untitled h1
untitled h2

Protocol NMTRC 008 NCT01802567 A Feasibility Trial using Molecular-Guided Therapy for the Treatment of Patients with Relapsed and Refractory Childhood Cancer Version 2.0 June 12, 2013
untitled h1
contact information study chair

| Giselle Sholler, MD NMTRC Chair 100 Michigan Avenue NE MC 272  Grand Rapids, MI 49503 Tel: (616) 267-0332  Fax: 616-391-2785  giselle.sholler@helendevoschildrens.org   |                                                                                                                                                                                                                                                    |              |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------|
| Study Arms:  Neuroblastoma Study Chair:                                                                                                                                 | Giselle Sholler, MD NMTRC Chair 100 Michigan Avenue NE MC 272  Grand Rapids, MI 49503 Tel: (616) 267-0332  Fax: 616-391-2785  giselle.sholler@helendevoschildrens.org                                                                              |              |
| Neuroblastoma Study Vice Chair:                                                                                                                                         | Nehal Parikh, MD  Assistant Professor of Pediatrics  Connecticut Children's Medical Center Division of Hematology / Oncology 2J 282 Washington Street Hartford, CT 06106 Tel: 860-545-9630  Fax: 860-545-9622  Nparikh@ccmckids.org                |              |
| Brain Tumor Chair:                                                                                                                                                      | Amy Lee Bredleau, MD Director, Pediatric Brain Tumor Program Assistant Professor, Sullivan Scholar Medical University of South Carolina 135 Rutledge Avenue, MSC 558 Charleston, SC 29425 Phone: 843-876-2872  Fax: 843-792-8912  bredlau@musc.edu |              |
| Brain Tumor Vice Chair:                                                                                                                                                 | Don Eslin, MD  MDACCO  Pediatric Hematology/Oncology 92 West Miller Street, MP 318 Orlando, FL 32806                                                                                                                                               |              |
|                                                                                                                                                                         | Tel: 321-841-8588 Don.Eslin@orlandohealth.com                                                                                                                                                                                                      |              |
| Version 2.0 Final                                                                                                                                                       | Page 2 of 268                                                                                                                                                                                                                                      | Confidential |
| Issued 12 June 2013                                                                                                                                                     |                                                                                                                                                                                                                                                    |              |  
Rare Tumor Vice Chairs: Melinda Merchant, PhD, MD  
Pediatric Oncology Branch National Cancer Institute 10 Center Drive Bldg 10 Room 1W-3952 Bethesda, Maryland 20892 Tel: 301.443.7955 merchanm@mail.nih.gov  
Rare Tumor Chair: Jacqueline Kraveka, DO
Assistant Professor of Pediatrics Medical University of South Carolina 135 Rutledge Avenue, Room 480 Division of Pediatric Hematology-Oncology MSC 558 Charleston, SC 29425 Tel: 843-792-2957 Fax: 843-792-8912 kravekjm@musc.edu Kathleen A. Neville, M.D., M.S.  
Children's Mercy Hospitals and Clinics Divisions of Pediatric Clinical Pharmacology and Pediatric Hematology/Oncology 2401 Gillham Road Kansas City, MO 64108 kaneville@cmh.edu Safety Officer: Dr. Timothy Triche Department of Pathology and Laboratory Medicine Children's Hospital Los Angeles 4650 Sunset Blvd., Mailstop 133 Los Angeles, CA 90027 Ph: 323-361-8898 Fax: 323-361-4945 triche@usc.edu
untitled h1
study committee members

Genevieve Bergendahl, RN Clinical Program Director NMTRC 100 Michigan Avenue NE MC 272 Grand Rapids, MI 49503 Alyssa VanderWerff Clinical Program Coordinator NMTRC 100 Michigan Avenue NE MC 272 Grand Rapids, MI 49503 Emily Arndt Clinical Project Coordinator NMTRC 100 Michigan Avenue NE MC 272 Grand Rapids, MI 49503 Akshita Dutta, MS Computational Biologist NMTRC 100 Michigan Avenue NE MC 272 Grand Rapids, MI 49503 William Ferguson, MD Pediatric Hematology-Oncology Cardinal Glennon Children's Medical Ctr Saint Louis University School of Medicine 1465 S. Grand Blvd.  
St. Louis, MO
William Roberts, MD Pediatric Hematology/Oncology Rady Children's Hospital San Diego 3020 Children's Way MC5035 San Diego, CA 92123-4282 Don Eslin, MD
MDACCO Pediatric Hematology/Oncology 92 West Miller Street, MP 318 Orlando, FL 32806 Tel: (616) 267-0335 Fax: 616-391-2785 E-Mail: Genevieve.bergendahl@helendevoschildrens.or g Tel: (616) 267-0327 Fax: 616-391-2785 E-Mail: Alyssa.VanderWerff@helendevoschildrens.org Tel: (616) 267-0326 Fax: 616-391-2785 E-Mail: emily.arndt@helendevoschildrens.org Tel: (616) 267-0309 Fax: 616-391-2785 E-Mail: Akshita.dutta@helendevoschildrens.org Tel: (314) 577-5638 Fax: (314) 268-4081 fergusws@slu.edu Tel: (858) 966-5811 Fax: (858) 966-8035 wroberts@chsd.org Tel: 321-841-8588 Don.Eslin@orlandohealth.com Jacqueline Kraveka, DO Assistant Professor of Pediatrics Medical University of South Carolina Division of Pediatric Hematology-Oncology 135 Rutledge Avenue, Room 480 MSC 558 Charleston, SC 29425 Joel Kaplan, DO, MPH
Levine Children's Hospital 1000 Blythe Blvd Charlotte, NC 28203 Deanna Mitchell, MD
Michigan State University Helen DeVos Children's Hospital 100 Michigan NE
Grand Rapids, MI 49503 Nehal Parikh, MD
Assistant Professor of Pediatrics Connecticut Children's Medical Center Division of Hematology / Oncology 2J
282 Washington Street Hartford, CT 06106 Kathleen A. Neville, M.D., M.S. Children's Mercy Hospitals and Clinics Divisions of Pediatric Clinical Pharmacology and Pediatric Hematology/Oncology 2401 Gillham Road Kansas City, MO 64108 Randal K. Wada, MD Associate Professor of Pediatrics and Nursing University of Hawaii Kapiolani Medical Center for Women and Children 55 Merchant Street, 26th Floor Honolulu, HI 96813 Tel: 843-792-2957 Fax: 843-792-8912 kravekjm@musc.edu Tel: 704-381-9900 Fax: 704-381-9901 Joel.Kaplan@carolinashealthcare.org Tel: 616-391-2086 Deanna.Mitchell@devoschildrens.org Tel: 860-545-9630 Fax: 860-545-9622 Nparikh@ccmckids.org kaneville@cmh.edu Office/Lab: (808) 586-2963 tbear@cc.hawaii.edu Melinda Merchant, PhD, MD
Pediatric Oncology Branch National Cancer Institute 10 Center Drive Bldg 10 Room 1W-3952 Bethesda, Maryland 20892 Francis Eshun, MD Center for Cancer and Blood Disorders Phoenix Children's Hospital 1919 Thomas Road Phoenix, AZ 85016 Valerie I. Brown, MD, PhD Assistant Professor of Pediatrics Division of Pediatric Oncology/Hematology Leader, Translational Biology Monroe Carrell Jr. Children's Hospital at Vanderbilt Vanderbilt-Ingram Cancer Center 397 Preston Research Building 2220 Pierce Ave Nashville, TN 37232 Sharon K Lockhart, MD
COG Principal Investigator Clinical Research Director, Dell Children's Blood and Cancer Center 1301 Barbara Jordan Blvd, Ste 401 Austin, Texas 78723 Mark Fluchel, MD
Primary Children's Hospital Division of Pediatric Hematology/Oncology 100 North Mario Capecchi Drive, 4th Floor Salt Lake City, UT 84113 Takamaru Ashikaga, Ph.D.  
Medical Biostatistics
& Bioinformatics Facility UVM College of Medicine 27 Hills Building 105 Carrigan Drive Burlington, VT 05405 Tel: 301.443.7955 merchanm@mail.nih.gov feshun@phoenixchildrens.com Phone: 615-875-6129 Fax: 615-936-1767 Email: valerie.brown@vanderbilt.edu 512-628-1900 office 512-656-1801 cell 512-628-1901 fax slockhart@sfcaustin.com Phone: 801-662-4700 Fax: 801-662-4705 email: mark.fluchel@imail.org Tel: (802)656-2526 Fax (802)656-0632 Takamaru.Ashikaga@uvm.edu Jeffrey Bond, PhD Vermont Cancer Center UVM College of Medicine Given E-315 89 Beaumont Avenue Burlington, VT 05405 Gina K. Hanna, PharmD
Hematology Oncology/Investigational Drugs Clinical Pharmacy Specialist Nemours Children's Hospital 13535 Nemours Parkway Orlando, Florida 32827 Tel: (802) 656-4608 jeffrey.bond@uvm.edu Tel: 407.567.4815 ghanna@nemours.org Stacy Epstein, Pharm.D., BCOP
Clinical Pharmacy Specialist, Pediatric Blood and Marrow Transplantation Carolinas Medical Center Levine Children's Hospital 1000 Blythe Boulevard Charlotte, North Carolina 28203 Karen Gradischer Pharmacist Connecticut Children's Hospital 282 Washington Street Hartford, CT 06106 Amy Lee Bredleau, MD
Director, Pediatric Brain Tumor Program Assistant Professor, Sullivan Scholar Medical University of South Carolina 135 Rutledge Avenue, MSC 558 Charleston, SC 29425 Jeff Trent Translational Genomics Research Institute (TGen) President and Research Director 445 N 5th Street Phoenix, AZ 85004 Matt Huntelman Translational Genomics Research Institute
(TGen) Associate Professor, Neurogenomics Division 445 N 5th Street Phoenix, AZ 85004 Tel: 704-381-7173 Fax: 704-381-7171 Stacy.Epstein@carolinashealthcare.org Tel: (860) 545-9935 Kgradis@ccmckids.org Phone: 843-876-2872 Fax: 843-792-8912 602-343-8419 jtrent@tgen.org 602-343-8730 mhuentelman@tgen.org Spyro Mousses PhD Vice President, Office for Innovation Professor & Director, Center for BioIntelligence Translational Genomics Research Institute (TGen)
445 N. 5th Street, Suite 600 Phoenix, AZ 85004 602-343-8444 (O) 602-653-5512 (M) 602-343-8457 (F)
smousses@tgen.org 602-343-8819 jcarpten@tgen.org John Carpten Translational Genomics Research Institute (TGen) Deputy Director of Basic Science 445 N 5th Street Phoenix, AZ 85004 Jeff Keifer Translational Genomics Research Institute
(TGen) Research Associate Professor 445 N 5th Street Phoenix, AZ 85004 602-343-8667 jkiefer@tgen.org Jason Corneveaux Translational Genomics Research Institute
(TGen) Bioinformatician II
445 N 5th Street Phoenix, AZ 85004 602-343-8714 jcorneveaux@tgen.org
untitled h1
investigator signature sheet

I have read the attached protocol amendment and agree that it contains all the necessary details for performing the study.  
I will provide copies of the amended protocol and of the preclinical and clinical information on the test article, which was furnished to me by the Sponsor, to all members of the study team responsible to me who participate in the study. I will discuss this material with them to assure that they are fully informed regarding the test article and the conduct of the study.  
Once the protocol has been approved by the Institutional Review Board (IRB)/Independent Ethics Committee (IEC), I will not modify this protocol without obtaining the prior approval of the Sponsor and of the IRB/IEC. I will submit the protocol modifications and/or any informed consent form (ICF) modifications to the Sponsor and the IRB/IEC, and approval will be obtained before any modifications are implemented.  
I understand the protocol and will work according to it, the principles of Good Clinical Practice (GCP) [current International Conference of Harmonization (ICH) guidelines], and the Declaration of Helsinki (1964) including all amendments up to and including the Scotland revision (2000) and notes of clarification added in 2002 and 2004.  
Investigator's Signature Date Investigator's Printed Name Investigational Site Name
untitled h1
table of contents

