 months (from start of nivolumab/placebo) will be greater in the nivolumab arm compared to the placebo arm but will not impact long-term QOL outcome at 15 months (from start of nivolumab/placebo).  
To describe the levels and change in baseline at each assessment of the EQ-5D index and EQ-5D VAS in each of the study arms
rtog foundation rtog 3505
background

Health related quality-of-life (QOL) is an important endpoint in clinical trials to assess the overall disease burden and treatment effect from the patient's perspective using patient reported outcomes (PROs). In this study, consolidative nivolumab/placebo will follow CRT. If the consolidative nivolumab arm shows superior OS compared to placebo, a new standard of care will be defined for locally advanced NSCLC, provided that the QOL and toxicity profiles are acceptable to the patient. Past efforts at treatment intensification such as concurrent chemotherapy cetuximab plus platinum-based CRT or induction chemotherapy regimens, have failed to improve survival yet have shown substantial increases in toxicity, unplanned treatment breaks or delays in therapy, all of which can lead to inferior QOL outcome and potential decreased efficacy, such that trials investigating further refinement of concurrent CRT regimens need to factor in QOL with toxicity outcome. The effects of nivolumab immunotherapy following standard CRT in lung cancer on QOL are currently unknown. Hence, this study will provide a unique and critical opportunity to characterize the effects of this novel treatment on QOL. It is anticipated consolidative nivolumab will improve OS with minimal overlapping acute toxicities of nivolumab and CRT. Obtaining QOL data at baseline, during treatment, and follow up after treatment provides baseline prognostic data as well as reporting the effect of treatment on HRQOL
from the patient's perspective. Baseline QOL data was found to be a prognostic variable in addition to clinical factors in predicting survival in cancer patients (Quinten 2014, Urba 2012) in a pooled analysis of 7417 cancer patients entered on randomized trials. These data suggest that obtaining baseline QOL data are of superior prognostic significance than obtaining physician graded performance scale scores such as Karnofsky performance scales or ECOG scales prior to initiation of treatment. The primary QOL and PRO objective in this study is to measure functional, physical and lung cancer specific QOL in lung cancer patients using the change from baseline of FACT-TOI at 15 months (using each patient as his or her own control). A clinically meaningful between group change from baseline is defined as FACT-TOI of ≥5 points between arms. The FACT-TOI instrument will also measure longitudinal QOL in both arms and will be administered at the following time points: baseline, prior to nivolumab/placebo, at 3, 6, 9 and 12 months during nivolumab/placebo treatment, and at 15 and 18 months from start of nivolumab/placebo.
rtog foundation rtog 3505
fact-toi

In order to analyze the difference in QOL between all arms, we plan to use a brief, validated instrument that is user friendly and has clinical relevance. FACT-TOI is a measure that sums the functional well-being (FWB), physical well-being (PWB), and the lung cancer subscale (LCS) of the Functional Assessment of Cancer Therapy - Lung
(FACT-L) QOL instrument, which has been extensively used for measuring QOL in patients with lung cancer. In a review of literature reported that the FACT-L scale has been used in more than 5,000 patients and has been found to be sensitive to changes in performance status, treatment response. FACT has been translated into many languages and is available free of charge to institutions with the completion of an agreement to share data, accessible at http://www.facit.org/translation/licensure.aspx. The full FACT-L questionnaire can be completed in less than 10 minutes. This instrument has not only been shown to be prognostic for survival, but also sensitive to changes in QOL on serial evaluations throughout treatment. Importantly, the FACT-TOI has been associated with clinically meaningful changes in patients with lung cancer. The lung cancer sub-scale (LCS) consists of 9 items, involving lung cancer specific symptoms. All items are rated on a 5-item (point) Likert Scale, from 0 (not at all) to 4 (very much). It has been determined that a 5-point difference on the FACT-TOI is associated with a meaningful difference in clinical and subjective indicators. Thus, a difference of 5 points will be considered clinically significant. Handling of missing data will be in accordance with the FACIT Administration and Scoring Guidelines, described at www.facit.org.
rtog foundation rtog 3505
fatigue in nsclc

Fatigue in lung cancer patients receiving consolidative nivolumab is caused by immune mediated effects combined with RT dose volume factors and chemotherapy toxicities. In melanoma studies investigating nivolumab, the most common toxicity described is fatigue. Other toxicities include dermatologic toxicity, gastrointestinal, symptoms (diarrhea/colitis, decreased appetite). Rare immunologic mediated toxicities from immunotherapy including nivolumab and ipilumumab have been described and include pneumonitis and endocrinopathies (pituitary, hypothalamus, thyroid, and adrenal disease). These immune-mediated toxicities may present with nonspecific symptoms such as fatigue, headache, mental status changes, abdominal pain, change in bowel habits, or hypotension. In lung cancer, fatigue is one of the most common and distressing symptoms affecting up to 60% of patients. With concurrent chemoradiation, the majority of patients experience fatigue usually peaking during the first and second weeks after completion of CRT, which can remain higher than baseline in approximately half of patients long-term (Spratt 2012). Because approximately 25% of patients undergoing nivolumab monotherapy experience fatigue, the effect of consolidative nivolumab following CRT on patient-reported fatigue warrants study. Hence an exploratory objective is to evaluate patient reported fatigue using the PROMIS fatigue short form in patients receiving nivolumab following chemoradiation for lung cancer. PROMIS-Fatigue: A Novel Short Form Fatigue Scale The National Institutes of Health (NIH) Patient-Reported Outcomes Measurement Information System (PROMIS) Roadmap initiative (www.nihpromis.org) was initiated. PROMIS is a 5-year cooperative group program of research designed to develop, validate, and standardize item banks to measure patient-reported outcomes (PROs) relevant across common medical conditions, including cancer [Cella, 2007; Garcia, 2007]. Integral to the work of this group, includes the creation of a PROMIS-derived fatigue short form (using limited questions to minimize patient burden) that was developed for ease of use in oncology populations. While the psychometric properties of this 7-question short fatigue scale have been validated in the general population [Garcia, 2007; Lai, 2008], validation in patients with cancer is ongoing. A "cross-walk" has been successfully developed between the PROMIS fatigue item bank and the PROMIS-Cancer fatigue item bank that produced the short form measure. These two item banks, sharing 54 common items, were linked by equating item parameters using items that held stable psychometric properties between the cancer and general population populations in which they were tested. Results showed that cancer patients reported more severe fatigue (1/3 standard deviation more severe, but the same scale characteristic curve slope) than the general population, which matches clinical expectations [Cella, 2008]. The PROMIS fatigue will be completed at baseline, prior to nivo/placebo, at 3, 6, 9 and 12 months during nivolumab/placebo treatment, and at 15 and 18 months from start of nivolumab/placebo.
rtog foundation rtog 3505
quality-adjusted survival euroqol (eq-5d-5l)

The EuroQol (EQ-5D) is a well-accepted instrument to measure general QOL and costutility analysis (Pickard 2007) and will be used to assess quality-adjusted survival for this study. It is a two-part questionnaire that the patient can complete in 5 minutes (Schulz 2002) and has been translated into multiple languages. The first part consists of 5 items covering 5 dimensions, including mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. Each dimension can be graded on 3 levels including: 1-no problem, 2moderate problems, and 3-extreme problems. There are 243 potential health states. The second part is a visual analogue scale (VAS) valuing current health state, measured on a 20 cm, 10 point-interval scale. Either the index score or the VAS score can be used in the quality-adjusted survival analysis (Wu 2002). The benefit of measuring quality-adjusted survival is that the product, quality-adjusted survival, can be compared to the outcomes of other interventions across disease sites and can be used by health policy makers to rank interventions. The EQ5D will be used to evaluate the effect of adding nivolumab to chemoradiation on quality-adjusted survival. Protocol-eligible patients will be included in the quality-adjusted survival analysis only if they have provided baseline and at least 1 subsequent measurement. Patients will complete the EQ-5D-5L version at the following time points: baseline, prior to starting nivolumab/placebo phase, at 3, 6, 9 and 12 months during nivolumab/placebo treatment, and at 15 and 18 months, 2, 3, 4, and 5 years from start of nivolumab/placebo.
rtog foundation rtog 3505
12. modality reviews 12.1 radiation

The Radiation Oncology Co-Chair, James Urbanic, M.D. and delegates, will perform an RT Quality Assurance Review after the ACR Core Laboratory/RTQA has received complete data for the first 20 cases enrolled. Dr. Urbanic will perform the next review after the ACR Core Laboratory/RTQA has received complete data for the next 20 cases enrolled. Subsequent reviews will continue in a periodic and timely fashion. The final cases will be reviewed within 3 months after this study has reached the target accrual or as soon as the ACR Core Laboratory/RTQA has received complete data for all cases enrolled, whichever occurs first. The goal of the review is to evaluate protocol compliance. The review process is contingent on timely submission of radiotherapy treatment data as specified in Section 13.2. The scoring mechanism is: Per Protocol, Variation Acceptable, and Deviation Unacceptable.  
12.2 Drug Quality Assurance Reviews The Principal Investigator/Medical Oncology, David Gerber, MD and Medical Oncology Co-Chair, Corey Langer, M.D., will perform a Chemotherapy Assurance Review of all patients who receive or are to receive chemotherapy in this trial. The goal of the review is to evaluate protocol compliance. The review process is contingent on timely submission of chemotherapy treatment data as specified in Section 13. The scoring mechanism is: Per Protocol, Acceptable Variation, Unacceptable Deviation, and Not Evaluable. Drs. Gerber and Langer will perform a Quality Assurance Review after RTOG Headquarters has received complete data for the first 20 cases enrolled. Drs. Gerber and Langer will perform the next review after RTOG Headquarters has received complete data for the next 20 cases enrolled. The final cases will be reviewed within 3 months after this study has reached the target accrual or as soon as RTOG Headquarters has received complete data for all cases enrolled, whichever occurs first.
rtog foundation rtog 3505
13. data and records

This study will utilize Medidata Rave. See the Data Management Plan in the studyspecific guide on the RTOG Foundation 3505 protocol page of the RTOG website, www.rtog.org, for further details.  
13.1 Summary of Dosimetry Digital RT Data Submission (Submit to ACR Core Laboratory/RTQA see the study-specific guide on the RTOG Foundation 3505 protocol page of the RTOG website, www.rtog.org, for details of TRIAD.)  
| Item                                  | Due                          |
|---------------------------------------|------------------------------|
| Treatment Planning Data exported from | Within 1 week of start of RT |
| planning system and uploaded to TRIAD |                              |
rtog foundation rtog 3505
all digital rt data must

| will include the following:  Planning CT                                                                                                                                                                                                                                                                                                                 | Indicate "RT Digital Data" in  Submission Type in TRIAD for all  planning files                          |                                |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------|--------------------------------|
|                                                                                                                                                                                                                                                                                                                                                          | RT Structure File - all required  structures included and labeled per  tables in Section 5.2.5 and 5.2.6 |                                |
|                                                                                                                                                                                                                                                                                                                                                          | RT Dose File (one file for total  prescription dose)                                                     |                                |
|                                                                                                                                                                                                                                                                                                                                                          | RT Plan File                                                                                             |                                |
|                                                                                                                                                                                                                                                                                                                                                          | PET Scan - if used to aid                                                                                | Indicate "Imaging-Baseline" in |
| contouring for treatment plan                                                                                                                                                                                                                                                                                                                             | Submission Type in TRIAD                                                                                 |                                |
| Notes:  Upon submission of all digital RT data via TRIAD, complete an online Digital Data  transmission form (DDSI) located on the RTOG Foundation 3505 websitehttps://www.rtog.org/clinicaltrials/rtogfoundationstudies/rtogfoundationstudy350 5.aspx  RTOG 3505 DVH Analysis Worksheet  Note: Data will not be processed until DDSI form is received |                                                                                                          |                                |  
13.2 Summary of Diagnostic Imaging Digital Data Submission (Submit to ACR Imaging Corelab; see the study-specific guide on the RTOG Foundation 3505 **protocol page of the** RTOG website, www.rtog.org, for details of TRIAD.))
rtog foundation rtog 3505
all digital diagnostic imaging data

| Item                                                                   | Due (via TRIAD) 1                                                                                                                                                  |
|------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Contrast-enhanced CT of the Chest to                                   | Within two weeks of acquisition                                                                                                                                    |
| include liver and adrenals (Baseline)2 Contrast-enhanced CT/MRI of the | Within two weeks of acquisition                                                                                                                                    |
| Brain (Baseline) FDG-PET/CT Whole-Body (Baseline)                      | Within two weeks of acquisition                                                                                                                                    |
| Contrast-enhanced CT of the Chest to  include liver and adrenals (postchemoRT)2                                                                        | Within two weeks of acquisition                                                                                                                                    |
| Contrast-enhanced CT of the Chest to  include liver and adrenals (FollowUp)2                                                                        | Within two weeks of acquisition  Every three months for the first  two years (e.g., Months 3, 6, 9,  etc.)  Every six months for years 3-5  Annually thereafter |  
| Contrast-enhanced CT of the Chest to  include liver and adrenals in timepoints of site-determined PD2                   | Within 3 days of acquisition    |
|-------------------------------------------------------------------------------------------------------------------------|---------------------------------|
| FDG-PET/CT Whole-Body (Follow-up;                                                                                       | Within two weeks of acquisition |
| perform as clinically indicated)3 Contrast-enhanced CT/MRI of the  Brain (Follow-up; perform as clinically  indicated)3 | Within two weeks of acquisition |
| Notes:                                                                                                                  |                                 |  
Notes:
1*All images should be accompanied by an Image Transmittal Worksheet (ITW).* 2If IV contrast is contraindicated, MRI Chest may be submitted in place of a contrast-enhanced CT
Chest, along with a non-contrast-enhanced CT Chest.  
3 Patients with uncertain progression may require repeat imaging at 6 weeks rather than 3 months if disease status remains uncertain.
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14. statistical considerations 14.1 study

Patients will be enrolled and randomized 1:1 between two arms. The treatment allocation scheme described by Zelen (1974) will be used. Patients will be stratified by Zubrod Performance Status (0 vs. 1); histology (squamous vs. non-squamous), as there may be a trend toward greater benefit of nivolumab in squamous histology (Topalian 2012); and tumor PD-L1 status.
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14.2 study endpoints

14.2.1 Primary endpoints:
Overall survival (OS)
OS is defined as the time between the date of randomization and the date of death due to any cause. OS will be censored on the last date a subject is known to be alive. Progression free survival (PFS) based on Independent Radiology Review Committee (IRRC) according to RECIST 1.1 PFS is defined as the time between the date of randomization and the first date of documented progression, as determined by IRRC according to RECIST 1.1, or death due to any cause, whichever occurs first. Please refer to Section 4 "Central Imaging Review" for details on IRRC PFS data generation and collection, and Section 14.3.2 for detailed definition of PFS events and censoring.  
14.2.2 Secondary endpoints:
Toxicities Functional Assessment of Cancer Therapy - Trial Outcome Index for lung cancer (FACTTOI) at 15 months OS and PFS in patients with (1) PD-L1-positive, (2) PD-L1-negative and PD-L1 not evaluable/undetermined tumors 14.2.3 Exploratory endpoints biomarker and biomarker correlatives proportion of patients alive at 12 and 24 months proportion of patients progression free at 12 and 24 months using investigational site assessments according to RECIST 1.1  
PROMIS fatigue at 3 months Descriptive analysis of the EQ-5D utilities and the EQ-VAS by study arm before and after progression.  
PFS based on investigator assessment.
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14.3 primary objectives study design

The primary hypotheses of this study are (1) nivolumab increases OS with a hypothesized hazard ratio of 0.7; (2) nivolumab increases IRRC-determined PFS with a hypothesized hazard ratio of 0.667 (control arm is the reference level). In particular, assuming OS and PFS are exponentially distributed (at least approximately), the alternative hypothesis for OS is that patients receiving the experimental regimen will have a median survival time of at least 34.3 months, while those receiving the control regimen will have a median survival time of 24 month. Similarly, the alternative hypothesis for PFS assumes the median times for PFS will be 12 and 18 months for the control and experimental arms, respectively.  
14.3.2 Statistical Analysis Plan for Primary Endpoints The primary analysis will be performed on an intent-to-treat (ITT) basis, such that all randomized cases will be included in the treatment arm to which they were randomized regardless of what treatment the patients actually received. This is the primary dataset for analyses of demography, protocol deviations, baseline characteristics, and efficacy outcome research. The study accounts for two primary endpoints: OS and PFS. Overall (2-sided) alpha is 0.05, which is split with 0.01 for evaluating PFS and with 0.04 for evaluating OS. The primary OS analysis will be conducted using a two-sided log-rank test stratified by histology, Zubrod and PD-L1 status in all randomized patients. Hazard ratio (HR) and corresponding two-sided 96% confidence interval (CI) will be estimated using a Cox proportional hazard model, with treatment group as a single covariate, stratified by the above stratification factors. The stratification factors will be obtained at the time of randomization. Results from an unstratified analysis will also be provided. The rates at various timepoints (i.e., every 6 months after randomization) and medians of OS for each arm will be estimated using the Kaplan-Meier method (1958). The associated 95%
confidence interval (CI) will be calculated using Greenwood's formula and based on a log-log transformation applied on the survival function. The primary PFS analysis will be conducted using the same methods and stratification factors as OS analysis, except that the hazard ratio (HR) and corresponding two-sided 99% confidence interval (CI) will be estimated using a stratified Cox proportional hazard model. PFS is defined as the time between the date of randomization and the first date of documented progression, regardless of discontinuation of study drug, or death due to any cause, whichever occurs first. Progressive disease (PD) is determined by IRRC according to RECIST 1.1. Subjects who die without a reported progression will be considered to have the PFS event on the date of their death. Subjects who do not progress or die will be censored on the date of their last evaluable tumor assessment as determined with radiographic evidence. Subjects who do not have any on-study tumor assessments and do not die will be censored on their date of randomization. For the primary PFS analysis, subjects who have palliative local therapy or initiate systematic anti-cancer therapy (aka new therapy) without a prior reported progression or a progression confirmed by IRRC, whichever occurs first, will be censored on the date of their last evaluable tumor assessment as determined with radiographic evidence prior to the permanent discontinuation of treatment or the initiation of subsequent anti-cancer therapy or palliative local therapy.  
Two sensitivity analyses will be performed to assess the robustness of the primary PFS analysis: 1. Sensitivity analysis 1 is the same as the primary analysis except that for patients with a PFS event who miss two or more scheduled assessments immediately prior to the PFS event, data will be censored at the last tumor assessment as determined with radiographic evidence prior to the missed visits.  
2. Sensitivity analysis 2 is the same as the primary analysis except that it considers permanent discontinuation of study treatment (per Section 6.4) or initiation of palliative local therapy or systematic anti-cancer therapy, whichever occurs earlier, to be a PD event for subjects without a documented PD or death.  
| Table 3: Censoring rules for Primary and Sensitivity Analyses of PFS Situation Primary Analysis Sensitivity  Sensitivity  Analysis 1 Analysis 2 No PD and no  death; new  therapy is not  initiated Censored at last  Censored at last  Censored at last  disease assessment disease assessment disease assessment  if still on study  therapy; otherwise  progressed at  treatment  discontinuation No PD and no  death; new  therapy is  initiated Progressed at date  of new therapy or  treatment  discontinuation PD or death  documented after  ≤1 missed disease  assessments Censored at last  disease assessment  prior to new  therapy Censored at last  disease assessment  prior to new  therapy Progressed at date  of documented PD  or death Progressed at date  of documented PD  or death Progressed at date  of documented PD  or death PD or death  documented after  ≥2 missed disease  Progressed at date  of documented PD  or death Censored at last  disease assessment  immediately prior  Progressed at date  of documented PD  or death   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
The censoring rules for primary and sensitivity analyses are summarized in Table 3.  
assessments to the ≥2 missed disease assessments To determine whether the treatment effect is consistent across various subgroups, the estimate of the between-group treatment effect (with a nominal 95% CI) for the primary endpoints will be estimated and plotted within each category of the following classification variables:
Age category (≤65, >65 years)
Sex (female, male)
Race (white, non-white) ECOG status (0, 1)
Histology (squamous, non-squamous)
Smoking status (never, former, current)
The consistency of the treatment effect will be assessed descriptively via summary statistics by category for the classification variables listed above.
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14.3.3 sample size and power