| Contact Information: ________________________________________________________ 2 TABLE OF CONTENTS ____________________________________________________ 10 PROTOCOL SYNOPSIS ___________________________________________________ 13 TABLE OF PROCEDURES AND ASSESSMENTS _____________________________ 17   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 1                                                                                                                                                                                                                                                                                                         | Background ______________________________________________________ 18 1.1 Study Disease ________________________________________________ 18 1.2 Rationale____________________________________________________ 18                                                                                                                                                                                                                                                                                                                                                                         |
| 2                                                                                                                                                                                                                                                                                                         | STUDY OBJECTIVES _____________________________________________ 32 2.1 Primary Objectives ____________________________________________ 32 2.2 Secondary Objectives __________________________________________ 32                                                                                                                                                                                                                                                                                                                                                                         |
| 3                                                                                                                                                                                                                                                                                                         | STUDY DESIGN __________________________________________________ 32 3.1 Subject Enrollment and Sample Procurement and Data Processing ____ 32 3.2 RNA Microarray Analysis ______________________________________ 33 3.3 DNA Mutation Panel __________________________________________ 33 3.4 Data Analysis and Predictive Methodologies _______________________ 33 3.6 Safety Evaluations ____________________________________________ 40 3.7 Efficacy Evaluations __________________________________________ 41 3.8 Biological Studies _____________________________________________ 41 |
| 4                                                                                                                                                                                                                                                                                                         | SUBJECT SELECTION ____________________________________________ 41 4.1 Inclusion Criteria _____________________________________________ 41 4.2 Exclusion Criteria ____________________________________________ 42                                                                                                                                                                                                                                                                                                                                                                        |
| 5                                                                                                                                                                                                                                                                                                         | STUDY PROCEDURES AND ASSESSMENTS _________________________ 42 5.1 Enrollment of Subjects_________________________________________ 42 5.2 Screening ___________________________________________________ 43 5.3 Study Procedures/Study Interventions ____________________________ 44                                                                                                                                                                                                                                                                                                      |  
6 Protocol Drugs *____________________________________________________* 47  
| 7   | Efficacy Assessments _______________________________________________ 49 7.1 Tumor Assessments/Scans______________________________________ 49 7.2 Scan Submission:_____________________________________________ 49 7.3 Response Criteria _____________________________________________ 49   |
|-----|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
8 Sample Size Justification____________________________________________ 51  
| 9   | Laboratory Evaluations _____________________________________________ 52 9.1 Specimens to be Collected, Schedule and Amount ___________________ 52 9.2 Tumor collection and correlative biology studies. ___________________ 52 9.3 Detailed Procedure For Sending Subject Samples: __________________ 53 9.4 Correlative Biology Studies _____________________________________ 58 9.5 Storage _____________________________________________________ 58 9.6 Plan for Communication of Samples and Data _____________________ 59 9.7 Plan for Communication of Collaborating Sites ____________________ 60   |
|-----|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
10 ADVERSE EVENT REPORTING ____________________________________ 60 11 SUBJECT WITHDRAWAL AND TRIAL DISCONTINUATION ___________ 64  
| 11.1 Criteria for Subject Off-Therapy________________________________ 64 11.2 Criteria for Subject Off-Study __________________________________ 64 11.3 Trial Discontinuation_________________________________________ 64   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| 12   | DATA ANALYSIS _________________________________________________ 64 12.1 Data Quality Assurance_______________________________________ 64 12.2 Data Safety Monitoring Board (DSMB) __________________________ 65 12.3 Process and Feasibility Analysis ________________________________ 65 12.4 Secondary Data Analysis ______________________________________ 65   |
|------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
References ________________________________________________________________ 69 Appendix I: Performance Status/Scores **_______________________________________** 73 Appendix II: Biology Studies Collection and Shipping forms ______________________ 74 Appendix III: Pharmacopeia Library _________________________________________ 76 Appendix IV: Intervention Insights Whole Body Reference Set ___________________ 266 Appendix V: OncInsights Method Summary Diagram __________________________ 268  
| 13   | ADMINISTRATIVE PROCEDURES _________________________________ 65 13.1 Subject Informed Consent _____________________________________ 65 13.2 Ethical Conduct of the Study and IRB/IEC Approval _______________ 66 13.3 Monitoring _________________________________________________ 66 13.4 Pre-Study Documentation _____________________________________ 67 13.5 Confidentiality ______________________________________________ 67 13.6 Source Documents ___________________________________________ 67 13.7 Record Retention ____________________________________________ 68   |
|------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| PROTOCOL SYNOPSIS PROTOCOL TITLE   | A Feasibility Trial using Molecular-guided therapy for the treatment of  patients with relapsed and refractory childhood cancers                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |              |
|------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------|
| PROTOCOL NUMBER                    | NMTRC 008                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |              |
| PHASE OF DEVELOPMENT               | Feasibility                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   |              |
| OBJECTIVES                         | Primary Determine feasibility of using tumor samples to assess genomic mRNA  expression arrays and DNA Mutation Panels using predictive modeling  to make real-time treatment decisions for children with  relapsed/refractory cancers.  Secondary - To continue to evaluate the safety of allowing a molecular tumor  board to determine individualized treatment plans - To determine the activity of treatments chosen based on: - Overall response rate (ORR)  - Progression free survival (PFS)  - (Voluntary) To explore the relationship between tumor  phenotype/genotype and response by permitting use of tumor tissue  in a correlative biologic study - To compare PFS interval to PFS intervals of previous  chemotherapy regimens since relapse for each subject.                                                                                                                                                                                                                                                                                               |              |
| STUDY DESIGN                       | A prospective open label, multicenter study to evaluate efficacy of  molecularly guided therapy in patients with relapsed or refractory  childhood cancers A total of 48 evaluable subjects with childhood cancer that are  refractory to or have relapsed on conventional therapy will be treated with molecular guided therapy.  Subjects will be evaluated in 3 strata: - Stratum 1: 16 subjects with refractory/relapsed neuroblastoma - Stratum 2: 16 subjects with relapsed or refractory brain tumors Stratum 3: 16 subjects with refractory or relapsed rare tumors Guided therapy will allow the use of any therapeutic combination (up  to 4 agents) provided it includes medications contained in the study  report. All subjects will be followed for survival, disease response,  progression and safety. All subjects will be treated according to the  discretion of the treating oncologist and study committee (minimum 3  oncologists and one pharmacist). Extent of disease will be measured  and assessed for changes throughout the course of the study. |              |
| Version 2.0 Final                  | Page 13 of 268                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                | Confidential |
| Issued 12 June 2013                |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |              |  
| ELIGIBILITY         | Inclusion Criteria 1. Subjects must have histologically proven neuroblastoma, brain  tumor, or rare tumor and confirmation of refractory or recurrent  disease with histologic confirmation at diagnosis or at the time of  recurrence/progression  2. Subjects must be age >12 months at enrollment 3. Subjects must be age ≤ 21 years at initial diagnosis 4. Subjects must have measurable disease as demonstrated by  residual abnormal tissue at a primary or metastatic site measuring  more than 1 cm in any dimension by standardized imaging (CT or  MRI); tumor must be accessible for biopsy. Subjects with bone  marrow only disease expected to be >75% tumor are eligible to  enroll. 5. Current disease state must be one for which there is currently no  known curative therapy  6. Lansky or Karnofsky Score must be more than 50 7. Subjects without bone marrow metastases must have an ANC >  750/μl. 8. Adequate liver function must be demonstrated, defined as: a. Total bilirubin ≤ 1.5 x upper limit of normal (ULN) for age  AND b. ALT (SGPT) < 10 x upper limit of normal (ULN) for age 9. A negative serum pregnancy test is required for female  participants of child bearing potential (≥13 years of age or after  onset of menses) 10. Both male and female post-pubertal study subjects need to agree  to use one of the more effective birth control methods during  treatment and for six months after treatment is stopped. These  methods include total abstinence (no sex), oral contraceptives  ("the pill"), an intrauterine device (IUD), levonorgestrol implants  (Norplant), or medroxyprogesterone acetate injections (Depoprovera shots). If one of these cannot be used, contraceptive foam  with a condom is recommended. 11. Informed Consent: All subjects and/or legal guardians must sign  informed written consent. Assent, when appropriate, will be  obtained according to institutional guidelines Exclusion Criteria 1. Subjects who have received any cytotoxic chemotherapy within  the last 7 days prior to enrollment and 14 days prior to study  treatment start date.  2. Subjects who have received any radiotherapy to the primary  sample site within the last 14 days (radiation may be included in  treatment decision after biopsy). 3. Subjects receiving anti-tumor therapy for their disease or any  investigational drug concurrently.                |              |
|---------------------|----------------|--------------|
| Version 2.0 Final   | Page 14 of 268 | Confidential |
| Issued 12 June 2013 |                |              |  
| 4.                                                                                                                                                                                                                                                                                                                                                   | Subjects with serious infection or a life-threatening illness  (unrelated to tumor) that is > Grade 2 (NCI CTCAE V4.0), or  active, serious infections requiring parenteral antibiotic therapy.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |              |     |       |         |            |           |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------|-----|-------|---------|------------|-----------|
| 5.                                                                                                                                                                                                                                                                                                                                                   | Subjects                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    | with         | any | other | medical | condition, | including |
| malabsorption syndromes, mental illness or substance abuse,  deemed by the Investigator to be likely to interfere with the  interpretation of the results or which would interfere with a  subject's ability to sign or the legal guardian's ability to sign the  informed consent, and subject's ability to cooperate and  participate in the study |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |              |     |       |         |            |           |
| Additional criteria: Subjects willing to participate in the correlative biologic studies will  sign an additional consent form to provide tumor tissue.                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |              |     |       |         |            |           |
| ESTIMATED NUMBER OF  PATIENTS/GEOGRAPHIC  REGIONS                                                                                                                                                                                                                                                                                                    | 48 evaluable (53-59 total) relapsed/refractory pediatric cancer patients16 neuroblastoma tumors, 16 brain tumors, and 16 rare tumors.  Patients will enroll at Arnold Palmer Children's Hospital (MD  Anderson Cancer Center Orlando) ,St. Louis University/Cardinal  Glennon Children's Medical Center, Helen DeVos Children's Hospital,  Levine Children's Hospital, Connecticut Children's Medical Center,  Mercy Children's Hospital, Rady Children's Hospital of San Diego, Medical University of South Carolina, Monroe Carrell Jr. Children's  Hospital at Vanderbilt, Phoenix Children's Hospital, Primary  Children's Hospital, Kapiolani Medical Center for Women and  Children, and at Dell Children's Hospital.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |              |     |       |         |            |           |
| LENGTH OF STUDY                                                                                                                                                                                                                                                                                                                                      | Accrual to this study is estimated to be approximately 2 years.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |              |     |       |         |            |           |
| STUDY ASSESSMENTS                                                                                                                                                                                                                                                                                                                                    | Refer to Table of Assessments for timing of study procedures.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |              |     |       |         |            |           |
| CRITERIA FOR                                                                                                                                                                                                                                                                                                                                         | Feasibility Measures: Primary objective                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |              |     |       |         |            |           |
| EVALUATION                                                                                                                                                                                                                                                                                                                                           | The definition of feasibility for this study will include: "Enrollment  onto study, RNA expression profile completed, DNA Mutation  Panel completed, genomic analysis and report generation, tumor  board held with treatment decision, treatment review completed  and start of treatment by 21 days post biopsy/surgical resection  date, and then completion of 1 cycle of therapy." Safety Measures: Secondary objectives - Safety analysis will be conducted on all subjects who have received  at least one dose of therapy, and will include the frequency of all  reported adverse events and laboratory abnormalities as well as  frequency of dose interruptions, dose reductions and treatment  discontinuation. |              |     |       |         |            |           |
| Version 2.0 Final                                                                                                                                                                                                                                                                                                                                    | Page 15 of 268                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | Confidential |     |       |         |            |           |
| Issued 12 June 2013                                                                                                                                                                                                                                                                                                                                  |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             |              |     |       |         |            |           |  
Efficacy Measures: Secondary objectives  
| -                      | Response:  To determine the overall response rate (ORR) by the presence of  radiologically assessable disease by cross-sectional CT or MRI  imaging and/or by MIBG or PET scans.   Duration of response, defined as the period of time from when  measurement criteria are met for complete response (CR) or  partial response (PR), whichever is first recorded, until the first  date that recurrent or progressive disease (PD) is objectively  documented (taking as reference for PD the smallest  measurements recorded since the treatment started)  The assessment of response will include the initial measurable  targets and will be performed after cycle 2, then after every other  cycle.   |
|------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| -                      | A subject will be defined as a responder if CR/PR is observed at  any time of the treatment                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  |
| -                      | Clinical response will be seen if stable disease and decrease in  tumor markers by ≥ 50% or CR of bone marrow.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               |
| -                      | Time to progression, defined as the period from the start of the  treatment until the criteria for progression are met taking as  reference the screening measurements                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
| STATISTICS/SAMPLE SIZE | 48 evaluable subjects                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        |
| ESTIMATE               |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
untitled h1
table of procedures and assessments