The study accounts for two primary endpoints: OS and PFS. Overall (2-sided) alpha is 0.05, which is split with 0.01 for evaluating PFS and with 0.04 for evaluating OS. The study is event-driven and plans to randomize 550 patients with 1:1 ratio into the experimental and control arms. Assuming a rate of 16.7% due to ineligibility and dropout between initial registration and randomization, a total of 660 patients will be enrolled to ensure 550 patients will be randomized after completion of chemoradiation. Assuming a uniform monthly accrual rate of 14.4 patients (12 eligible patients to be randomized), and minimal accrual in the first 6 months for ramp-up, the accrual is projected to last 52 months (4.3 years) with additional 25 months follow up. The overall study is projected to last 71 months (5.9 years) since the first patient first visit (FPFV), or 77 months (6.4 years) since the study activation to reach the required number of events. If a higher rate of ineligible and drop-out is observed during the study conduct, the protocol may be amended to ensure at least 550 eligible patients to be randomized.  
The null hypothesis for IRRC-assessed PFS is that both arms will have a median survival time of 12 months. The alternative hypothesis for PFS is that patients receiving the experimental regimen will have a median survival time of at least 18 months, corresponding to a 33% relative hazard reduction (HR=0.667). There is only one (final) PFS analysis planned and will be conducted after at least 363 PFS events are observed between the experimental and control arms. The analysis is projected to occur after 50 months from first patient first visit (FPFV) and 4 months after the completion of planned accruals. If the initial accruals are slower than expected such that 363 PFS events are observed prior to the completion of planned patient accruals, then the analysis may be delayed for 6 months to allow all randomized subjects to have a reasonable amount of follow-up information for the primary PFS analysis. With at least 363 PFS events, the study has ≥90% power to detect a hazard ratio of 0.667 for PFS at alpha=0.01 (2-sided) at this planned final PFS analysis. A p-value less than 0.01 (1-sided 0.005) using stratified log-rank test for PFS approximately corresponds to an empirical hazard ratio of 0.763 (or approximately at least 15.7 months of median PFS in experimental vs. 12 months in control, if PFS in both arms follow exponential distributions approximately). The null hypothesis for overall survival is that both arms will have a median survival time of 24 months. The alternative hypothesis for overall survival is that patients receiving the experimental regimen will have a median survival time of at least 34.3 months, corresponding to a 30% relative hazard reduction (HR=0.7). Using a 2-sided test with alpha of 0.04 and two planned OS analyses, a total of 355 deaths (complete information of OS) and 550 analyzable patients are required to detect the hypothesized survival difference with 90% power. There is one interim OS analysis and one final OS analysis is planned. The interim analysis will be conducted at the same time of the final PFS analysis. Based on the proportion of information available at the interim OS
analysis, the Lan-DeMets implementation of O'Brien-Fleming boundary will be used to determine the critical value for the interim analysis. It is projected at the time of PFS final analysis, projected to occur after 50 months from first patient first visit (FPFV) and 4 months after the completion of planned accruals, approximately 230 OS events or 65% cumulative information for OS will be observed. The final OS analysis will occur when complete information for overall survival, i.e., observing at least 355 deaths, is available. The final OS analysis is projected to occur after 71 months from first patient first visit (FPFV). The trial will be declared positive if either the overall or progression-free survival comparison of the treatments is statistically significant favoring the experimental arm at 0.04 and 0.01 (2-sided), respectively. For OS analysis, the boundary for statistical significance at the interim analysis and the final analysis will be determined based on the Lan-DeMets implementation of O'Brien-Fleming use function. For example, with 2sided alpha of 0.04 and using stratified log-rank test, if 230 deaths have occurred at the time of the interim analysis, the statistical significance will be declared p≤0.008 (1-sided 0.004, approximately corresponds to an empirical hazard ratio of 0.704); and if 355 deaths have occurred at the time of the final OS analysis, statistical significance will be declared if p≤0.0376 (1-sided 0.0188, approximately corresponds to an empirical hazard ratio of 0.802).  
If the long-term clinical effect ("cure rate") is present around 3 to 3.5 years since randomization, the study is anticipated to last 2 additional years of follow-up to observe the required number of events, i.e., the entire study duration may be 101 months (8.4 years) or longer. The sample size and power calculation were performed in the software EAST.
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14.4 study monitoring

14.4.1 Interim Analysis for the DMC
The RTOG Foundation Data Monitoring Committee (DMC) will review the study twice a year with respect to patient accrual and safety after enrollment of the first patient. An interim study summary report will be prepared at each meeting accordingly until the initial study results have been released. In general, the interim reports will contain information about 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 endpoint.
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14.4.2 interim analysis for safety

To ensure the safety of this trial, the rates of protocol-specified adverse events between the arms will be compared after 13.3% and 26.7% of the eligible and evaluable patients who receive any protocol treatment (40 and 80 on each arm) have been accrued to the trial and observed for at least 180 days after the end of chemoradiation treatment (or die within 180 days). The As-Treated (AT) patient population will be used for the analysis of safety data in this study. The AT population consists of all randomized subjects who received at least one dose of study treatment (nivolumab or placebo). Subjects will be included in the treatment group corresponding to the trial treatment they actually received for the analysis of safety data. With a total of 160 patients approximately evenly receiving nivolumab and placebo, we will have at least 85% power to detect the following difference in the rates of protocolspecified AE occurring within 180 days after the end of concurrent chemoradiation treatment at the significance level of 0.05 (2-sided): 5% vs.25%, 10% vs. 30%, and 15%
vs. 35%. Using the Lan-DeMets implementation of O'Brien-Fleming boundary, if the pvalue related with this comparison is less than 0.003 and 0.049 (2-sided) the trial accrual
(if applicable) will be suspended due to safety concerns. In the event of excessive protocol-specified adverse events such that the trial accrual suspension is warranted, RTOG Foundation DMC will hold a special meeting to review the study data and provide a recommendation as to whether the study may continue, whether amendment(s) to protocol should be implemented, or whether the study should be stopped. The difference in rates between the two treatment arms and their two-sided 95% CI, as well as associated odds-ratios will be estimated using the Cochron-Mantel-Haenszel (CMH) method of weighting (Agresti 2002), adjusting for the stratification factors. The difference in rate will also be compared using Cochron-Mantel-Haenszel (CMH) test, adjusting for the stratification factors, when 80 and 160 eligible and evaluable AT patients available from both treatments. Protocol-specified adverse events are defined as following: The development of grade 3 or higher adverse events definitely, probably, or possibly related to treatment for the following adverse events within 180 days after the end of concurrent chemoradiation treatment:  
| ChemoRT Toxicity     | Notes                                                                             |                                |
|----------------------|-----------------------------------------------------------------------------------|--------------------------------|
| Cardiac Disorders    | Grade 3 or higher: Acute coronary  syndrome, Atrial fibrillation, Atrial flutter, |                                |
| RTOG Foundation 3505 | 80                                                                                | Version Date: January 30, 2017 |  
| Conduction disorder, Pericardial effusion,  Pericarditis, Restrictive cardiomyopathy   |                                                                                                                                                                                                                                                                                                                                             |
|----------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Gastrointestinal Disorders                                                             | Grade 4 or higher: Dysphagia,  Esophagitis, Esophageal fistula,  Esophageal obstruction, Esophageal  perforation, Esophageal stenonsis,  Esophageal ulcer, Esophageal  hemorrhage                                                                                                                                                           |
| Injury, Poisoning, and Procedural                                                      | Grade 3 or higher: Dermatitis radiation,                                                                                                                                                                                                                                                                                                    |
| Complications                                                                          | Fracture (limited to rib fractures only)                                                                                                                                                                                                                                                                                                    |
| Nervous System Disorders                                                               | Grade 3 or higher: Brachial plexopathy,  Recurrent laryngeal nerve palsy, Myelitis                                                                                                                                                                                                                                                          |
| Respiratory, Thoracic, and Mediastinal                                                 | Grade 3 or higher: Atelectasis (grade 4-5                                                                                                                                                                                                                                                                                                   |
| Disorders                                                                              | only), Bronchopulmonary hemorrhage,  Mediastinal hemorrhage, Pleural  hemorrhage, Tracheal hemorrhage,  Bronchial fistula, Pulmonary fistula,  Bronchopleural fistula, Tracheal fistula,  Hypoxia (provided gr.3 is worse than  baseline), Bronchial obstruction, Tracheal  obstruction, Pleural effusion,  Pneumonitis, Pulmonary fibrosis |
| Skin and Subcutaneous Disorders                                                        | Grade 3 or higher: Skin ulceration (thorax  only)                                                                                                                                                                                                                                                                                           |
| Changes in PFTs per the RTOG  Pulmonary Toxicity Scale (see Section  5.2.14)           | Grade 3 or higher: FEV1 decline, Forced  Vital Capacity decline, DLCO decline                                                                                                                                                                                                                                                               |
| IMMUNE-MEDIATED Toxicity                                                               | Notes                                                                                                                                                                                                                                                                                                                                       |
| Skin Rash and Oral Lesions                                                             | Grade 3 or higher: Bullous dermatitis,  Erythema multiforme, Erythroderma,  Palmar-plantar erythrodysesthesia  syndrome, Papulopustular rash, Pruritus,  Purpura, Rash acneiform, Rash maculopapular, Skin ulceration, Stevens-Johnson  syndrome, Toxic epidermal necrolysis,                                                                                                                                                                                                                                                                                                                                             |
| Liver Function, AST/ALT/T. Bili                                                        | Grade 3 or higher                                                                                                                                                                                                                                                                                                                           |
| Renal Function, Serum Creatinine                                                       | Grade 3 or higher                                                                                                                                                                                                                                                                                                                           |
| Diarrhea/Colitis                                                                       | Grade 3 or higher;                                                                                                                                                                                                                                                                                                                          |  
| Pancreatitis, Amylase/Lipase     | Grade 3 with development of  symptomatic pancreatitis or DM; Grade 4  or higher                                                                                                                                        |
|----------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Pneumonitis                      | Grade 3 or higher                                                                                                                                                                                                      |
| Other GI (including N-V)         | Grade 3 and symptoms not resolving  within 7 days with symptomatic  treatment; Grade 4 or higher                                                                                                                       |
| Neurologic Events                | Grade 3 or higher; any CNS events  including aseptic meningitis, encephalitis,  or myopathy, peripheral demyelinating  neuropathy, cranial neuropathy (other than  peripheral n. VII), GB syndrome,  myasthenia gravis |
| Endocrine, Hypophysitis, Adrenal | Grade 3 or higher                                                                                                                                                                                                      |
| Insufficiency Uveitis/Ocular     | Grade 3                                                                                                                                                                                                                |
| Infusion Reaction                | Grade 3 or higher                                                                                                                                                                                                      |
| All Other Immune-Mediated Events | Grade 3 or higher                                                                                                                                                                                                      |
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14.4.3 interim analysis for efficacy

The RTOG Foundation Data Monitoring Committee (DMC) will review the interim analysis for superiority based on OS and the primary analysis of PFS. Both analyses are planned to occur and the results will be reviewed by DMC at the same time. If either the primary PFS analysis or the interim OS analysis is deemed to be positive per the respective rules, the study results will be unblinded and reported. Otherwise, the study will remain open and the results will continue to remain blinded. The primary PFS analysis is planned after 363 PFS events are observed between the experimental and control arms. The analysis is projected to occur after 50 months from first patient first visit (FPFV) and 4 months after the completion of planned accruals. If the initial accruals are slower than expected such that 363 PFS events are observed prior to the completion of planned patient accruals, then the analysis may be delayed for 6 months to allow all subjects to have a reasonable amount of follow-up for PFS analysis. The interim OS analysis will be based on the proportion of complete OS information available at the time of primary PFS analysis. It is projected that approximately 230 OS events or 65% cumulative information for OS (355 deaths) will be observed.
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14.5 accrual/study duration considerations

The study is event-driven and plans to randomize 550 patients with 1:1 ratio into the experimental and control arms. Assuming a uniform monthly accrual rate of 14.4 patients (12 eligible patients to be randomized), and minimal accrual in the first 6 months for ramp-up, the accrual is projected to last 52 months (4.3 years), and the study is projected to last 77 months (6.4 years) to reach the required number of events. The above projected study durations are based on the hypothesized design parameters. If the actual study parameters deviate from the hypothesized ones, the actual study duration may be different from the projection
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14.6 dose level guidelines na

14.7.1 Toxicities The As-Treated (AT) patient population will be used for the analysis of safety data in this study. The AT population consists of all randomized subjects who received at least one dose of study treatment (nivolumab or placebo). Subjects will be included in the treatment group corresponding to the trial treatment they actually received for the analysis of safety data. Exposure to nivolumab treatment and length of safety follow-up will be summarized. For each patient, the maximum severity reported will be used in the summaries. Adverse events will be summarized regardless of relationship to study drug as assessed by the investigator. All adverse events, adverse events leading to withdrawal of study drug, adverse events leading to dose reduction or interruption, Grade ≥ 3 adverse events, serious adverse events, and adverse events of special interest will be summarized. Deaths and cause of death will be summarized. 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, the analysis will be performed at the time of primary endpoint analysis.
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14.7.2 functional assessment of cancer

The QOL analyses will be performed based on all randomized patients. FACT-TOI complete rates will be summarized at each assessment point as the proportion of assessments actually received out of the expected number (i.e., the number of subjects still on treatment in follow-up). Baseline and FACT-TOI at each subsequent assessment, as well as their change from baseline will be summarized using descriptive statistics by treatment group as randomized. The summary at baseline and at each time point is based on all randomized subjects with a measurement at respective time point. The change from baseline analysis will only include subjects who have an assessment at baseline and at the subsequent time point. FACT-TOI deterioration rate at 15 months is defined as the proportion of randomized subjects who had 5 points or more decrease from baseline in FACT-TOI. FACT-TOI deterioration rate at 15 months and associated 95% confidence interval will be calculated for each treatment group, based on all randomized subjects. Clopper-Pearson method will be used for calculating 95% CI. Based on prior trials in locally-advanced NSCLC, we expect about 40% patients will remain for evaluation at 15 months. Based on RTOG 0617, the deterioration rate at 1 year was about 50%. Therefore, assuming a 60% deterioration rate 15 months in control arm, about 220 evaluable patients at 15 months will detect a 20% difference (e.g., 60% vs. 40%) in deterioration rate with about 85% power at a two-sided alpha of 0.05, after taking account of the potential treatment effects which may result in unequal number of patients between arms. The scores at baseline and subsequent time points, as well the associated changes from baseline for each treatment group will be compared using the two-sample t-test. If the parametric assumptions are not met, then the Mann-Whitney test will be used. Effect size of FACT-TOI changes at different time points will be calculated based on Cohen's d, i.e.,
dividing the difference between arms in mean score changes by the pooled standard deviation of the baseline score means. Longitudinal data analysis will also be performed to characterize the trend of scores over time across the two treatment groups using hierarchical formulation of the linear mixed model. The model will include treatment groups, stratification factors and time.
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14.7.3 os and pfs in

If the trial is declared positive based on either OS or PFS in the overall ITT population, a step-down procedure will be applied to OS and PFS in the ITT subgroups in the following order: (1) PD-L1 positive (both ≥1% and ≥50% will be assessed); (2) PD-L1 negative, (3) PD-L1 undetermined, and the overall 2-sided alpha 0.05 is recycled for each subgroup analysis. Within each PD-L1 status group, OS and PFS will be analyzed using a 2-sided log-rank test stratified by histology and Zubrod in all randomized patients. Hazard ratio (HR) and corresponding two-sided 95% confidence interval (CI) will be estimated using a Cox proportional hazard model, with treatment group as a single covariate, stratified by histology and Zubrod. Results from an unstratified analysis will also be provided. The rates at various timepoints (i.e., every 6 months after randomization) and medians of OS for each arm will be estimated using the KaplanMeier method (1958). The associated 95% confidence interval (CI) will be calculated using Greenwood's formula and based on a log-log transformation applied on the survival function. In the event that study is not declared positive based on either OS or PFS in the ITT population, the presence of qualitative interactions between PD-L1 status and nivolumab will be assessed by analyzing OS and PFS within each PD-L1 positive and negative subgroups separately for exploratory purposes. The statistical analysis plan will be same except that no formal hypothesis testing will be performed. In addition, when assessing the presence of quantitative interactions is warranted based on exploratory subgroup analyses, an overall global interaction test for OS and PFS will be conducted. This will be performed in the overall ITT population by comparing the fit of a Cox proportional hazards model including treatment, PD-L1 status, and treatment-by-PD-L1 status interaction terms, stratified by histology and Zubrod, with one that excludes the interaction terms at the 2-sided 10% significance level. If the fit of the model is not significantly improved with including the global interaction terms, then it will be concluded that the overall treatment effect is consistent across the PD-L1 status subgroups. If there are too few OS events and PFS events available for a meaningful analysis of a particular subgroup, e.g., less than 20 events in a subgroup, the aforementioned subgroup statistical analysis plan will not be performed for statistical inferences, and only descriptive summaries of OS and PFS will be provided.
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14.8 exploratory hypothesis and endpoints

14.8.1 Biomarker analysis Exploratory biomarker analyses will be performed in an effort to understand the association of these markers with study drug response, including efficacy and/or adverse events. 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 available.  
14.8.2 Proportion of patients alive and progression-free at 12 and 24 months The proportion of patients alive and progression-free at 12 and 24 months will be estimated by the Kaplan-Meier methodology for each arm, and the 95% CI will be calculated using Greenwood's formula and based on a log-log transformation applied on the survival function in the ITT population.
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14.8.3 promis fatigue

Baseline and PROMIS at each subsequent assessment, as well as their change from baseline will be summarized using descriptive statistics by treatment group as randomized. The summary at baseline and at each time point is based on all randomized subjects with a measurement at respective time point. The change from baseline analysis will only include subjects who have an assessment at baseline and at the subsequent time point. Changes in PROMIS fatigue at 3 months are of particular interest.
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14.8.4 eq5d