| Off  Therapy/ 30 Day  Follow-up                                                                        |                                          |                                                                  |                         |    |    |    |    |
|--------------------------------------------------------------------------------------------------------|------------------------------------------|------------------------------------------------------------------|-------------------------|----|----|----|----|
| Informed consent                                                                                       | X                                        |                                                                  |                         |    |    |    |    |
| Demographics                                                                                           | X                                        |                                                                  |                         |    |    |    |    |
| Medical history                                                                                        | X                                        |                                                                  |                         |    |    |    |    |
| Concurrent meds                                                                                        | X                                        | X                                                                | X                       | X  | X  | X  | X  |
| Physical exam                                                                                          | X                                        | X                                                                | X                       | X  | X  | X  | X  |
| Vital signs                                                                                            | X                                        | X                                                                | X                       | X  | X  |    |    |
| Height                                                                                                 | X                                        |                                                                  |                         |    |    |    |    |
| Weight                                                                                                 | X                                        | X                                                                | X                       | X  |    |    |    |
| Karnofsky or Lansky  play score                                                                        | X                                        | X                                                                | X                       | X  | X  |    |    |
| CBC w/diff,                                                                                            | X                                        | X                                                                | X                       | X  | X  | X  | X  |
| Serum chemistry *                                                                                      | X                                        | X                                                                | X                       | X  | X  |    |    |
| Adverse events                                                                                         | X                                        | X                                                                | X                       | X  | X  | X  | X  |
| Urine catecholamines (NB only) or  other tumor marker (as  available)                                  | Cycle  1 Day  15                         |                                                                  |                         |    |    |    |    |
| Cycle  1 Day 1                                                                                         | Cycle  1 Day 8                           | Cycle 1 Day 22 (if  applicable)                                  | Subsequent Cycles Day 1 |    |    |    |    |
| PreStudy                                                                                                        |                                          |                                                                  |                         |    |    |    |    |
| Procedure                                                                                              | X                                        | X                                                                | X                       | X  |    |    |    |
| surgical resection &/or  diagnostic biopsy                                                             | X                                        |                                                                  |                         |    |    |    |    |
| MRI or CT                                                                                              | X                                        | Radiologic measurements will be performed every 8 weeks or every |                         |    |    |    |    |
| MIBG/PET (NB only)                                                                                     | X                                        | other cycle (whichever occurs first)                             |                         |    |    |    |    |
| Bone Marrow (NB or                                                                                     | X                                        | Should be repeated every 8 weeks or every other cycle (whichever |                         |    |    |    |    |
| any subject suspected                                                                                  | occurs first) if positive at study entry |                                                                  |                         |    |    |    |    |
| to have BM disease ) ^ B-HCG                                                                           | X                                        |                                                                  |                         |    |    |    |    |
| EKG (as indicated)                                                                                     | X                                        |                                                                  |                         |    |    |    |    |
| * Total bilirubin, BUN, creatinine, electrolytes, AST, ALT, LDH, Tumor Specific Marker  (if available) |                                          |                                                                  |                         |    |    |    |    |  
^Standard of Care Bone Marrow. (Separate consent required for additional research samples.  
Collect per section 9.3.1.3)
1.1 Study Disease Relapsed or refractory neuroblastoma, brain tumors, and rare childhood tumors.
untitled h1
1.2 rationale lethality of relapsed/refractory

Neuroblastoma is the most common extracranial solid tumor in children, with 700 new diagnoses projected for 2011. It accounts for 7% to 10% of childhood cancers [1, 2]. Whereas the prognosis for infants with neuroblastoma is generally good, currently only 30% of children diagnosed after 12-15 months of age survive despite aggressive multimodal therapies [3, 4]. Even with high-dose chemotherapy (HDC) followed by hematopoietic stem cell transplantation (HSCT) and maintenance therapy with retinoic acid the 5-year event-free survival remains below 50% [5, 6].  
Long-term survival of patients who are treated with conventional therapies following relapse is <5%. As such, neuroblastoma accounts for 15% of all pediatric cancer deaths in the United States
[7]. Consequently, the evaluation of new drugs is strongly needed in this disease.  
Recent evidence has established the genetic heterogeneity of the disease and revealed the existence of several major molecular subsets that collectively may provide prognostic value for future disease management [8] [9]. While the poor prognosis for older neuroblastoma patients underscores the need for new treatment strategies, the elucidation of specific biologic subsets of neuroblastoma suggests a way to improve disease management. The current standard-of-care treatments for relapsed neuroblastoma include a variety of Phase II or Phase I studies that generally have only modest response rates (10%-35%) [4]. Even in responding patients, tumors often go on to further rapid relapses and novel strategies to treat this patient population are urgently needed. There are currently few treatment options from which pediatric oncologists can select with any degree of confidence to improve the management of multiply recurrent neuroblastoma patients. The current strategy is to add salvage therapies based on the anecdotal experience of the treating physician, which often leads to drug-related toxicity but may or may not extend life. The identification of agents that target specific molecular pathways associated with the development and/or progression of neoplastic diseases holds promise. Improved approaches that identify in a more rational (data-driven) fashion combinations of existing agents that are likely to be effective should result in a survival benefit in the clinical setting, while avoiding the toxicity associated with agents that are unlikely to be beneficial. [10].
untitled h1
cns tumors

CNS tumors are the most common solid tumors in children, with 3000-40 00 new diagnoses each year. CNS tumors account for approximately 25-30% of all childhood cancers. Though many of these CNS tumors are quite treatable, the 5-year survival for pediatric CNS tumors is only 71%[11]. Patients who have relapsed and refractory CNS tumors are those most likely to succumb to their disease, in spite of aggressive chemotherapy and radiation, often followed by HSCT[12, 13]. Therefore, new treatment strategies are needed for control of CNS tumors. There is a growing focus on treating CNS tumors based on the biology of the disease[14, 15] rather than with "one size fits all" therapy. Risk stratification in CNS tumors based on biology is increasingly understood to be a priority. For example, medulloblastoma has been broken into 4 biologic categories with different genetic mutations and different clinical outcomes[16-18].  
Similar to neuroblastoma, these differences in the biology and clinical outcome of patients with medulloblastoma suggest new strategies to improve disease management. The current standardof-care treatments for relapsed CNS tumors include Phase I and Phase II therapies that often have few, if any, responding patients[19, 20]. Again reflecting the challenges in neuroblastoma, responding patients with high grade CNS tumors often experience multiple relapses and are in dire need of novel treatment strategies. Improved approaches that can inform treatment regimens based on the biology of the disease could result in a survival benefit in this challenging patient population.
untitled h1
rare cancers

Although improvements in the past 40 years have led to markedly improved survival rates of approximately 80% overall for pediatric cancers, patients with relapsed, rare and advanced stage tumors still have a very poor prognosis. Soft tissue sarcomas account for ~ 7% of pediatric cancers, with 850-900 new cases annually in the United States [21]. Rhabdomyosarcoma, the most common soft tissue sarcoma in children and adolescents, affects nearly 350 children in the United States each year [21]. Patients with metastatic rhabdomyosarcoma have a poor prognosis with an overall survival rate of ~30% [22]. The non-rhabdomyosarcoma soft tissue sarcomas affect ~500 children each year. Patients with metastatic non-rhabdomyosarcoma soft tissue sarcomas have long-term survival < 20% [23]. Malignant bone tumors (mainly Ewing's Sarcoma and Osteosarcoma) account for 6% of childhood cancers, with ~400 cases of osteosarcoma and 200 cases of Ewing's Sarcoma diagnosed annually [21]. Patients with localized osteosarcoma, who have a poor response to pre-operative chemotherapy have a survival rate of around 50%, while patients with metastatic osteosarcoma or Ewing's sarcoma disease at presentation, have a survival of < 20% [24, 25]. Renal Cancers account for ~6% of pediatric cancer diagnoses, with 95% of cases being Wilms Tumor, which has a very favorable prognosis [21]. However, rare and high risk renal tumors such as metastatic anaplastic Wilms tumor, clear cell sarcoma of the kidney, renal cell carcinoma, and malignant rhabdoid tumor have an inferior outcome, with survival of < 20% [26, 27]. For all these patients survival remains poor, despite use of doseintensified multi-agent chemotherapy and stem cell transplantation. Therefore, new approaches such as targeted therapies are warranted.
untitled h1
molecular networks as the drug

It is now firmly established that cancer results from perturbations in the molecular networks within cellular systems that disturb the normal homeostatic state [28-30]. Fluctuations in these networks can result from genetic or epigenetic cellular events and/or changes in the molecular constitution of the tumor microenvironment, which collectively dictate the phenotype of the biological system. The molecular networks engaged during tumor development and/or progression are complex. Molecular networks have evolved to provide the ultimate level of cellular plasticity, allowing cells to adapt to or exploit extracellular cues within the local milieu
[31]. The complexity of a tumor system is further exaggerated by the inherent genomic instability seen in many neoplasms, which leads to an accelerated micro-evolutionary process that results in further cellular and tumor sub-system heterogeneity [32, 33]. This variability combined with the adaptability of many molecular pathways provides a somewhat predictable and highly probable path to resistance for any given agent that targets a subset of cellular systems within a tumor's molecular and genetic repertoire [34]. A fundamental challenge in the area of targeted cancer treatment is how to identify optimal therapeutic combinations that can treat heterogeneous tumors that are both highly adaptive and that exhibit significant inter- and intra-patient variation [28, 3537]. Our proposal outlines an approach by which we can utilize our expanding knowledge of molecular networks and the mechanisms of action of a growing pharmacopeia [38, 39] in conjunction with standardized biomarker assessments to deliver targeted combinations of effective therapies to neuroblastoma patients.
untitled h1
practice and promise of biomarkers

setting to identify neuroblastoma patients most likely to respond to a specific therapy. For example, activation of the ALK gene through sequence variation mutations has been identified in a subset of neuroblastoma patients, and small-molecule inhibition of the ALK-encoded receptor tyrosine kinase caused cytotoxicity in affected neuroblastoma cell lines [40]. Such studies, in conjunction with a large body of in vitro and in vivo data, have further demonstrated that the efficacy of specific treatment modalities is dependent upon the molecular constitution of the tumor, and that the observed variations in tumor response to current therapies is attributable in large part to disease heterogeneity at the molecular level. While individual biomarkers may be predictive of responses to specific therapies, especially in the context of front-line treatment, a different approach to the management of patients with refractory disease is needed in order to identify effective treatments from the catalog of available agents. Advances in informatics and molecular technologies, coupled with our expanding knowledge of molecular networks and mechanism of action of the existing pharmacopeia, provide a great opportunity in translational medicine to develop a model that more accurately predicts tumor response. The studies outlined in this proposal are a step in the development of personalized oncology, in which each patient is truly treated individually based upon the systematic molecular analysis of their disease. Our project is focused on testing the merits of a specific data-driven predictive method through which any number of drugs in our current knowledge base has a chance of being recommended, of these only the FDA approved drugs listed in Appendix III will be used. Although this novel approach to personalized therapeutics brings with it to the concern that toxicities that may arise from novel combinations of agents, it is strongly felt by the investigators that the logical interpretation of molecular data by a committee of highly qualified physicians and pharmacists in order to identify drug candidates for consideration in the treatment of a patient population with few alternative options is a worthwhile endeavor. The systematic biomarker-driven approach outlined in this proposal, by tailoring a combination of drugs targeting the specific molecular composition of a tumor—irrespective of tumor classification or anatomical origin— provides a feasible alternative to the conventional approach that targets specific organs or tissues without consideration of the underlying tumor biology.
untitled h1
validation of gene expression as

The determination of gene transcript abundance through gene expression profiling has been frequently exploited in biomarker research. The mRNA analyte provides an estimation of the dynamic molecular events within a biological specimen. Moreover, various technologies that permit gene expression profiling have provided a systematic and high-throughput means to assess multiple mRNA transcripts simultaneously within a biological sample on a genomic scale, and have created an abundance of empirical datasets across cohorts of moderately characterized biological specimens [41]. Within the field of cancer research, gene expression profiles derived from human tumor specimens have been used to identify molecular subtypes associated with tumor cell behavior, patient outcome and response to a wide range of therapies [42]. For example, gene expression signatures have been utilized to create a connectivity map in which the genomic consequences of drug exposure can be aligned with de-regulated genes within a tumor specimen to identify compounds that may reverse the tumorigenic genotype [43]. Screening of a large number of potential therapeutic agents in a panel of cancer cell lines with a baseline gene expression profile has permitted the association between a gene expression signature and predicted response to various drugs [44, 45]. Thus, a wealth of publicly available genomic datasets and predictive methodologies coupled with increasing evidence demonstrating the utility of gene expression signatures as a valid biomarker argues for increased efforts towards implementing a more systematic approach to molecular-based theranostics in the prospective clinical setting. We propose to integrate the results of different predictive methodologies based upon the consistent input of tumor-derived gene expression data, to provide consolidated information to the treating physician regarding treatment options with predicted efficacy. The predictive methods under evaluation include the rudimentary analysis of expression of target genes, as well as the more sophisticated analysis of molecular network topology and signature based methods based upon empirical datasets. Throughout the project, we will seek to establish the potential predictive value of standardized gene expression data and the described methodologies for predicting efficacious agents for refractory or relapsed neuroblastoma patients and determine which, if any, of these individual predictive methodologies holds the most promise for further development as a diagnostic standard. Additionally, while beyond the scope of this specific proposal, the tumor resource created will also be used to assess the potential predictive value of emerging molecular technologies that assess other molecular aberrations associated with differential therapeutic response (such as single nucleotide polymorphisms, DNA sequencing, gene amplifications/deletions, gene promoter methylation, protein modifications, etc [28, 30].
untitled h1
pivotal pilot study highlights feasibility

In conjunction with community hospitals and physician groups in West Michigan, the Van Andel Research Institute under the direction of Dr. Webb recently completed a proof-of-feasibility study in which a heterogeneous group of fifty late stage cancer patients were enrolled to determine the feasibility and potential impact of providing physician's with molecular-based therapy predictions and to provide an early assessment of the first generation predictive methodologies. In this feasibility study, clinical investigators worked closely with Dr. Webb to coordinate a multidisciplinary team that included individuals from adult and pediatric oncology, surgery, interventional and non-interventional radiology, pathology, pharmacy, clinical research, statistics, bioinformatics, and clinical and basic science laboratories. Table 1 shows a sample of patients enrolled in this first pilot study, and highlights the patients whose physicians utilized the personalized report providing an early indication of the potential value of the information provided. In brief, 50 patients were consented to the study in which surgical tumor specimens excised as part of a standard of care diagnostic procedure were processed in a real-time fashion to obtain a gene expression profile from each qualified tumor specimen. Consistent with current practices, a series of essential quality control (QC) standards were implemented to ensure assay validity which included criteria regarding the proportion of viable tumor cells within surgical specimens, RNA quality, cDNA synthesis and amplification, GeneChip hybridization, data normalization and analysis. After obtaining a gene expression profile on the tumor specimen, researchers utilized the XB-BIS system to generate a standardize report for physicians to utilize in determining patient drug regimen. It is important to note that our current procedures permit a < 3 week turnaround from receipt of an individual tumor sample to the generation of a standardized report predicting drug efficacy. Our ability to provide real time feedback in less than 3 weeks is critical to permit the consideration of the predicted therapies in the care of the individual patient, and is concomitant with the wash out period between the  
| patient's prior treatment and administration of a new regimen.   |                            |               |                   |     |     |     |
|------------------------------------------------------------------|----------------------------|---------------|-------------------|-----|-----|-----|
| Age at                                                           | Report                     | Report        | | Clinical Beneft |     |     |     |
| Diagnosis                                                        | Gender                     | Specimen Type |                   |     |     |     |
| Enrolment                                                        | Generated Information Used | Observed      |                   |     |     |     |
| Neuroblastoms                                                    | Bone Marrow Aspirat        | Yes           | Yes               |     |     |     |
| Hodgkin Lymphoma                                                 | u                          | 19            | Peripheral Blood  | Yes | No  |     |
| Synovial Sarcoma                                                 | u                          | 16            | Surgical          | Yes | No  |     |
| n                                                                |                            |               |                   |     |     |     |
| Clear Cell Sarcoma of the Kidney                                 | Surgical                   | Yes           | Yes               | Yes |     |     |
| Osteosarcome                                                     | M                          | 17            | Surgical          | Yes | No  |     |
| Lung Cancer                                                      | M                          | Pleural Fluid | No                |     |     |     |
| Osteosarcoma                                                     | u                          | No            |                   |     |     |     |
| Surgical                                                         |                            |               |                   |     |     |     |
| Wilms' Tumor                                                     | N                          | Surgical      | No                |     |     |     |
| Gastric Cancer                                                   | Ascies Fluid               | YES           | Yes               |     |     |     |
| F                                                                | Asciles Fluid              |               |                   |     |     |     |
| Gastric Cancer                                                   | 50                         | Yes           | Yes               | No  |     |     |
| Malignant Melanoma                                               | M                          | 56            | Surgical          | Yes | Yes | No  |
| Colon Cancer                                                     | v                          | Needle Biops  | Yes               |     |     |     |
| Ewing's Sarcom                                                   | u                          | Yes           |                   |     |     |     |
| Surgical                                                         | Yes                        | No            |                   |     |     |     |
| I                                                                | 70                         |               |                   |     |     |     |
| Lung Cancer                                                      | No                         |               |                   |     |     |     |
| Colon Cancer                                                     | N                          | Surgical      | Yes               | Yes | No  |     |
| Esophageal Cance                                                 | M                          | 48            | Needle Biopsy     | No  |     |     |
| Inflammatory Myofibroblastic Tumor                               | M                          | Needle Biopsy | Yes               | Yes | No  |     |
| Breast Cancer                                                    | Surgical                   | No            |                   |     |     |     |
| Mantie Cell Lymphoma                                             | E                          | 62            | Needle Biopsy     | Yes | No  |     |
| Rhabdomyosarcoma                                                 | Surgical                   | Yes           | No                |     |     |     |
| Malignant Melanom                                                | M                          | 29            | Surgical          | Yes | No  |     |
| Bone Marrow Aspirate                                             | Yes                        | Yes           | Yes               |     |     |     |
| v                                                                |                            |               |                   |     |     |     |
| Ewing's Sarcon                                                   | Surgical                   | Yes           |                   |     |     |     |
| Gastro Esophageal Junction Cancer                                | M                          | Pleural Fluid | No                |     |     |     |
| Malignant Melanoma                                               | No                         |               |                   |     |     |     |
| Surgical                                                         |                            |               |                   |     |     |     |
| Breast Cancer                                                    | Surgical                   | No            |                   |     |     |     |
| Non Small Cell Lung Cancer                                       | F                          | Surgical      | Yes               | Yes | No  |     |
| Lung Cancer                                                      | u                          | Surgical      | Yes               |     |     |     |
| Breast Cance                                                     | Surgical                   | Yes           | Yes               | No  |     |     |
| n                                                                |                            |               |                   |     |     |     |
| Pancreatic Cancer                                                | 58                         | Needle Biopsy | Yes               | Yes | Yes |     |
| Osteosarcoma                                                     | Surgical                   | Yes           | Yes               | No  |     |     |
| Colon Cancer                                                     | i                          | No            |                   |     |     |     |
| I I                                                              |                            |               |                   |     |     |     |
| Malignant Melanoma                                               | 56                         | Surgical      | Yes               | NO  |     |     |
| Colon Cancer                                                     | edle Biops                 | Yes           | No                |     |     |     |
| Merkel Cell Carcinomi                                            | M                          | 49            | Surgical          | Yes | Yes | No  |
| n                                                                |                            |               |                   |     |     |     |
| Lymphangioma                                                     | Surgical                   | Yes           | No                |     |     |     |
| Small Bowel Cance                                                | Needle Biopsy              | Yes           | Yes               | No  |     |     |
| Hepatoblastom                                                    | M                          | Surgical      | Yes               | Yes | Yes |     |
| Breast Cancer                                                    | F                          | 60            | Needle Blopsy     | Yes |     |     |
| Renal Cell                                                       | Needle Biopsy              | No            |                   |     |     |     |
| Colon Cancer                                                     | F                          | 45            | Surgical          | Yes | Yes | No  |
| Pancreatic Cance                                                 | M                          | Needle Biopsy | Yes               | No  |     |     |
| Osteosarcoma                                                     | l                          | Surgical      | Yes               |     |     |     |
| Non Small Cell Lung Cancer                                       | F                          | 50            | Surgical          | Yes | Yes | No  |
| Plasmacyto                                                       | M                          | Surgical      | Yes               |     |     |     |
| Ovaria                                                           | u                          | 50            | Surgical          | Yes | Yes | Yes |
| Non Small Cell Lung Cancer                                       | 63                         | Surgical      | Yes               | Yes | Yes |     |
| Renal Cel                                                        | v                          | YES           |                   |     |     |     |
| Surgical                                                         | Yes                        | Yes           |                   |     |     |     |
| Non Small Cell Lung Cancel                                       | M                          | 63            | Surgical          | Yes | Yes | Yes |
| Lymphoms                                                         | M                          | 80            | Surgical          | Yes | Yes | No  |
| Table I: Sample of patient feasibility assessments.              |                            |               |                   |     |     |     |  
While this first 50-patient study was designed to determine the feasibility of the overall approach in different tumor types and to establish the necessary logistical workflow between the multiple disciplines, some anecdotal signs of tumor response and physician-reported patient benefit were observed (Table 1). As the Table illustrates, useful data was generated on 39/50 patients.  Of these, 24 out of the 34 cases utilized the report information.  Treatment decisions were based on the reported data for 24 patients, and 9 patients experienced tumor responses (37.5% response Version 2.0 Final Page 22 of 268 Confidential rate) determined through objective assessment of tumor burden (radiological imaging and/or biomarker endpoints, manuscript in preparation). While these findings remain anecdotal due to the uncontrolled nature of the study, they provide significant impetus to move ahead with the disease focused trial design outlined in this proposal. Moreover, these preliminary studies have demonstrated the feasibility of the approach in the real time prospective clinical setting.
untitled h1
use of transcriptional analysis to

In the proof-of-feasibility study, the specific predictive methodologies were being developed and refined throughout, and led to the standardized methods outlined in this proposal. The most rudimentary method of transcriptional analysis relates to assessment of target gene expression. In some cases, the over-expression of a specific drug target at the transcriptional level is associated with efficacy of an inhibitor against the corresponding protein target; for example overexpression of *Her2* indicates use of trastuzumab in breast cancer [46]. Despite concerns over the discordance between transcript expression and protein target activity (which is partially addressed through development of our network topology method described below), the simple assessment of target transcript abundance showed early promise in the pilot study. For example, a complete response was observed in a female adolescent with acute lymphoblastic leukemia (ALL) following the administration of a multi-kinase inhibitor that was selected based upon a significant over-expression of one of the drugs known targets (*Flt-3)* at the transcriptional level (z-score >+10). Thus, while we now also incorporate a more sophisticated analytical method to infer target activation status based upon analysis of network topology (see below) and will also be testing the utility of well published signature-based methods for selecting drug candidates[43-45, 47, 48] simple target gene expression remains a rational method for identification of drug candidates. The knowledge base utilizes the existing pharmacopeia's postulated molecular mechanisms of action and contains molecular entities that are targeted by drugs, many of which are approved for human use. This includes several targets of particular relevance to neuroblastoma including VEGFA-VEGFR, TRK, mTOR, ALK, HDAC etc [8]. The transcriptional status of each of these targets is reported using the standardized z-score where the relative level of each target within an individual's tumor is determined through a comparison with a whole body reference set.  
Novel Molecular Network Topology Analysis to Improve Utility of Targeted Pharmacopeia.  
A novel method developed in partnership with GeneGo over the past few years relates to the analysis of molecular network topology [49]. As stated above, the expression of a target gene does not necessarily equate to activation of the target protein. Nonetheless, we investigated the possibility of utilizing known protein-protein and protein-small molecule interactions within GeneGo's MetaCore™ database (over 200,000 interactions) in conjunction with transcriptional data to infer key network targets that if modulated with a targeted agent, may disrupt the disease system(s) identified within each patient's tumor. As with all methodologies in this proposal, tumor-derived transcriptional data (normalized to a reference set of normal whole body samples) provides the consistent input to the algorithm. This specific methodology was applied retrospectively to the data generated during the first 50-patient feasibility study and anecdotally corroborated the potential of this systematic approach for identifying key target hubs within perturbed molecular networks. For example, in the leukemic cells of the pediatric patient with ALL described above, *Flt-3* was identified as a likely input point to an integrated transcriptional network. This patient responded well to a targeted therapy that inhibits multiple kinases including Flt-3. In an adult patient with metastatic non-small cell lung carcinoma, analysis of network topology highlighted *Egfr* as a likely input to a highly connected transcriptional network, and subsequent treatment with erlotinib showed some efficacy (patient exhibited a partial response).  
This methodology has also been tested more systematically against specific public domain datasets. For example, Dr. Webb's group applied the methodology to a public dataset derived from the transcriptional profiling of skin from psoriasis patients. They were quickly able to identify several key targets and some associated targeted agents using this approach, many of which have been validated in the psoriasis literature. Collectively, we believe that the approach to the identification and subsequent analysis of neuroblastoma networks outlined in this proposal provides an exciting tool for systematically selecting network-based targeted regimens for the treatment of refractory or relapsed neuroblastoma patients. The systematic and automated approach described in this proposal is readily applicable to uncovering high quality therapeutic targets, and holds great promise for developing network-based combinational treatment strategies for a wide range of diseases especially for diseases such as neuroblastoma which have a low survival rate and are in need of new therapies. [2, 30].  
Methods to be used in generating this reports are; Drug Target Expression Algorithm, Network Target Activity (Systems Biology Method, GeneGo); Drug Response Signatures Algorithm (Connectivity Map), and Drug Sensitivity Signatures Algorithm (PGSEA). These are furthered explained in section 3.  
Reference Set: Whole body bank of normal tissue gene expression levels are used as the reference set for the normalization calculations. Whole body reference is chosen to provide a wide variance of gene expression for comparison in order to better identify signal from tumor tissue. This reference set will also help to decrease risk for toxicity as it will not identify targets that are highly expressed in normal tissues.
untitled h1
normalization calculations

A z-score is calculated using the mean gene expression level from the reference set to the same gene's expression level from the patient's tumor. A z-score is a quantitative measure of the number of standard deviations above or below the mean of the observed value (patient's gene) in comparison to the same observed value (the same gene in the reference set). Positive z-scores reflect the number of standard deviations that the observation being evaluated is above the mean
(over-expressed) and conversely negative z-scores represent the number of standard deviations that the observation being evaluated is below the mean (under-expressed).
untitled h1
drug knowledgebase

The drug knowledgebase is comprised of publically available, peer-reviewed and clinically relevant pharmacological data. Numerous sources contribute to the knowledgebase including GeneGo, DailyMed, DrugBank, UpToDate, Entrez Gene, Entrez Pubmed, PharmaGKB,
MedTrust Online and MedTrack.
untitled h1
preclinical model for neuroblastoma patients

Neuroblastoma cells can be isolated from either tumor biopsies or the bone marrow of patients using flow cytometry panel (GD2, CD81, CD9, CD56, CD34, CD45) or ficoll gradient isolation
[50]. The bone marrow cells have been sorted and mRNA isolated for expression arrays. The patient cells have been cultured in our lab resulting in both adherent neuroblastoma cells and neurospheres. These cells have been grown for drug testing in vitro and injected into mice for in vivo tumor formation. This will be an important correlative study to continue to use in comparison to clinical response in patients.
untitled h1
analysis of oncinsight's drug prediction