EQ-5D consists of the EQ-5D descriptive system and the EQ visual analogue scale (EQ VAS). The EQ-5D descriptive system comprises the following 5 dimensions: mobility, self-care, usual activities, pain/discomfort and anxiety/depression. Each dimension has 5 levels: no problems, slight problems, moderate problems, severe problems and extreme problems. The EQ VAS records the subject's self-rated health state on a 100-point vertical, visual analogue scale (0 = worst imaginable health state; 100 = best imaginable health state.  
Subjects' overall health state on a visual analog scale (EQ-VAS) at each assessment time point will be summarized using descriptive statistics by treatment group, as randomized. Proportion of subjects reporting problems for the five EQ-5D dimensions at each assessment time point will be summarized by level of problem and by treatment group, as randomized. Percentages will be based on number subjects assessed at assessment time point.  
14.8.5 PFS based on investigator assessment PFS based on investigator assessment (inv-PFS) is defined similarly to the co-primary endpoint, IRRC-PFS, except that progressive disease (PD) is determined by investigator's assessment based on RECIST criteria. The intention of this analysis is to illustrate if the estimation and inferential results based on inv-PFS are concordant with those based on IRRC-PFS in order to reassure the applicability of the study results in clinical practice. The analysis will be considered as exploratory and descriptive in nature. In addition to analyzing inv-PFS using the methods described in Section 14.3.2 for PFS primary analysis, the source of PFS events (PD vs. death) based on investigators' assessment and IRRC will be summarized separately, and the censoring times distributions of inv-PFS and IRRC-PFS will be summarized separately.  
REFERENCES
Ahn JS, Ahn YC, Kim JH, et al. Multinational randomized phase III trial with or without consolidation chemotherapy using docetaxel and cisplatin after concurrent chemoradiation in inoperable stage III non-small-cell lung cancer: KCSG-LU05-04. *J Clin Oncol*. 33(24):2660-6, 2015. Anscher MS, Kong FM, Andrews K, et al. Plasma transforming growth factor beta1 as a predictor of radiation pneumonitis. *IJROBP*. 41:1029-35, 1998. Anscher MS, Marks LB, Shafman TD, et al. Using plasma transforming growth factor beta-1 during radiotherapy to select patients for dose escalation. *J Clin Oncol*. 19:3758-65, 2001. Belani CP, Choy H, Bonomi P, et al. Combined chemoradiotherapy regimens of paclitaxel and carboplatin for locally advanced non-small-cell lung cancer: a randomized phase II locally advanced multi-modality protocol. *J Clin Oncol*. 23:5883-91, 2005. Borghaei H, et al. Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer. *N Engl J Med*. 2015 Sep 27. Epub ahead of print: http://www.ncbi.nlm.nih.gov/pubmed/26412456 Bradley JD, Paulus R, Komaki R, et al. Standard-dose versus high-dose conformal radiotherapy with concurrent and consolidation carboplatin plus paclitaxel with or without cetuximab for patients with stage IIIA or IIIB non-small-cell lung cancer (RTOG 0617): a randomised, two-bytwo factorial phase 3 study. *Lancet Oncology*. 16(2):187-199, 2015.  
Brahmer JR, Drake CG, Wollner I, et al. Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. *J Clin Oncol*. 28:3167-75, 2010. Brahmer JR, Hom L, Antonia SJ, et al. Nivolumab (anti-PD-1; BMS-936558; ONO-4538) in patients with non-small cell lung cancer (NSCLC): overall survival and long-term safety in a phase 1 trial. International Association for the Study of Lung Cancer 15th Annual World Conference on Lung Cancer. Abstract MO18.03. 2013. Brahmer J, Reckamp KL, Baas P, et al. Nivolumab versus docetaxel in advanced squamous-cell non-small cell lung cancer. *N Engl J Med* 373:123-35, 2015. Butts CA, Socinski MA, Mitchell PL, et al. Tecemotide (L-BLP25) versus placebo after chemoradiotherapy for stage III non-small cell lung cancer (START): a randomised, double-blind, phase 3 trial. *Lancet Oncol* 15:59-68, 2014. Carthon BC, Wolchok JD, Yuan J, et al. Preoperative CTLA-4 blockade: Tolerability and immune monitoring in the setting of a presurgical clinical trial. *Clin Cancer Res*. 16:2861-71, 2010. Cella D, Yount S, Rothrock N, et al. PROMIS Cooperative Group. The Patient-Reported Outcomes Measurement Information System (PROMIS): Progress of an NIH Roadmap cooperative group during its first two years. *Med Care*. 2007; 45(5 Suppl 1):S3-S11. Cella D, Lai J, Garcia SF, et al. The Patient Reported Outcomes Measurement Information System-Cancer (PROMIS-Ca): Cancer-specific application of a generic fatigue measure. [Abstract]. J Clin Oncol. 2008; 26, 6537. Chen Y, Hyrien O, Williams J, et al. Interleukin (IL)-1A and IL-6: Applications to the predictive diagnostic testing of radiation pneumonitis. *IJROBP*. 62:260-6, 2005. Demaria S, Ng B, Devitt ML, Babb JS, Kawashima N, Liebes L, Formenti SC. Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated. *IJROBP.* 58:862-70, 2004. Deng L, Liang H, Burnette B, et al. Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice. *J Clin Invest*. 2014 Jan 2. Epub ahead of print: http://www.jci.org/articles/view/67313 Ezer N et al. Cisplatin vs. carboplatin-based chemoradiotherapy in patients >65 years of age with stage III non-small cell lung cancer. *Radiother Oncol*. 2014;112: 272-8. Fu XL, Huang H, Bentel G, et al. Predicting the risk of symptomatic radiation-induced lung injury using both the physical and biologic parameters V(30) and transforming growth factor beta. IJROBP. 50:899-908, 2001. Garcia SF, Cella D, Clauser SB, et al. Standardizing patient-reported outcomes assessment in cancer clinical trials: a patient-reported outcomes measurement information system initiative.J Clin Oncol. 2007;25(32): 5106-12. Review. Erratum in: *J Clin Oncol*. 2008; 26(6):1018. Glasziou PP, Simes RJ, Gelber RD. Quality adjusted survival analysis. *Stat Med*. 1990; 9(11):1259-76. Hanna N, Neubauer M, Yiannoutsos C, et al. Phase III study of cisplatin, etoposide, and concurrent chest radiation with or without consolidation docetaxel in patients with inoperable stage III nonsmall-cell lung cancer: The Hoosier Oncology Group and U.S. Oncology. *J Clin Oncol.* 26:575560, 2008. Hara R, Itami J, Komiyama T, et al. Serum levels of KL-6 for predicting the occurrence of radiation pneumonitis after stereotactic radiotherapy for lung tumors. *Chest*. 125:340-4, 2004. Hodi FS, Butler M, Oble DA, et al. Immunologic and clinical effects of antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in previously vaccinated cancer patients. *Proc Natl* Acad Sci U S A. 105:3005-10, 2008. Hodi FS, Dranoff G. The biologic importance of tumor-infiltrating lymphocytes. J Cutan Pathol.
rtog foundation rtog 3505
2010 37(suppl 1) 48-53.