Total RNA preparations from each of SK-N-AS and SH-SY5Y from Dr. Khan's laboratory were analyzed using Affymetrix's HG-U133 Plus 2 platform. Raw MAS5 data was converted using Intervention Insight's whole body expression data, a collection of 45 different adult tissues supplied by Asterand. Dr. Khan's laboratory supplied Intervention Insights with CEL file data from Affymetrix's HG-U133 Plus 2 platform generated in their laboratory to be used as a comparator of cross-laboratory expression data. An Intervention Insights, OncInsights report of predicted drugs was prepared exactly as described elsewhere in the protocol.  
Fifty-six compounds were tested at high and low concentrations in a cell growth assay for each of SK-N-AS and SH-SY5Y (determined as percent growth at 24 hrs post drug treatment)[51]. Of the fifty-six compounds tested, the OncInsights system could select up to eighteen drugs. Of the eighteen drugs selectable, nine were selected (50%, selected either as sensitive or insensitive).  
Seven drugs were selected in the analysis of SK-N-AS and nine drugs were selected in the analysis of SH-SY5Y. Thirty two overlaps were comparable [SK-N-AS high concentration (7); SK-N-AS low concentration (7); SH-SY5Y high concentration (9) and SH-SY5Y low concentration (9)]. For SK-N-AS, 79% agreement was observed and 21% disagreement was observed. For SH-SY5Y, 61% agreement was observed and 39% disagreement was observed.  
Combined, OncInsights accurately predicted growth sensitivities for SK-N-AS and SH-SY5Y 63% of the time.
untitled h1
analysis of oncinsight's drug prediction

The National Cancer Institute (NCI) has established the Pediatric Preclinical Testing Program
(PPTP) for testing drugs against in vitro and in vivo childhood cancer models to aid in the prioritization of drugs considered for early phase pediatric clinical trials. Drugs tested in xenograft models and predicted to be effective were evaluated using the OncInsights report as a comparison.  
AZD2171 Response in Rhabdoid Kidney Tumor FGFR2 overexpression has been identified to affect a variety of bone, skin and cancer pathologies[52]. In this PPTP xenograft sample of a rhabdoid kidney tumor (KT-16), a complete response was shown when treating the tumor with AZD2171 (Recentin®). The mRNA from the xenograft KT-16 was analyzed using Affymetrix's HG-U133 Plus 2 platform and converted using Intervention Insight's whole body expression data. The OncInsights platform using the Drug Target Expression Method identified AZD2171 as a potential therapeutic option, based on the overexpression of FGFR2 (Z=3.978) from this mouse xenograft.  
  
shown in the PPTP Rhabdoid Kidney xenograft, KT-16. (Right). The Drug Target Expression Method identifies ADZ2171 as a potential therapy based on overexpression of FGFR2.  
Dasatinib Response in B-Cell ALL
Several recent papers have demonstrated the effectiveness of dasatinib in the treatment of patients with ALL[53-55]. In this PPTP mouse xenograft of B-cell ALL (ALL-4), treatment with dasatinib resulted in a complete response. The mRNA from the xenograft  ALL-4 was analyzed using Affymetrix's HG-U133 Plus 2 platform and converted using Intervention Insight's whole The OncInsights platform showed that three of the seven targets of body expression data.  
dasatinib were found to be activated in this tumor using the network-based Transcription Factor Activity Method.  
  
square) in this mouse xenograft of B-cell ALL (ALL-4). Of the seven targets of dasatinib, three
untitled h1
dna mutation panel and use

Spectrum Health Molecular Diagnostics offers a CLIA certified targeted next generation sequencing test utilizing multiplex PCR with ion semiconductor sequencing (The Ion AmpliSeq™ Cancer Hotspot Panel v2) designed to amplify 207 amplicons covering approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes. This includes the 739 COSMIC mutations from 46 genes in the first Ion AmpliSeq™ Cancer Panel along with added hotspot mutations from significant cancer genes. The test can provide important individual information regarding tumor development and progression, and a more reliable prediction of personalized cancer therapies. This Cancer Gene Mutation Panel is designed to target 739 mutations in the following 46 key cancer genes: ABL1, AKT1, ALK, APC, ATM,
BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR, KIT, KRAS, MET,
MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET,
SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL, EZH2, GNA11, GNAQ, and IDH2. This mutation panel is designed to detect targeted mutations only. The 46 genes are not sequenced in their entirety. Mutations outside the targeted regions will not be detected. The limit of detection is 5% at 500X coverage.  
This is a test currently available at centers such as Baylor College of Medicine as the "Cancer Gene Mutation Panel - Targeted Mutation Detection by Next Generation Sequencing". While it is currently available for patients, our study will examine the safety and predictive value for tumor boards of this test in pediatrics.
untitled h1
a pilot trial testing the

The primary objective of this pilot study was to evaluate the feasibility of using predictive modeling based on genome-wide mRNA expression profiles of neuroblastoma tumor biopsies to make real-time treatment decisions. Feasibility was defined as the completion of the following in a two week time period: tumor biopsy, quality RNA extraction, mRNA U133 2+ Affymetrix gene chip hybridization, analysis utilizing a series of predictive methodologies, report generation, tumor board review with formulated treatment plan, and medical monitor review. Subjects were not treated on this study. There were 5 subjects enrolled between April-June 2010 with multiply relapsed or refractory neuroblastoma. Subjects had received between 2-13 previous relapsed therapies and were between 2-6.5 years post diagnosis. All subjects had soft tissue disease which was able to be biopsied. All biopsies were adequate by pathology evaluation (>75% viable tumor)
and RNA quality (>6.5 RIN). Gene chips were completed in 3-7 days, report generation was 1-5 days, tumor board was 1-3 days, medical monitor sign off was 1 day. The total time was 10-12 days for all subjects. The tumor board which consisted of 4-10 pediatric oncologists from sites across the US and a pediatric oncology pharmacist was able to create individualized therapy regimens for all subjects. Correlative biology specimens were obtained and grown in culture.  
Mice xenografts of 3/5 subjects were established. Cultures and xenografts are able to be used for validation studies of predicted drug sensitivity. In conclusion it is feasible to obtain real-time genomic profiling for molecularly guided therapy for use in treatment decision making.
untitled h1
a feasibility trial using molecular-guided

The primary objective of this feasibility study was to evaluate the feasibility and safety of using predictive modeling based on genome-wide mRNA expression profiles of neuroblastoma tumor biopsies to make real-time treatment decisions. Feasibility was defined as "enrollment onto study, quality mRNA obtained, gene chip completed, tumor board held, medical monitor review and approval, start of treatment by 21 days post biopsy/surgical resection date, and completion of 1 cycle of therapy." There are 11 subjects enrolled to date with multiply relapsed or refractory neuroblastoma. Subjects were between 1-7 years post diagnosis. All subjects had soft tissue disease which was able to be biopsied. All biopsies were adequate by pathology evaluation (>75% viable tumor) and RNA quality (>6.5 RIN). 2 subjects were deemed ineligible due to tumor type after biopsy. Gene chips were completed in 3-8 days (95% CI: 3.8 - 6.8), report generation took 0-3 days (95% CI: 0.0 - 1.5), tumor board took 1-6 days (95% CI: 1.6 - 4.2),
medical monitor sign off took 1-2 days (95% CI: 0.8 - 1.4). The total time from date of biopsy to tumor board was 6-11 days (95% CI: 7.5 - 10.2) for all subjects and 7-20 days to treatment (95% CI: 8.9 - 16.1). The tumor board which consisted of 4-15 pediatric oncologists from sites across the US and a pediatric oncology pharmacist was able to create individualized therapy regimens for all subjects. Correlative biology specimens were obtained and grown in culture. Mice xenograft establishment is ongoing. Cultures and xenografts are able to be used for validation studies of predicted drug sensitivity. In conclusion it is feasible to obtain real-time genomic profiling for molecularly guided therapy for use in treatment decision making.  
The study endpoint showing that 9/14 patients were feasible to enroll, biopsy, genomic analysis, tumor board review and patient treatment was met. All regimens were found to be safe by the medical monitor proving that the molecular tumor board was able to make safe decisions similar to hospital tumor boards making treatment decisions without molecular information on the patients. It was felt that the pharmacy review for evaluating drug-drug interactions and deciding on laboratory and safety testing was an important part of the tumor board (this is not usually present at hospital tumor boards) and will continue to be incorporated in this and future trials.  
All patients tolerated therapy well and were treated without any adverse events >grade 2 related to study drugs.
untitled h1
neuroblastoma gene target analysis platform

Five core needle biopsies were processed for the reproducibility study: MGT 2, 3, 4, 7, and 8. One of these, MGT 4, was subsequently found to be a neuroendocrine carcinoma. Since five such samples are required, completion of the reproducibility study requires one additional patient.  
Background and Goal The Neuroblastoma Gene Target Analysis Platform (IDE \#
G100111) includes a process that, based on core needle biopsies of a neuroblastoma solid tumor, provides genome-wide expression profiles, comparative gene expression profiles based on a reference collection of normal tissues, and a set of drug therapies, each of which has a score (Figure 1). We proposed to study reproducibility of the IDE by applying it to triplicate samples obtained by dissecting each of five solid tumors. The primary goal of the reproducibility study is to evaluate the variation among sets of drug therapies associated with the same tumor. Secondary goals are 1) to compare the variation among sets of drug therapies from the same tumor with variation between tumors from distinct patients and 2) to evaluate variation associated with earlier steps in the process, in particular, expression profiles and comparative expression profiles (Figure 1).  
Design The design employed (Figure 2) was a hierarchical component that allows us to quantify variation within biopsies and to evaluate it within the context of the variation between biopsies as well as a cross-classification by GeneChip batch that allows us to evaluate batch effects. Methods Affymetrix GeneChip oligonucleotide array technology is used to obtain gene expression profiles,
�Eg|g ∈ G�, where G is the set of probe sets on the GeneChip. The OncInsights service calculates comparative expression profiles, �Zg�, from the sample mean and sample standard deviation, Figure 1. The IDE identifies drug therapies based on a solid tumor  
  
  
biopsy. Expression profiles and comparative expression profiles are intermediate results.  
��E�g R, sg R��, of a Reference collection of normal tissue expression profiles using Zg = Eg−E�g R
sg R .  
The OncInsights service uses comparative expression profiles to obtain a set of drug therapies, Dpr, for each patient, p, and replicate biopsy, r, as well as scores, �Sprd�, for each drug, d in Dpr.  
The reproducibility of the identification of a drug, d ∈ Dpr, is defined as the proportion of other drug sets associated with the same patient that identify the drug  
$$\mathrm{F_{prd}=\frac{1}{\left|R_{p}\right|-1}\sum_{r^{\prime}\in R_{p},r^{\prime}\neq r}H\big(d\in D_{pr^{\prime}}\big)}$$ isn that equals 1 when its argument is true.
where H is an indicator function that equals 1 when its argument is true and 0 otherwise, Rp is the set of replicates, and �Rp� is the size of Rp (3, the number of replicates). Fdpr = 1 when drug d is identified on all replicate lists and Fprd = 0 when it is not identified on any other list. We consider the average reproducibility associated with each patient, F�p, that is, the average of Fprd over replicates and drugs,  
$$\mathrm{F_{p}=\sum_{n=1}^{\infty}\frac{1}{|\mathrm{D_{n}}|}\;A}$$
$\mathrm{F_{prd}}$.
�Dpr� � Fprd r d∈Dpr as well as the average reproducibility of drugs scoring above a threshold, F�(S), that is, the average of Fdpr over patients, replicate drug lists, and drugs, conditional upon the drug achieving a minimum score  
$$\Gamma\frac{1}{\left|\mathrm{d}{\colon}\mathrm{S}_{\mathrm{prd}}>\mathrm{S}\right|}_{\mathrm{d}{\colon}\mathrm{S}}$$  
$${\bar{\mathrm{F}}}(S)={\frac{1}{\mathrm{|I|}}}$$  
|P|
�� 1
�d: Sprd > S� � Fprd rp d:Sdpr>S
We note that a method that uniformly identifies every drug would not be an acceptable method yet would exhibit perfect reproducibility. We therefore evaluate the variation among drug lists associated with the same patient in the context of the variation among drug lists from different patients. Methods developed by Gower, Anderson, and their coworkers [56-58] and the Jaccard distance allow us to use the framework familiar from univariate ANOVA. More precisely, these methods allow us to test the null hypothesis that the variation among sets lists associated with different patients can be accounted for by the variation among drug sets associated with the same patient as well as to partition the variation into components so that their magnitudes can be compared.  
  
Distance-based nonparametric multivariate analysis of variance [57, 58] allowed us to reject the null hypothesis that variation between biopsies can be accounted for by the variation within biopsies (p=0.001). That the variation among expression profiles associated with the same biopsy is small compared with the variation between expression profiles associated with different biopsies is apparent from Principal Component Analysis (Figure 3).
untitled h1
sets of drug therapies

Similarly, the variation among drug sets associated with the same biopsy is small compared with the variation among drug lists associated with different biopsies (p=0.001). The reproducibility averaged over patients, replicates, and drugs is 0.68. As the threshold score increases the reproducibility increases to 1 (Figure 4).
untitled h1
conclusion

Collectively, the early preclinical and current clinical data suggests that this integrated approach is highly feasible and safe in neuroblastoma and that it is reproducible among repeat samples within patients. It is also clear that an investigation of this approach in other targeted patient populations is warranted. Brain tumors, and rare solid tumors are logical diseases in which to evaluate this approach, based upon the analogous need to develop a more rational-based approach to therapeutic selection in these difficult to treat tumors. Furthermore, the ongoing collaborative efforts between the co-investigators of this proposal in identifying novel molecular-based treatments for pediatric cancers and provides further justification to develop this analysis further to include DNA mutation analysis in tumor board decision making.
untitled h1
2 study objectives

2.1 Primary Objectives Determine feasibility of using tumor samples to assess genomic mRNA expression arrays and DNA mutation panels using predictive modeling to make real-time treatment decisions for children with relapsed/refractory cancers.  
2.2 Secondary Objectives
♦ To continue to determine the safety of allowing a molecular tumor board to determine individualized treatment plans
♦ To determine the activity of treatments chosen based on:
- Overall response rate (ORR)
- Progression free survival (PFS)
♦ To explore the relationship between tumor phenotype and response by permitting use of tumor tissue in a correlative biologic study
♦ To compare PFS interval to PFS intervals of previous chemotherapy regimens since relapse for each subject.
untitled h1
3 study design

This is an open label, multicenter prospective feasibility study in patients with refractory or recurrent childhood cancers.
untitled h1
3.1 subject enrollment and sample

Eligible subjects will be enrolled onto this study at each site following a registration process that includes receipt of a signed subject consent form and a copy of the required baseline laboratory tests. Subjects will undergo a scheduled surgical resection and/or diagnostic biopsy procedure.  
Common sample identifiers will be provided by the NMTRC Lead Study Coordinator to the participating site and will be used by all parties throughout the project to ensure sample, data and report alignment between participating organizations. At the time of tissue resection or biopsy, two fresh tumor samples will be committed for this specific research study (referred to as
"Primary Samples"), and prepared immediately per Appendix II and section 9.3. Primary Samples will be sent to Clinical Reference Laboratory (CRL) and Spectrum Health Molecular Diagnostics. Samples will be reviewed by local pathology to confirm >75% tumor and >75%
tumor cell viability which is required for microarray analysis. In the absence of an adequate tumor specimen from the enrolled subject, this would represent a non-feasible outcome for this subject on the study. Tumor samples will also be sent for research purposes (referred to as
"Secondary Samples.") A portion of the tumor tissue biopsy will be shipped overnight to the Neuroblastoma Translational Research Laboratory on dry ice.  
Upon arrival of the tumor specimens at the Clinical Reference Laboratory (CRL), Spectrum Health, the Neuroblastoma Translational Research Laboratory, and TGen, the samples will be logged and immediately processed according to details in Section 9.  
3.2 **RNA Microarray Analysis**
Cells or tumor samples will have RNA isolated and amplified for hybridization to an Affymetrix U133 Plus 2.0 GeneChip®. This step will be performed by molecular technologists in conjunction with the Clinical Reference Laboratory (CLIA certified lab) in Lenexa, KS. The RNA extraction, amplification, hybridization, and scanning procedures adhere to strict standard operating procedures to ensure that results are highly accurate and reproducible. The operation includes stringent controls over quality including measurements of RNA quality to minimize the chance that inaccurate data will be utilized in the study. By default, a failure in an individual step will result in a re-run of that specific step if suitable material remains. A second failure will result in exclusion of the subject from the study unless an additional fresh surgical specimen can be obtained. Standard metrics include the 260/280 ratio and the Agilent 2100 bioanalyzer (Pico Chips) which provides a measure RNA degradation as an RNA integrity number (RIN). To pass QC, the 260/280 ratio must exceed 1.8 and the RIN number must be greater than 6.5 which indicates substantially intact RNA. In the isolation, purification, amplification, and labeling processes, a sole individual will handle all samples to limit operator errors. Positive mutation findings from the Ion Torrent Panel will be verified using Sanger Sequencing. While all attempts will be made to have this data available to the tumor board, due to the potential length of time to process Sanger Sequencing samples, these results may not be available until after the tumor board has met and the patient has started protocol treatment. If Sanger Sequencing results differ from Ion Torrent results and the subject has already started protocol treatment, this information will be communicated to the treating site Principal Investigator who will then communicate this information to the patient and a mutual decision about continued treatment will be made.
untitled h1
3.3 dna mutation panel

Ion Torrent Deep Amplicon Sequencing of Tumor Samples: Deoxyribonucleic acid will be extracted from tumor tissues and quantitated using the Qubit2 fluorometer (Invitrogen). Ten nanograms of DNA will be used for multiplex PCR of a panel covering 739 mutations in 46 cancer-related genes (Ion AmpliSeq Cancer Panel, Life Technologies). Subsequent processing of samples will be performed according to the manufacturer's protocol. Library constructions of the amplicons and subsequent enrichment of the sequencing beads will be performed using the OneTouch system. Sequencing will be done on the 314 chip with 10 megabases capacity using the Ion Torrent Personal Genome Machine (Life Technologies) as per the manufacturer's protocol. Data analysis, including alignment to the hg19 human reference genome and base calling, is done using built-in software. De-identified Data will be sent to the NMTRC via secure e-mail for creation of the DNA Mutation Panel Drug Prediction Report.  
3.4 **Data Analysis and Predictive Methodologies**
Each subject will be assigned a unique anonymous identifier by the Data Manager at the Neuroblastoma and Medulloblastoma Translational Research Consortium (NMTRC), which will be used to track the "Subject" derived "Samples" and performed "Experiments". At each entity level, pertinent data can be associated; for example, primary and secondary clinical endpoints can ultimately be associated with the Subject and the histopathological assessment of the donated Sample, and experimental parameters with each Experiment. For the proposed work it should be adequate to exchange sample information in tables that provide for easy identification of the subject identifier. In addition, the normalized Affymetrix GeneChip data will be associated with the Experiment for further analysis as described below. Upon exchange of the normalized GeneChip data and all associated experimental parameters, an additional QC check will be performed to ensure all critical experimental steps passed the required threshold of acceptance. While quality control parameters are checked by the CLIA certified laboratory, a secondary assessment provides an additional level of quality assurance. If no failures are noted upon this review, a complete analysis of the molecular data will proceed.
untitled h1
3.4.1 intervention insights oncinsights mrna

The following describes the Intervention Insights OncInsights service which utilizes data generated from the RNA Microarray analysis (as described in section 3.2 above). A consistent step for the OncInsights methodologies outlined in this proposal is the conversion of normalized probe set intensities to a relative measure of transcript abundance. While many studies use an arbitrary fold change threshold relative to a reference sample as a means to determine significant changes in gene expression, we prefer to utilize a standardized z-score method (described below in Signal Pre-Preprocessing and Z-Score Calculation) to determine statistically significant differences in each neuroblastoma sample relative to a whole body reference. This method assigns a z-score to each probe set based upon the number of standard deviations from the normal sample population mean. This provides a good assessment of transcript "differences" relative to a whole body reference set which may not necessarily be associated with large fold changes in transcript expression. Having established a consistent data input of relative transcript abundance in each subjects tumor specimen, the following analytical methods will be applied to the standardized data to predict drug efficacy.  
Predicted Therapeutic Compounds: At this time, there are currently greater than 300 drugs within the OncInsights system each with a published and defined molecular target. For the purposes of this protocol, the tumor board will only use the drugs listed in Appendix III.
untitled h1
drug knowledgebase

The drug knowledgebase is comprised of publically available, peer-reviewed and clinically relevant pharmacological data. Numerous sources contribute to the knowledgebase including GeneGo, DailyMed, DrugBank, UpToDate, Entrez Gene, Entrez Pubmed, PharmaGKB,
MedTrust Online and MedTrack.  
Whole Body Reference Set: A whole body bank of forty-five normal tissue gene expression levels are used as the reference set for the normalization calculations. A whole body reference was chosen to provide a wider variance of tissue specific gene expression for comparison in order to best identify expression differences from tumor tissue. The reference also helps to decrease toxicity risk by not identify targets that are highly expressed in normal tissues.
untitled h1
signal pre-processing z-score calculation and

Signal Pre-Processing. Signal data from the Affymetrix U133 2.0 Plus GeneChip is normalized using Affymetrix's MAS5.0 Normalization Algorithm. Using Affymetrix Annotation version 32 (released August 2011), probesets which map to more than one gene (potential cross-hybridizing)
are removed from the analysis. Probesets marked "present" by Affymetrix P/M/A algorithm are included in the OncInsights method calculations. In addition and to include down regulated genes, probesets marked "absent" or "marginal" are included in the OncInsights method based on the following logic: probesets with a tumor signal value less than the mean reference signal value and the reference probeset is marked "present" by Affymetrix P/M/A logic 50% or more of the time in the whole body reference set. All probesets which pass the pre-processing thresholds as described above are evaluated for inclusion in the OncInsights report.  
Z-Score Calculation. The z-score is calculated for all included probesets using the whole body reference mean and whole body reference standard deviation as follows:
untitled h1
x z m s -

Where x is the signal intensity, µ is the mean probeset intensity from the reference standard and σ is the standard deviation of the mean of the reference. Interpretation of the z-score is straightforward and has no special case. Positive z-scores represent the number of standard deviations that the observation being evaluated is above the mean; conversely negative z-scores represent the number of standard deviations below the mean. Observations with high, positive zscores are said to be "up-regulated" or "over-expressed" whereas observations with low, negative z-scores are said to be "down-regulated" or "under-expressed". Finally, it is important to note that the z-score represents the individual z-score relative to the same probeset in the normal reference group and not its z-score among all other probesets within the total expression data.  
Sibling Probeset Logic. Within the Affymetric Annotation file, numerous probesets map to the same Entrez Gene, these probesets are called siblings. To merge multiple probeset measurements
(converted to Z-score), the OncInsights systems using an industry accepted logic as follows:
A probeset maps to a single Entrez Gene, the z-score value is assigned to the Entrez Gene. Two probesets map to a single Entrez Gene, the maximum observed z-score value is assigned to the Entrez Gene.  
Three or more probesets map to a single gene, the median observed z-score vaule is assigned to the Entrex Gene, Upon conclusion of these methods, the OncInsights system has a list of genes with their associated z-score value. These genes and their measured z-score values are used in all subsequent OncInsights methods except the Drug Response Signatures Algorithm which uses only probeset values as described below.
untitled h1
biomarker based rules (sensitive and

The method employs predefined and published rules maintained in the drug knowledgebase in which a drug effect has been associated with an expression of a genomic marker. This method not only predicts drugs which would be suited to treat a specific condition (sensitive), it also flags drugs which are predicted to be insensitive given a subject's tumor biomarker expression
(insensitive). Genes with z-score values within the rule range (positive or negative z-score values) trigger display of the therapeutic compound. As an example, one rule within the BioMarker Based Algorithm refers to AR, androgen receptor, and bicalutamide. When tumor AR z-score is ≥ +3.0 (three standard deviations above the reference mean) the rule is trigger and bicalutamide is included in the OncInsights report. This is an example of a therapeutic compound that is "sensitive" when "over-expressed". Other rules trigger "sensitive" when "underexpressed" as when tumor ERCC1, excision repair cross-complementing repair deficiency, z-score ≤ -3.0 (three standard deviations below the reference mean) triggers carboplatin as Page 35
Issued 12 June 2013
"sensitive". While simple and practical in nature, one concern with this methodology is that over-expression of a drug target can represent a means by which tumors develop resistance to a targeted agent [35]. In addition, BioMarker Based Rules can trigger "insensitive" when gene expression is "over-expressed" or "under-expressed". Currently there are 30 sensitive rules and 12 insensitive rules within the BioMarker Based Rules knowledgebase. Each rule is confirmed against two or more publically available sources in which the therapeutic agent (drug) has an effect on the gene target.
untitled h1
drug target expression algorithm

Genes with z-scores ≥ +3 (three standard deviations above the reference mean) are submitted to the method and therapeutic compounds which meet the rule requirement are selected and displayed in the OncInsights report. This is analogous to the current approaches of detecting Her2 and *Egfr* gene amplification or over-expression prior to treatment with the appropriate targeted inhibitors [59, 60]. For this approach, we utilize public domain knowledge of the existing pharmacopeia and their molecular mechanisms of action [38, 39]. For example, one rule within the Drug Target Expression Algorithm refers to ESR1, estrogen receptor 1, and tamoxifen. When tumor ESR1 z-score expression is ≥ +3.0 the rule is triggered and tamoxifen is included in the OncInsights report. Currently, 163 rules are contained within the Drug Target Expression Algorithm rule set. Each rule is confirmed against two or more publically available sources in which the therapeutic agent (drug) has an inhibitory effect on the gene target.
untitled h1
network target activity (systems biology