Investigator Brochure: Nivolumab (v.14). Bristol-Myers Squibb. 30 June 2015. Ivanova A, Qaqish BF, and Schell MJ. Continuous toxicity monitoring in phase II trials in oncology. *Biometrics*. 61: 540-545, 2005. Iyengar P, Gerber DE. Locally advanced lung cancer: An optimal setting for vaccines and other immunotherapies. *Cancer J.* 19:247-62, 2013 Kelly K, Chansky K, Gaspar LE, et al. Phase III trial of maintenance gefitinib or placebo after concurrent chemoradiotherapy and docetaxel consolidation in inoperable stage III non-small-cell lung cancer: SWOG S0023. *J Clin Oncol*. 26:2450-6, 2008. Ku GY, Yuan J, Page DB, et al. Single-institution experience with ipilimumab in advanced melanoma patients in the compassionate use setting: lymphocyte count after 2 doses correlates with survival. *Cancer*. 116:1767-75, 2010. Lai J-S, Cella D, Choi SW, et al.Developing a fatigue item bank from the patient-reported outcomes measurement information system (PROMIS FIB version 1). Presented at the International Conference on Survey Methods in Multinational, Multiregional, and Multicultural Contexts (3MC). June 25-28, 2008. Larici AR, del Ciello A, Maggi F, et al. Lung abnormalities at multimodality imaging after radiation therapy for non-small cell lung cancer. *Radiographics*. 31:771-89, 2011. PMID 21571656 Le DT, et al. PD-1 blockade in tumors with mismatch-repair deficiency. *NEJM*. 372: 2509-20, 2015. Lee Y, Auh SL, Wang Y, et al. Therapeutic effects of ablative radiation on local tumor require CD8+ T cells: changing strategies for cancer treatment. *Blood*. 114:589-95, 2009. Li QZ, Karp DR, Quan J, et al. Risk factors for ANA positivity in healthy persons. *Arthritis* Research & Therapy. 27 :153-60, 2011. Liakou CI, Kamat A, Tang DN, et al. CTLA-4 blockade increases IFNgamma-producing CD4+ICOShi cells to shift the ratio of effector to regulatory T cells in cancer patients. *Proc Natl* Acad Sci U S A. 105:14987-92, 2008. Lynch TJ, Bondarenko I, Luft A, et al. Ipilimumab in combination with paclitaxel and carboplatin as first-line treatment in stage IIIB/IV non-small-cell lung cancer: results from a randomized, double-blind, multicenter phase II study. *J Clin Oncol*. 30:2046-54, 2012. Mitchell P, et al. Tecemotide in unresectable stage III non-small cell lung cancer in the phase III START study: Updated overall survival and biomarker analyses. *Ann Oncol*. 26:1134-1142, 2015.  
O'Brien PC, Fleming TR. A multiple testing procedure for clinical trials. *Biometrics*.35:549–556, 1979. Pickard AS, et al. Health utilities using the EQ-5D in studies of cancer. *Pharmacoeconomics.* 25(5):365-84, 2007. Postow MA, Callahan MK, Barker CA, et al. Immunologic correlates of the abscopal effect in a patient with melanoma. *NEJM*. 366:925-31, 2012. Quinten C, Martinelli F, Coens C, et al. A global analysis of multitrial data investigating quality of life and symptoms as prognostic factors for survival in different tumor sites. *Cancer*. 120(2):302-11, 2014.. Rizvi NA, et al. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. *Science*. 348: 124-8, 2015. Rizvi NA, Antonia SJ, Chow LQM, et al. A phase I study of nivolumab (anti-PD-1; BMS-936558, ONO-4538) plus platinm-based doublet chemotherapy (PT-doublet) in chemotherapy-naive nonsmall cell lung cancer (NSCLC) patients (pts). *J Clin Oncol*. 31 (suppl; abstract 8072), 2013.  
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.* 23(28): 7199-7206, 2005. Santana-Davila R et al. Cisplatin and etoposide versus carboplatin and paclitaxel with concurrent radiotherapy for stage III non-small-cell lung cancer: an analysis of Veterans Health Administration data. *J Clin Oncol.* 2015;33: 567-74. Santin AD, Hermonat PL, Ravaggi A, et al. Effects of concurrent cisplatinum administration during radiotherapy vs. radiotherapy alone on the immune function of patients with cancer of the uterine cervix. *IJROBP*. 48:997-1006, 2000. Socinski M, et al. CheckMate 026: A phase 3 trial of nivolumab vs investigator's choice (IC) of platinum-based doublet chemotherapy (PT-DC) as first-line therapy for stage iv/recurrent programmed death ligand 1 (PD-L1)-positive NSCLC. *Ann Oncol*. 27 (suppl_6):552-587, 2016. Schulz MW, et al. A comparison of techniques for eliciting patient preferences in patients with benign prostatic hyperplasia. *J Urol.* 168(1):155-9, 2002. Sun J-M, Zhou W, Choi Y-L et al. PD-L1 expression and survival in patients with non-small cell lung cancer (NSCLC) in Korea. *J Clin Oncol* 32:5s (abstr 8066), 2014. Talmadge JE, et al. Inflammatory cell infiltration of tumors: Jekyll or Hyde. Cancer Metastasis Rev. 2007; 26: 373-400. Tan EM, Feltkamp TI, Smolen JS, et al. Range of antinuclear antibodies in "healthy" individuals. Arthritis Rheum. 40:1601-11, 1997. Taube JM, Anders RA, Young GD, et al. Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. *Sci Transl Med.* 4(127):127ra37, 2012. Topalian SL, Hodi FS, Brahmer JR, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. *NEJM*. 366:2443-54, 2012. Uchida A, Mizutani Y, Nagamuta M, et al. Effects of X-ray irradiation on natural killer (NK) cell system. I. Elevation of sensitivity of tumor cells and lytic function of NK cells. Immunopharm Immunotoxicol. 11:507-19, 1989. Urba S, Gatz J, Shen W, et al. Quality of life scores as prognostic factors of overall survival in advanced head and neck cancer: analysis of a phase III randomized trial of pemetrexed plus cisplatin versus cisplatin monotherapy. *Oral Oncol*. 2012;48(8):723-9. doi: 10.1016/j.oraloncology.2012.02.016. Epub 2012 Mar 11. Wandstrat AE, Carr-Johnson F, Branch V, et al. Autoantibody profiling to identify individuals at risk for systemic lupus erythematosus. *Journal of Autoimmunity*. 27:153-60, 2006. Wang L et al. Randomized phase II study of concurrent cisplatin/etoposide or paclitaxel/carboplatin and thoracic radiotherapy in patients with stage III non-small cell lung cancer. *Lung Cancer*. 2012;1: 89-96. Weber JS, O'Day S, Urba W, et al. Phase I/II study of ipilimumab for patients with metastatic melanoma. *J Clin Oncol*. 26:5950-6, 2008. Wu AW, Jacobson KD, Frick DL, et al. Validity and responsiveness of the EQ-5D as a measure of health-related quality of life in people enrolled in an AIDS clinical trial. *Qual Life Res*. 2002; 11(3):273-82. Yuan J, Gnjatic S, Li H, et al. CTLA-4 blockade enhances polyfunctional NY-ESO-1 specific T cell responses in metastatic melanoma patients with clinical benefit. Proc Natl Acad Sci U S A.105:20410-5, 2008. Zelen H. The randomizatino and stratification of patients to clinical trials (MI\#7506064). J Chronic Disease. 27:365-375, 1974. Zhang L, Wang L, Yang M, et al. Association of DNA repair and inflammatory cytokines gene polymorphisms with radiation-induced toxicity in lung cancer. *IJROBP*. 69:S60-61, 2007.
rtog foundation rtog 3505
appendix i nivolumab (bms-936558) pharmacy

The objective of this procedure manual is to provide the study site with clear and detailed information for the storage, handling, preparation and administration of Nivolumab used in the RTOG 3505/CA209-333 study
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2 bms clinical product contact

If concerns regarding the quality or appearance of the study drug, or questions regarding administration arise, do not dispense the study drug and contact the Bristol-Myers Squibb immediately:
Questions with regard to drug preparation and pharmacy manual content: Peter Trimboli, RPh Pharmacy Services, Drug Supply Management Bristol-Myers Squibb Research and Development Telephone: 609-252-4862 Email: pharmacyservices@bms.com 3 NIVOLUMAB INJECTION 3.1 DESCRIPTION  
| Product Name                       | Nivolumab Injection (BMS 936558), 100 mg/vial (10 mg/mL)  Packaging: Vials assembled into white dispensing boxes containing  100 mg vials.                                         |
|------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Product description  and Packaging | Vials: 10 cc Type I glass vial. 20 mm stopper and seal, respectively.  Appearance: Clear to opalescent, colorless to pale yellow liquid,  light (few) particulates may be present. |
| Product Ingredients                | Each vial contains 100 mg Nivolumab                                                                                                                                                |  
Continued on next page
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appendix i (continued) 3.2 handling

As with all injectable drugs, care should be taken when handling and preparing Nivolumab.  
Whenever possible, Nivolumab infusions should be prepared in a laminar flow hood, glovebox, or safety cabinet using standard procedures for the safe handling of intravenous agents applying aseptic techniques. Gloves are required. If nivolumab solution comes in contact with the skin or mucosa, immediately and thoroughly wash with soap and water.
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dose preparation and administration

Visually inspect drug product solution for particulate matter and discoloration prior to administration. Nivolumab is a clear to opalescent, colorless to pale yellow solution. Discard the vial if the solution is cloudy, discolored, or contains extraneous particulate matter other than a few translucent-to-white, proteinaceous particles. Do not shake the vial.
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preparation

Withdraw the required volume of Nivolumab and transfer into an intravenous container.  
Dilute Nivolumab with either 0.9% Sodium Chloride Injection, USP or 5%
Dextrose Injection, USP to prepare an infusion with a final concentration ranging from 1 mg/mL to 10 mg/mL (see example below)
Mix diluted solution by gentle inversion. Do not shake.  
Discard partially used vials or empty vials of Nivolumab.
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storage of infusion

The product does not contain a preservative. After preparation, store the Nivolumab infusion either:
at room temperature for no more than 4 hours from the time of preparation. This includes room temperature storage of the infusion in the IV container and time for administration of the infusion or  
under refrigeration at 2°C to 8°C (36°F to 46°F) for no more than 24 hours from the time of infusion preparation.
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do not freeze. administration

Administer the infusion over 30 minutes through an intravenous line containing a sterile, nonpyrogenic, low protein binding in-line filter (pore size of 0.2 micrometer to 1.2 micrometer). Do not coadminister other drugs through the same intravenous line. **Flush the intravenous line at**
end of infusion with appropriate amount of diluent (e.g. 15-20 ml) to ensure that the total dose is administered. Total infusion and flush time should equal to 30. Dose selection for Nivolumab should be assigned per subject as outlined in the clinical protocol study drug dosing section of the clinical protocol. The examples below use a total volume of 60  
ml to allow for a practical administration rate, however *other volumes may be used for* Nivolumab preparation so as long as the final concentration remain between 1 - 10 mg/ml *(e.g.*
100 ml).  
NOTE: When Nivolumab *Placebo is required, placebo shall be considered an equal volume of* drug diluent alone (NS, D5W) which is standard to site practice for mixing active Nivolumab (60 ml, or 100 ml) and should be infused at the same rate (30 minutes). Preparation example 240 mg dose; Nivolumab 240 mg (24 ml *Nivolumab 10mg/ml solution, local rounding rules applicable) may be* mixed with 36 ml NS or D5W to a total volume = 60 ml which can be infused over 30 minutes at 2 ml/min *followed by flush.*
Preparation example 480 mg dose; Nivolumab 480 mg (48 ml *Nivolumab 10mg/ml solution, local rounding rules applicable) may be* mixed with 12 ml NS or D5W to a total volume *= 60 ml which can be infused over 30 minutes at* 2 ml/min *followed by flush.*
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3.3 product storage and stability

Nivolumab 100 mg vials for injection should be refrigerated at 2°C to 8°C (36°F to 46°F).  
Protected from light and **should not be frozen**. Do not use beyond the expiration date on the vial. Protect the vials from light by storing in the original package until time of use. Vials do not contain preservative and thus are intended for single use only and should be discarded after use and product reconciliation. Nivolumab should not be infused concomitantly in the same intravenous line with other agents.  
No physical or biochemical compatibility studies have been conducted to evaluate the coadministration of Nivolumab with other agents.
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4 site temperature excusions and

Drug must be stored under the proper conditions as listed on the clinical supply label. If any temperature excursions are encountered during on site storage, please report these to BMS for assessment as outlined in the Bristol-Myers Squibb, "Investigational Medicinal Product (IMP)
handling at Investigational Sites: Shipment, Receipt, Storage, Use Date Extension and Return/Destruction" guideline using the Temperature Excursion Response Form. See Form 1 Proper storage conditions must be maintained during movement of inventory within an  
investigational site. Storage conditions for medications requiring storage at 2ºC to 8ºC (36ºF46ºF) must be maintained throughout the transport with documentation maintained within the site files. Where controlled storage conditions (for example, temperature, relative humidity, light, etc.) are required during transit, the necessary environmental controls must be in place to ensure that the drug product remains within the acceptable temperature range. Temperature monitoring devices such as min max device must be implemented during transit.
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5 product receipt accountability and

2. Carefully inspect kits ensuring all of the supplies were received in good condition, correct quantity received, and all of the container ID \#s were received as noted on packing slip.  
3. Sign and date (date of receipt) packing slip and file with study-specific documents.  
4. Log in all supplies in each shipment on the appropriate Inventory log Form
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accountability

It is the responsibility of the investigator to ensure that a current disposition record of investigational product accountability and reconciliation is maintained at each study site where study drug is inventoried and dispensed. In addition, records or logs must comply with local regulations and guidelines for the conduction and handling of clinical supplies should include but not limited too:  
Amount received and placed in storage area Amount currently in storage area Label ID number or batch number Amount dispensed to and returned by subject, including unique subject identifiers Amount transferred to another area/site for dispensing or storage Non-study disposition (e.g. lost, wasted)
Amount destroyed at study site, if applicable Amount returned to the Sponsor, if applicable Dates and initials of person responsible for Investigational Product (IP)
Dispensing/accountability, as per the Site Signature and Delegation Log.
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study drug destruction

Study drugs can be destroyed on site if local policies allow to do so. It is the Investigator's  
responsibility to ensure that arrangements have been made for the disposal, procedures for proper disposal have been established according to applicable regulations, guidelines and institutional procedures, and appropriate records of the disposal have been documented.
form 1 bristol-myers squibb temperature
study drug destruction

Section A. To be completed by the site at the time of Site Storage Temperature Excursion:
Protocol Number: **Site Number/Investigator Name/Country:**  
| Description of Drug Products involved in Excursion: Batch number (s) printed on label:                                | Container numbers:    |                        |
|-----------------------------------------------------------------------------------------------------------------------|-----------------------|------------------------|
| Description of Excursion (temperature highs/lows and duration): Below label storage lower limit …...°C Duration:…………. | Low extreme t°: …..°C |                        |
| Above label storage upper limit ….. °C                                                                                | Duration:………...       | High extreme t°: …..°C |
| Reason for excursion:                                                                                                 |                       |                        |  
Has the issue been resolved?  
| Have these specific containers been involved in a previous excursion?        | NO                 | YES                                 |
|------------------------------------------------------------------------------|--------------------|-------------------------------------|
| If yes, please provide: Batch number (s):                                    | Container numbers: | Temperature highs/lows and duration |
| When is the next planned patient visit when these supplies may be dispensed? |                    |                                     |  
Excursion information submitted by: _________________________________________ Date: ____________
*Print/Signature/Title of site staff* Section B. Usage Decision to be made by Bristol-Myers Squibb:
Temperature excursion details for the products listed above have been evaluated. Usage decision is based on the temperature data that were made available by the investigational site. Conclusion (and comments):
All products are suitable for continued use
______________________________________________________________________________________ ______________________________________________________________________________________
All products are NOT **suitable for further dispensation. Please remove supplies from available inventory and work with your**
Site Manager/Site Monitor to have supplies destroyed and IVRS updated if applicable. ______________________________________________________________________________________ ______________________________________________________________________________________
Assessment completed by:_______________________________________________ Date ___________
Print/Signature/Title Send completed form to: Peter Trimboli, RPh at pharmacyservices@bms.com Templates as provided via separate attachment or below may serve as a sample for dosage calculations and may be printed out and used to document any calculations and infusion times as required by the clinical study team. *Content in this attachment may be added or omitted.*  
| Drug Dose and Volume Calculator  for IV Infusion                                                                                                                                                                                         |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Drug Product : Protocol number Patient name or ID:                                                                                                                                                                                       |
| Preparation date and time:                                                                                                                                                                                                               |
| Drug Strength (mg/mL) Subject body weight (kg) 1Dose in mg Volume of Drug (mL) Total Volume of infusion (mL) Volume of Diluent (mL) NS or D5W Infusion duration (min) Infusion Rate (mL/min) Infusion hang time Infusion completion time |  
Note: If required, flush line with separate volume of same diluent (e.g. 15-20 ml) as outlined in the study pharmacy manual
appendix ii
1.1 definitions associated with response

The presence of at least one measurable lesion. If the measurable disease is restricted to a solitary lesion, its neoplastic nature should be confirmed by cytology/histology.
appendix ii
1.1.2 measurable lesions

Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as >20 mm by chest x-ray, as >10 mm with CT scan, or >10 mm with calipers by clinical exam. All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters).
appendix ii
1.1.3 non-measurable lesions

All other lesions (or sites of disease), including small lesions (longest diameter <10 mm or pathological lymph nodes with ≥ 10 to <15 mm short axis), are considered non-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed by CT or MRI), are considered as non-measurable.  
Note: Cystic lesions that meet the criteria for radiographically defined simple cysts should not be considered as malignant lesions (neither measurable nor non-measurable) since they are, by definition, simple cysts.  
'Cystic lesions' thought to represent cystic metastases can be considered as measurable lesions, if they meet the definition of measurability described above. However, if non-cystic lesions are present in the same subject, these are preferred for selection as target lesions. 1.1.4 Malignant lymph nodes 1.1.1.1 To be considered pathologically enlarged and measurable, a lymph node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). At baseline and in follow-up, only the short axis will be measured and followed.
appendix ii
1.1.5 baseline documentation of "target"

All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, should be identified as target lesions and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. It may be the case that, on occasion, the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected. A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters. If lymph nodes are to be included in the  
sum, then only the short axis is added into the sum. The baseline sum diameters will be used as reference to further characterize any objective tumor regression in the measurable dimension of the disease.
appendix ii
non-target lesions.

All other lesions (or sites of disease) including any measurable lesions over and above the 5 target lesions should be identified as non-target lesions and should also be recorded at baseline. Measurements of these lesions are not required, but the presence, absence, or in rare cases unequivocal progression of each should be noted throughout follow-up.  
| Evaluation of target lesions Complete Response (CR)   | Disappearance of all target lesions. Any  pathological lymph nodes (whether target or nontarget) must have reduction in short axis to <10  mm.                                                                                                                                                                                                                                                                                                                                                                                                    |
|-------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Partial Response (PR)                                 | At least a 30% decrease in the sum of the  diameters of target lesions, taking as reference the  baseline sum diameters                                                                                                                                                                                                                                                                            |
| Progressive Disease (PD)                              | At least a 20% increase in the sum of the diameters  of target lesions, taking as reference the smallest  sum on study (this includes the baseline sum if that  is the smallest on study). In addition to the relative  increase of 20%, the sum must also demonstrate an  absolute increase of at least 5 mm. (Note: the  appearance of one or more new lesions is also  considered progression). |
| Stable Disease (SD)                                   | Neither sufficient shrinkage to qualify for PR nor  sufficient increase to qualify for PD, taking as  reference the smallest sum diameters while on  study                                                                                                                                                                                                                                         |  
| Evaluation of non-target lesions Complete Response (CR)   | Disappearance of all non-target lesions and  normalization of tumor marker level. All lymph  nodes must be non-pathological in size (<10 mm  short axis) Note: If tumor markers are initially above the  upper normal limit, they must normalize for a  subject to be considered in complete clinical  response.   |
|-----------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Non-CR/ Non-PD                                            | Persistence of one or more non-target lesion(s)  and/or maintenance of tumor marker level above  the normal limits                                                                                                                                                                                                 |
| Progressive Disease (PD)                                  | Appearance of one or more new lesions and/or  unequivocal progression of existing non-target  lesions. Unequivocal progression of non-target  lesions should not normally trump target lesion  status. It must be representative of overall disease  status change, not a single lesion increase.                  |  
* Although a clear progression of "non-target" lesions only is exceptional, the opinion of the treating physician should prevail. Evaluation of best **overall** response The best overall response is the best time point response recorded from the start of the treatment until disease progression/recurrence (taking as reference for PD the smallest measurements recorded since the treatment started). In general, the participant's best response assignment will depend on the achievement of both measurement and confirmation criteria.  
| Table 3: Response Assignment Target Lesions Non-Target lesion   | New Lesion          | Overall  response   |    |
|-----------------------------------------------------------------|---------------------|---------------------|----|
| CR                                                              | CR                  | No                  | CR |
| CR                                                              | Non-CR/ Non-PD      | No                  | PR |
| Not evaluated                                                   | No                  | PR                  |    |
| PR                                                              | Non-CR/ Non-PD/ not | No                  | PR |
| evaluated                                                       |                     |                     |    |
| SD                                                              | Non-CR/ Non-PD/ not | No                  | SD |
| evaluated                                                       |                     |                     |    |
| PD                                                              | Any                 | Yes or No           | PD |
| Any                                                             | PD*                 | Yes or No           | PD |
| Any                                                             | Any                 | Yes                 | PD |  
101 *In exceptional circumstances, unequivocal progression in non-target lesions may be accepted as disease progression.  
APPENDIX II (continued)
Subjects with a global deterioration of health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be classified as having "symptomatic deterioration." Every effort should be made to document the objective progression even after discontinuation of treatment. In some circumstances, it may be difficult to distinguish residual disease from normal tissue. When the evaluation of complete response depends on this determination, it is recommended that the residual lesion be investigated (fine needle aspirate/biopsy) to confirm the complete response status.
appendix ii
1.1.7 definitions for response evaluation

The time between initiation of therapy and first documentation of PR or CR.
appendix ii
1.1.7.2 duration of response

Duration of overall response—the period measured from the time that measurement criteria are met for complete or partial response (whichever status is recorded first) until the date that recurrent or progressive disease is objectively documented, taking as reference for progressive disease the smallest measurements recorded since treatment started.
appendix ii
1.1.7.3 duration of overall complete