A major consideration for the future of predictive therapeutics is the identification of active (or inactive) molecular networks within a tumor to permit network-directed therapeutic strategies [28, 30, 61]. In partnership with GeneGo (www.genego.com), we have developed a systems wide biology method based upon network topology for predicting drug target status and contribution within tumor-specific networks [62]. The method looks at biological pathways by constructing a shortest path network within a list of the subject's genes which are above a predefined threshold (z-score ≥ +2). Using these genes, a global network representing known geneomic pathways is constructed using knowledge contained in GeneGo's master database of publically available, peer-reviewed literature describing protein-protein interactions. These two networks are then compared to determine which genes may represent significant pathways with respect to the subject's tumor biology. Genes are "scored" using the comparison of the two networks. The method is designed to uncover regulatory proteins (kinases, transcription factors)
which may not be affected at the differential gene expression level, but drive down stream transcription activity through transcription factors. Statistically, this method evaluates the enrichment in transcriptional targets compared to the global protein network and evaluates whether there is a higher proportion of possible transcriptional targets in the gene set of interest than would be expected in a random collection of genes. This algorithm yields a robust analysis of tertiary expression of targets and downstream effects of regulation.
untitled h1
drug response signatures algorithm (connectivity

The method uses a publically available data set called The Connectivity Map developed at the Broad Institute [43, 48] and recently commercialized by private equity. In the original description of the method, Lamb et al were able to successfully predict cell lines that were responsive to estrogen receptor inhibitors [43]. Subsequently, the general approach has been applied to several datasets to hypothesize potential drug efficacy in the background of different disease contexts [42, 49]. It was developed to elicit gene expression signatures from human cells associated with drug exposure to identify a functional (disease state) connection. The top 500 genes ranked by z-score with a z-score ≥ 1.5 and the bottom 500 genes ranked by z-score with a z-score ≤ -1.5 are submitted to the OncInsights Drug Response Signature algorithm. The connectivity score is calculated from a nonparametric, rank-based pattern-matching strategy based on the Kolmogorov-Smirnov statistic with p-values estimated by permutation testing
(n=50,000 tests) as described previously [63, 64]. Upon completion of the analysis, drugs with p-values ≥ 0.05 (negative enrichment scores) are included in the report. A negative enrichment score implies that genes that are down-regulated by the drug are up-regulated in the tumor sample and vice versa. The implication is that the drug reverses the gene expression changes associated with the disease and thus may act to reverse the disease.
untitled h1
drug sensitivity signatures algorithm (pgsea)

The Drug Sensitivity Signatures algorithm reproduces the published implementation of the Parametric Gene Set Enrichment Analysis (PGSEA) using the NCI-60 cell line sensitivity signatures [44, 45]. A PGSEA drug signature maps over-expressed genes (determined pre-drug treatment) to drug sensitivity as measured by the half maximal inhibitory concentration (IC50) of the cell line studied. The drug signature is a collection of Entrez Gene IDs that were found to be over-expressed in drug sensitive cells. Based on this evidence, if a tumor's gene signature of over-expression is similar to a sensitive cell's gene signature of over-expression the drug may also be effective for the tumor cells. To evaluate potential sensitivity to a signature, a one sample t-test is applied to evaluate if the mean of the z-score values is significantly greater than zero. The analysis only tests for a difference in the positive z-score direction. A negative z-score trend even if it is significant is discarded. This approach is consistent with well published methods for inferring drug sensitivity utilizing the NCI 60 cell line dataset [45, 51].  
OncInsights Version.  
The study will use OncInsights version 1.4.7.0
untitled h1
3.4.2 dna mutation panel analysis

The following describes a process **which utilizes data generated from the DNA Mutation** Panel analysis (as described in section 3.3 above).
untitled h1
dna mutation panel drug prediction

The data from the Spectrum Health Ion Torrent Cancer Panel will be provided to the NMTRC.. A
knowledge database created from published research literature linking mutations with targeted drugs in cancers will provide a report for the tumor board showing the mutations and predicted therapeutics from the list provided in Appendix III. An in-house software application is implemented that produces reproducible, rules-based lists linking gene mutations with potential drug therapies. An electronic rule-capture interface is used to import a spreadsheet database, that contains genes, measurement types, and values for those measurements, and any other additional columns. Rules (for each gene) are assigned to the application by the investigators, without modifying the core software code. All conditions follow the basic form "<measurement> <verb> <value>". These verbs include <, <=, >, >=, equals, is present, is not present. Every rule may have any number of modifiers that effectively serve as sub-rules that associate the rule with other potential drug treatment for the parent rule. Report: The process of report generation begins with gene specific input with accompanying aberration data. If gene matches gene in the database then aberration is checked. This will generate a rule in table-format stating a direct relationship, indicate/do not indicate drug. If an aberration is not exactly matched it will trigger an inferred relationship, indicate/do not indicate drug. The generated report includes a PubMed ID for each rule, which will be included as both supplementary data and an outbound hyperlink. The software enables only authorized investigators to access the underlying system configuration. Users of this application are able to access the data only through the GUI "front-end", thereby locking the data relationships for the duration of an investigational trial. This software takes into account the following:
1. 100% reproducible output given the same input. 2. Simple yet sophisticated rules management.  
3. Cross reference data with several open source data sets.  
4. Generation of a clinically understandable, actionable report.  
Positive mutation findings from the Ion Torrent Panel will be verified using Sanger Sequencing. While all attempts will be made to have this data available to the tumor board, due to the potential length of time to process Sanger Sequencing samples, these results may not be available until after the tumor board has met and the patient has started protocol treatment. If Sanger Sequencing results differ from Ion Torrent results and the subject has already started protocol treatment, this information will be communicated to the treating site Principal Investigator who will then communicate this information to the patient and a mutual decision about continued treatment will be made.
untitled h1
3.5 treatment protocol decision 3.5.1

Treatments protocols will be generated from the tumor board meeting which will consists of three pediatric oncologists (minimum) and one pharmacist utilizing the information contained in a report generated on the basis of genomic analysis of the gene expression profile of the subject's tumor. Specific treatment details will consist of a regimen chosen from a guided list of agents implicated in critical molecular signaling pathways and/or from signature-based predictions of drug efficacy summarized in the guided therapy reports. All agents are listed in the current pharmacopoeia for human use, but will differ amongst individual subjects.
untitled h1
3.5.1.1 decision rules for the

1. All drugs with predicted efficacy will be reported to the tumor board with an associated predicted efficacy score and rank. At this time, FDA approved drugs predicted by one of the different predictive methodologies under evaluation will be included (Drug Target Expression, Biomarker-Based Rules Algorithm, Systems Biology Methods, Connectivity Map, Gene Set Enrichment Analysis, or DNA mutation panel). For these panels the analytical methods are independently applied to the standardized and normalized (relative to whole body reference set) gene expression data derived from the profiling of each subject's tumor. Each method is associated with an objective statistical threshold that must be exceeded in order for a specific drug to be predicted and reported for consideration. Multiple lines of evidence (e.g. multiple methods predicting the same drug, multiple targets for the same drug) will be considered during the review process. Molecular-based predictions of drug efficacy will be supplemented with evidence gathered from automated searching of the literature, clinical trials and the internet.  
2. Drugs must be FDA approved with established standard and safe dosing schedules (see drug dosing below Section 3.5.1.2). Those without known pediatric dosing will be excluded.  
3. Potential drug choices will be analyzed with regards to safety, mechanism, availability and cost. Focus will be on low-toxicity, targeted therapies.  
4. Drug combinations will be allowed, up to a maximum of 4 agents. Literature searches will be conducted to assemble data on previously established and tested regimens. These regimens will be given priority.  
5. A pharmacist will analyze potential drug interactions between the guided agents and the subject's routine medications and supplements. For drug interactions and known toxicities the following databases will be used: MicroMedex LexiComp, E-facts, Natural Medicines Database.  
Subjects' history and previously received treatments will be reviewed. Drugs which a subject has failed will be given low priority and used only if there is a rationale for synergy in combination therapy.
untitled h1
3.5.1.2 recommended priorities for determining

1. For a given proposed individualized combination of drugs the first priority to establish doses will be to identify the same combination of drugs in a peer-reviewed journal article or presented as a reviewed abstract, or which is part of an ongoing peer-reviewed clinical trial registered with clinical trials.gov.  
2. When a proposed individualized combination of drugs has not previously been reported, the process to establish doses will be to then identify individual members of the proposed combination that have been used in combination with other cytotoxic agents similar to those being considered for combination therapy. The source of information will be a peer-reviewed journal article or presented as a reviewed abstract, or which is part of an ongoing peer-reviewed clinical trial registered with clinical trials.gov.  
3. When a proposed individualized combination of drugs have no available combination data, dosing guidelines will start with the MTD determined from a phase I/II pediatric study. Reference information will be based upon either a peer-reviewed journal article or presented as a reviewed abstract, or which is part of an ongoing peer-reviewed clinical trial registered with clinical trials.gov. Doses will be reduced to compensate for potential additive toxicities of combination agents (refer to protocol section 3.5.1.1 \#5 that describes pharmacy programs that are used to evaluate potential interactions and combination dosing for all medications).  
Please note that many of the drugs that will be considered for proposed individualized combination therapy in this protocol are not considered cytotoxic agents and therefore will not have previously been specifically tested as part of conventional antineoplastic protocols.  
Examples of these medications may include anti-seizure or anti-cholesterol agents. These noncytotoxic agents will be used at standard doses based on recommendations of an experienced pediatric pharmacist (refer to protocol section 3.5.1.1 \#5 that describes pharmacy programs that are used to evaluate potential interactions and combination dosing for all medications). The final treatment regimen will be subjected to a 12 hour in depth review and evaluation for safety and then be signed off by one of the protocol pharmacists. The treatment memo will then be discussed with the subject and their parents (if the subject is < 18 years of age).
untitled h1
3.6 safety evaluations

The principal investigator and the study chairs will evaluate reported serious adverse events and other toxicities in real time. Study chair and PI meetings will occur at least monthly. The principal investigator will meet with the clinical research team at frequent and regularly scheduled intervals to determine treatment modifications and treatment based toxicities. Toxicity data must be submitted to the disease specific Study Chair at the end of each cycle of therapy. Any safety concern or new information that might affect either the safety or the ethical conduct of this trial will be immediately forwarded to the study chairs in written form. The study chairs will be responsible for informing the IRB and DSMB. If trends in toxicities are noted or stopping rules are met, the PI will temporarily suspend enrollment while reviewing the episodes with the IRB and DSMB. The DSMB will convene every 6 months for evaluation of all safety data. Study will be on hold for safety monitoring by DSMB review when:  
1. Any deaths deemed related to the study drug by the treating PI while on study or occurring less than 30 days after medications ended or 2 serious adverse events possibly related to protocol within 60 days.  
2. Any other reason that the NMTRC feels it is in the best safety interest of the subjects.  
Subjects will be required to either come off study or be dose reduced per treating physician standard of care if one of the following toxicities related to treatment regimen occurs. Toxicity will be graded according to the NCI-Common Terminology Criteria for Adverse Events (CTCAE
v4.0).
untitled h1
non-hematologic toxicity

∙ ≥Grade 3 non-hematological toxicity not resolved to <Grade 2 or baseline within 2 weeks excluding nausea, vomiting, anorexia, hypertension, electrolyte abnormalities corrected with oral supplementation ∙ Any Grade 4 non-hematological toxicity.
untitled h1
hematologic toxicity

∙ Grade 4 neutropenia lasting ≥14 days;
∙ Grade 4 neutropenia with Grade 4 infection 3.7 **Efficacy Evaluations**
At the times indicated in Section 5 and the Table of Procedures and Assessments, scans will be obtained to evaluate response data for subjects enrolled in this study. Efficacy will be assessed according to criteria outlined in Section 7 (Efficacy Assessments) to evaluate the activity and potential benefit of the chosen treatments protocol in this subject population.  
3.8 **Biological Studies**
Subjects will have an opportunity to participate in correlative biological studies on a voluntary basis. This study will be used to correlate in vivo and in vitro predictive drug responses. This study may be able to contribute to our knowledge of molecular determinants of response to therapy and/or development of biomarkers to help guide future therapy.
untitled h1
4 subject selection