The period measured from the time that measurement criteria are met for complete response until the first date that progressive disease is objectively documented.
appendix ii
1.1.7.4 objective response rate

The objective response rate is the proportion of all subjects with confirmed PR or CR according to RECIST v1.1, from the start of treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the start of treatment).
appendix ii
1.1.7.5 progression free survival

A measurement from the start of the treatment until the criteria for disease progression is met as defined by RECIST 1.1 or death occurs, taking as reference the smallest measurements recorded since the treatment started. Progression free survival will be measured from the date of initial treatment to the earliest date of disease progression, resection of measurable tumor or death for subjects who fail; and to the date of last contact for subjects who remain at risk for failure.
appendix ii
1.2 methods of measurement

All measurements should be taken and recorded in metric notation using a ruler or calipers. All baseline evaluations should be performed as closely as possible to the beginning of treatment and  
never more than 28 days before the beginning of the treatment. Imaging-based evaluation is preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be imaged but are assessable by clinical exam. The same imaging modality must be used throughout the study to measure disease.
appendix ii
1.2.1 ct and mri

CT and MRI are the best currently available and most reproducible methods for measuring target lesions. This guideline has defined measurability of lesions on CT scan based on the assumption that CT slice thickness is 5 mm or less. If CT scans have slice thickness greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness. MRI is also acceptable in certain situations (e.g. for body scans). Use of MRI remains a complex issue. MRI has excellent contrast, spatial, and temporal resolution; however, there are many image acquisition variables involved in MRI, which greatly impact image quality, lesion conspicuity, and measurement. Furthermore, the availability of MRI is variable globally. As with CT, if an MRI is performed, the technical specifications of the scanning sequences used should be optimized for the evaluation of the type and site of disease. Furthermore, as with CT, the modality used at follow-up should be the same as was used at baseline and the lesions should be measured/assessed on the same pulse sequence. It is beyond the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all scanners, body parts, and diseases. Ideally, the same type of scanner should be used and the image acquisition protocol should be followed as closely as possible to prior scans. Body scans should be performed with breath-hold scanning techniques, if possible. PET-CT. At present, the low dose or attenuation correction CT portion of a combined PET-CT is not always of optimal diagnostic CT quality for use with RECIST measurements. However, if the site can document that the CT performed as part of a PET-CT is of identical diagnostic quality to a diagnostic CT (with IV and oral contrast), then the CT portion of the PET-CT can be used for RECIST measurements and can be used interchangeably with conventional CT in accurately measuring cancer lesions over time. Note, however, that the PET portion of the CT introduces additional data which may bias an investigator if it is not routinely or serially performed.
appendix ii
1.2.2 chest x-ray

Lesions on chest x-ray are acceptable as measurable lesions when they are clearly defined and surrounded by an aerated lung (CT is preferable).
appendix ii
1.2.3 clinical examination

Clinically detected lesions will only be considered measurable when they are superficial (e.g. skin nodules and palpable lymph nodes). For skin lesions, documentation by color photography, including a ruler to estimate size of the lesion, is recommended. Photographs should be retained
appendix ii
1.2.4 cytology and histology

Cytologic and histologic techniques can be used to differentiate between complete and partial responses in rare cases (e.g. after treatment to differentiate residual benign lesions and residual malignant lesions in germ cell tumors). Cytologic confirmation of the neoplastic nature of any effusion that appears or worsens during treatment is required when the measurable tumor has met response or stable disease criteria.
appendix iii
instructions for ffpe and serum

oSlides should be shipped in plastic slide holder/slide box. Place a small wad of padding in top of the container. If you can hear slides shaking they can break during shipping.  
o Unstained slides must be stored at 4C after cutting and shipped to the Biospecimen bank with cold packs at 4C within 5 business days from cutting. The date that the slides were cut should be noted on the ST form.  
oFFPE Blocks can be shipped in a plastic block holder or wrapped with paper or placed in a paper envelope, and 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 blocks shaking they may break during shipping. During warm weather months the use of cold packs is recommended to prevent wax from melting.  
oSlides, 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**.  
o Urgent overnight shipments (central review) should always be sent by Courier. oIf patients consented to banking DO NOT ship FFPE specimens with a return request.  
We cannot accept or bank samples that we cannot keep. Always send what can be banked (duplicate H&Es, Blocks or punches as specified in the protocol). We can punch and return blocks if noted in the protocol. Punch kits can be requested from us by email (RTOG@ucsf.edu) for sites that wish to punch the block themselves.
appendix iii
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.  
oPlace 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.  
oSpecimens received thawed due to insufficient dry ice or shipping delays will be discarded and the site will be notified.  
oSend frozen specimens via overnight courier to the address above. Frozen 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 Biospecimen Bank at**
UCSF by e-mail: RTOG@ucsf.edu or phone: 415-476-7864 or Fax: 415-476-5271.  
| US Postal Service Mailing Address:  Use only for non-urgent ambient  specimens- FFPEs, slides, blocks:   | Courier Address (FedEx, UPS, etc.): For Frozen, Urgent or Trackable Specimens:                                    |
|----------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------|
| Biospecimen Bank at UCSF UCSF- Box 1800 2340 Sutter St, room S341 San Francisco, CA 94143-1800           | Biospecimen Bank at UCSF University of California San Francisco 2340 Sutter St, room S341 San Francisco, CA 94115 |  
Shipping Instructions **for FFPE and Serum:** (See Appendix III for processing and shipping information for peripheral blood)
Include all required specimen paperwork in pocket of biohazard bag. Check that the Specimen Transmittal (ST) Form has been completely filled out Check that all samples are labeled with the study and case number, and include date of collection as well as collection time point (e.g., pretreatment, post-treatment).
appendix iii
blood collection kit instructions

This Kit is for collection, processing, storage, and shipping of serum and whole blood for first 3 timepoints. Recurrence/ toxicity patients- site should request follow-up kits separately for these patients.  
|                                                                                                                                                 | Five 1 ml cryovials per timepoint.  (Twenty per kit)                                  |                                 |
|--------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------|---------------------------------|
|                                                                                                                                                 | Biohazard bag (4)                                                                     |                                 |
|                                                                                                                                                 | ST Form and Kit Instructions                                                          |                                 |
| Note: sites will have to provide their own  EDTA for whole blood and standard Red  Top tube for serum blood draw unless  specifically requested. |                                                                                      | Absorbent shipping material (4) |
|                                                                                                                                                 | 1 Styrofoam container (inner) and  Cardboard shipping (outer) box for  batch shipping |                                 |
|                                                                                                                                                 | UN1845 DRY Ice Sticker and  UN3373 Biological Substance  Category B Stickers          |                                 |
appendix iii
preparation and processing of serum

Label as many 1ml cryovials (5 to 8) for each time point as necessary for the serum collected. Label tubes with the RTOG Foundation study and case number, time point, collection date and time, and clearly mark cryovials "serum".  
Preparation and Processing of Serum (continued)
Process:
1. Allow one red top tube (not provided in kit) to clot for 30 minutes at room temperature.  
2. Spin in a standard clinical centrifuge at ~2500 RPM for 10 minutes at 4°C
(preferred). If sites are unable to process samples at 4°C 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 five (5) labeled cryovials. Make sure tubes are labeled as specified above.  
4. Place cryovials into biohazard bag and immediately freeze at -70 to -90C. Store frozen until ready to ship. See below for storage conditions.
appendix iii
whole blood for dna purple

Label as many 1ml cryovials (3 to 5) as necessary for the whole blood collected. Label them with the RTOG 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 labeled cryovials as are necessary for the blood collected (3 to 5). Make sure tubes are labeled as specified above.  
3. Place cryovials into biohazard bag and immediately freeze at -70 to -90 C. Store frozen until ready to ship on dry ice. See below for storage conditions PLEASE make sure EVERY SPECIMEN IS LABELED and include collection time point on ST Form.
PLEASE make sure EVERY SPECIMEN IS LABELED and include collection time point on ST Form.
appendix iii
freezing and storage

Freeze Serum and whole 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: Monday-Tuesday 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 Monday-Wednesday only; Canada: Monday-Tuesday only).  
Please indicate on Specimen Transmittal Form the storage conditions used and time stored.
appendix iii
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 required paperwork in sealed plastic bag taped on outside top of the Styrofoam box.  
Wrap frozen specimens of same time point together in absorbent shipping material and place specimens from separate time points or cases 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 dry ice stickers to outer cardboard box. Fill out the UN1895 Label COMPLETELY.
appendix iii
appendix iii (continued) preparation and

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 Serum Collection Kit, please e-mail RTOG@ucsf.edu or call (415) 476-7864.
appendix iii
shipping address

Courier Address (FedEx, UPS, etc.): For all Frozen Specimens Biospecimen Bank UCSF 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 Note: See Appendix IV for processing and shipping information for peripheral blood.
appendix iv
kit contents

5 Heparin Blood (green top) draw tubes per time point Tube holder Outer box for tube holder Study Specific ST Form Fed Ex Clinical Pak with label Processing:
1. Sites should only draw blood samples on Monday-Wednesdays and only ship MondayThursdays.  
2. Draw blood in 5 green heparin tubes. 3. Mix gently. 4. Place tubes in tube holder and outer box provided with Kit. 5. Seal up tube holder box and place in Fed Ex Clinical Pak provided 6. Ship the blood at room temperature on the same day for overnight delivery if possible. 7. Blood samples drawn too late in the day for overnight shipping, should be refrigerated overnight and shipped the following day for same day delivery.  
8. Ship same day to Hooper Laboratory- Label is provided on the Clinical Pak.  
Shipping **Address: Note:** Do NOT ship peripheral blood to Biospecimen Bank at UCSF.  
Hooper Laboratory Attn.: **Rhonda Kean (lab manager) or Craig Hooper, PhD** JAH 454 1020 Locust Street Philadelphia, PA 19107 For questions, contact: 215-503-1559