4.1 **Inclusion Criteria**
1. Subjects must have histologically proven neuroblastoma, brain tumor, or rare tumor and confirmation of refractory or recurrent disease with histologic confirmation at diagnosis or at the time of recurrence/progression 2. Subjects must be age >12 months at enrollment.  
3. Subjects must be age ≤ 21 years at initial diagnosis.  
4. Subjects must have measurable disease as demonstrated by residual abnormal tissue at a primary or metastatic site measuring more than 1 cm in any dimension by standardized imaging (CT or MRI); tumor must be accessible for biopsy. Patients with bone marrow only disease expected to be >75% tumor are eligible to enroll.  
5. Current disease state must be one for which there is currently no known curative therapy 6. Lansky or Karnofsky Score must be more than 50 7. Subjects without bone marrow metastases must have an ANC > 750/μl 8. Adequate liver function must be demonstrated, defined as:
a. Total bilirubin ≤ 1.5 x upper limit of normal (ULN) for age AND b. ALT (SGPT) < 10 x upper limit of normal (ULN) for age 9. A negative serum pregnancy test is required for female participants of child bearing potential
(≥13 years of age or after onset of menses)
10. Both male and female post-pubertal study subjects need to agree to use one of the more effective birth control methods during treatment and for six months after treatment is stopped. These methods include total abstinence (no sex), oral contraceptives ("the pill"), an intrauterine device (IUD), levonorgestrol implants (Norplant), or medroxyprogesterone acetate injections (Depo-provera shots). If one of these cannot be used, contraceptive foam with a condom is recommended.  
11. Informed Consent: All subjects and/or legal guardians must sign informed written consent.  
Assent, when appropriate, will be obtained according to institutional guidelines. Voluntary consent for optional biology studies will be included.  
4.2 **Exclusion Criteria**
1. Subjects who have received any cytotoxic chemotherapy within the last 7 days prior to enrollment and 14 days prior to study treatment start date.  
2. Subjects who have received any radiotherapy to the primary sample site within the last 14 days (radiation may be included in treatment decision after biopsy).  
3. Subjects receiving anti-tumor therapy for their disease or any investigational drug concurrently 4. Subjects with serious infection or a life-threatening illness (unrelated to tumor) that is >
Grade 2 (NCI CTCAE V4.0), or active, serious infections requiring parenteral antibiotic therapy.  
5. Subjects with any other medical condition, including malabsorption syndromes, mental illness or substance abuse, deemed by the Investigator to be likely to interfere with the interpretation of the results or which would interfere with a subject's ability to sign or the legal guardian's ability to sign the informed consent, and subject's ability to cooperate and participate in the study
untitled h1
5 study procedures and assessments

All subjects (or subjects' legal representatives) must provide written informed consent before any study specific assessments may be performed. The NMTRC will keep a "possible enrollment list" for all sites combined. Subjects will be allowed to enroll in the order that they are added to that list. Prior to consent of the subject, the NMTRC research coordinator will be contacted (via email) to place subject on this possible enrollment list. The NMTRC coordinator will then reply with study space availability. If a spot is not available at that time, the site will be contacted as soon as a spot does open up (based on the subjects order on the list). If a spot is available at the time, the potential subject will undergo consent and completion of all required screening procedures and certification of all inclusion and exclusion criteria by the Investigator. If the subject fits all enrollment criteria, the site will again contact the coordinator at the NMTRC for official enrollment confirmation and unique subject identifier assignment. In addition, a study enrollment form will be e-mailed or faxed to the coordinator at the NMTRC. A subject may NOT
be enrolled on trial until official approval from the NMTRC is received. When a spot becomes available, the first subject from the list will be contacted and will have 5 working days with which to enroll in the study. If this subject does not enroll by that time then the subject forfeits his/her spot and the next subject will be offered that spot.  
5.2 **Screening**
The Investigator is responsible for keeping a record of all subjects screened for entry into the study and subsequently excluded.
untitled h1
the following screening procedures must

1. Signed informed consent form. All subjects (or subjects' legal representatives) must provide written informed consent before any study specific assessments may be performed. Signed informed consent form for voluntary participation in correlative biologic analysis.  
2. Complete medical and surgical history, including documentation of the histologic evidence of malignancy and prior treatments for cancer. Include all other pertinent medical conditions and a careful history of all prior medical treatments; 3. Demographics; 4. Physical examination (including height and weight), noting all abnormalities and sites of palpable neoplastic disease; 5. Vital signs, including temperature, pulse rate, and blood pressure; 6. Karnofsky Performance status/Lansky Play status (Appendix I);
7. CBC with differential; 8. Serum electrolytes (sodium, potassium, chloride, bicarbonate), blood urea nitrogen
(BUN), creatinine, albumin, Bilirubin, LDH, ALT, and AST;
9. Serum pregnancy test for female subjects of child bearing potential (onset of menses or
≥13 years of age);
10. CT or MRI of measurable disease sites 11. Solid tumor biopsy for "Primary Sample" with remaining tumor tissue used in correlative biologic studies handled per section 9.3 and submitted per Appendix II.  
For subjects that cannot provide a solid tumor biopsy; a bone marrow core biopsy sample will replace the solid tumor sample as the "Primary Sample." Bone Marrow must have adequate tumor (defined as >75% tumor) in order to qualify as "Primary Sample."
12. Concomitant medications/therapies including documentation of steroid use and dose; 13. Confirmation of inclusion and exclusion requirements;
Following completion of all required screening procedures and certification of all inclusion and exclusion criteria by the Investigator, the coordinator at the NMTRC will be contacted (via e-mail or phone call), at which time the subject will be enrolled in the trial and a unique subject number assigned.
untitled h1
the following screening procedures must

1. Neuroblastoma subjects: MIBG scan (not required if subject's disease is previously determined to be non-avid). PET may be considered as alternative for MIBG. Other imaging appropriate for tumor type may also be considered.  
2. Any subject that is suspected to have possible bone marrow disease and for all neuroblastoma subjects: Bone marrow aspirate and biopsy. Additional bone marrow for correlative biologic studies will be handled per section 9.3 and submitted as per Appendix II.  
3. Baseline EKG (as indicated) 4. Neuroblastoma subjects: Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid
(HVA)
5. Specific Tumor Marker for defined disease (if available) 6. Completion (by study staff) of pre study case report forms 7. Any additional labs or testing required by the tumor board
untitled h1
5.3 study procedures/study interventions 5.3.1

Subjects will return to the enrolling clinic at Day 1 of cycle 1 for evaluations. The following evaluations will be conducted prior to starting study drug:
1. Physical examination (including body weight);
2. Karnofsky Performance status/Lansky Play status-Appendix I;
3. Vital signs, including weight, temperature, pulse rate, and blood pressure (sitting); 4. CBC with differential; 5. Serum electrolytes, BUN, creatinine, Bilirubin, ALT, AST, LDH;
6. Neuroblastoma subjects: Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid
(HVA) (may have been done within 3 days of treatment start/day 1)
7. Specific Tumor Marker for defined disease (if available) (may have been done within 3 days of treatment start/day 1)
8. Review and recording of concomitant medications; 9. Monitoring of AEs and review of concurrent illnesses 10. Completion (by study staff) of Case Report Forms.  
11. Any additional labs or testing required by the tumor board
untitled h1
5.3.2 study visits- cycle 1

Subjects will return to the enrolling clinic at Day 8 of cycle 1 for evaluations. The following evaluations will be conducted at those time points:
1. Physical examination; 2. CBC with differential; 3. Monitoring of AEs and review of concurrent illnesses 4. Review and recording of concomitant medications; 5. Completion (by study staff) of Case Report Forms. 6. Any additional labs or testing required by the tumor board
untitled h1
5.3.3 study visits- cycle 1

Subjects will return to the enrolling clinic at Day 15 of cycle 1 for evaluations. The following evaluations will be conducted at those time points:
1. Physical exam; 2. Karnofsky Performance status/Lansky Play status-Appendix I;
3. Vital signs, including temperature, pulse rate, and blood pressure (sitting);
4. CBC with differential; 5. Serum electrolytes, BUN, creatinine, Bilirubin, ALT, AST, LDH; 6. Review and recording of concomitant medications; 7. Monitoring of AEs and review of concurrent illnesses 8. Completion (by study staff) of Case Report Forms.  
9. Any additional labs or testing required by the tumor board
untitled h1
5.3.4 study visits- cycle 1

Subjects will return to the enrolling clinic at Day 22 of cycle 1 for evaluations. The following evaluations will be conducted at those time points:
1. Physical examination; 2. CBC with differential; 3. Monitoring of AEs and review of concurrent illnesses 4. Review and recording of concomitant medications; 5. Completion (by study staff) of Case Report Forms. 6. Any additional labs or testing required by the tumor board
untitled h1
5.3.5 completion of protocol defined

Subjects who receive one total cycle of treatment (21 or 28 days) will be considered as having completed the protocol defined therapy for feasibility. Additional treatment cycles may be delivered in a maintenance setting if there are no safety concerns, there is no disease progression, and/or there is an indication of clinical benefit. Maintenance monitoring will be conducted as described for Subsequent Cycles.
untitled h1
5.3.6 subsequent cycles (maintenance protocol

Drug administration will be according to guidelines in previous cycles with dose modifications if needed per Section 6.1.1. The following evaluations will be performed on day 1 of each cycle as indicated. Evaluations will be performed within 5 days prior to dosing unless otherwise indicated. Subsequent cycles (cycles 2 and beyond) may be given at the subject's home institution. If subjects are to receive subsequent cycles at a home institution, this will occur only at COG (Children's Oncology Group) member hospitals or those specifically approved by FDA.  
1. Physical examination; 2. Karnofsky Performance status/Lansky Play status-Appendix I; 3. Vital signs, including weight, BSA, temperature, pulse rate, and blood pressure (sitting);
(must be done on Day 1)
4. CBC with differential; 5. Serum electrolytes, BUN, creatinine, Bilirubin, ALT, AST, LDH;
6. Neuroblastoma subjects: Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid
(HVA)
7. Specific Tumor Marker for defined disease (if available)
8. Review and recording of concomitant medications; 9. Monitoring of AEs and review of concurrent illnesses (must be done on Day 1) 10. Completion (by study staff) of Case Report Forms. 11. Any additional labs or testing required by the tumor board
untitled h1
5.3.7 reevaluation during subsequent (maintenance)

Subjects will be assessed for disease at the end of cycle 2 and every other cycle or every 8 weeks, whichever occurs first, with the following (timing should be during the last week of the cycle):
1. CT or MRI of measurable disease sites (same as study done at study entry);
2. Neuroblastoma subjects only: MIBG scan or PET scan same as/if done at baseline evaluation.  
3. Neuroblastoma subjects and any other subject with suspected bone marrow disease with positive bone marrow diease at study entry: Bone marrow aspirate and biopsy; Monitoring of AEs and review of concurrent illnesses 4. Review and recording of concomitant medications; 5. Any additional labs or testing required by the tumor board 6. Additional imagining or studies may be done at any time if clinically indicated by symptoms, exam, or tumor markers.
untitled h1
5.3.8 off therapy/30 day follow-up

Subjects will be seen at either the study clinic or their home institution within 30 (+7) days after the last dose of treatment. Last dose of treatment is defined as the last day that the subject receives the combination of tumor board recommended treatments. The following evaluations will be conducted:
1. Physical examination (including body weight), focusing on an update of all previous abnormalities, any new abnormalities; 2. Vital signs, including temperature, pulse rate, blood pressure (sitting);
3. Karnofsky Performance status/Lansky Play status (Appendix I);
4. CBC with differential; 5. Serum electrolytes, BUN, creatinine, Bilirubin, LDH, ALT, AST;
6. Neuroblastoma subjects: Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid
(HVA)
7. Specific tumor Marker for defined disease (if available)
8. Review and recording of concomitant medications; 9. Monitoring of AEs and review of concurrent illnesses 10. Completion (by study staff) of Case Report Forms Any subject with a suspected study drug-related toxicity at the follow-up visit must be followed until all current drug-related adverse events have resolved to baseline or ≤ Grade 2 or stabilization of the event. This may require additional clinical assessments and laboratory tests. The follow-up results will be recorded on the appropriate page of the CRF, as well as in the subject's source documentation.  
6 **Protocol Drugs**
Specific treatment details will consist of a regimen chosen from a guided list of agents implicated in critical molecular signaling pathways and/or from signature-based predictions of drug efficacy summarized from the guided therapy report. All agents are listed in the current pharmacopoeia for human use, but will differ amongst individual subjects. The treatment regimen will consist of FDA approved drugs that have known dosing per Section 3.5.1.2. The treatment regimens will be discussed with families and will include review of known side effects per Pharmacopeia Library
(Appendix III) (information obtained from package insert and from MicroMedex LexiComp, Efacts, and Natural Medicines Database), serious adverse effects of possible new drug combinations, and any additional clinical monitoring that may be recommended by the tumor board. The family will be given the option to proceed with therapy and if the family decides to proceed with the tumor board's treatment decision they will be asked to sign a treatment specific memo.  
Drugs will be prescribed by the enrolling institution per treating hospital protocol and administered per FDA guidelines and tumor board recommendations.
untitled h1
6.1.1 treatment modifications

Dosing delays and modifications will be at the discretion of the treating Principal Investigator and their institutional pharmacist and based upon the selected therapy, subject response and practiced dosing regimens. Decisions will be made on the basis of clinical expertise of the physicians and pharmacists and with the subject's welfare being the principal concern. Informational websites such as drug bank (Micromedex, Lexicomp, E-facts, Natural Medicines Database) will be utilized to identify potential adverse events associated with individual treatments. The study pharmacist will be available to consult on decisions made with respect to changes and modification made due to potential drug related adverse events.  
6.2 **Concomitant Medications and Treatments**
All intercurrent medical conditions will be treated at the discretion of the Investigator according to acceptable community standards of medical care. All concomitant medications and treatments will be documented on the appropriate case report form.  
The following medications are not permitted during the trial:
- Any cytotoxic chemotherapy
- Any other investigational treatment
- Any other systemic anti-neoplastic therapy including, but not limited to, immunotherapy, hormonal therapy, targeted therapies, anti-angiogenic therapies, or monoclonal antibody therapy The following medications/treatments may be administered as follows:
- Any radiotherapy administered with palliative intent/pain control or recommended by tumor board as part of therapeutic regimen for best patient care.  
- Prophylactic filgrastim, pegfilgrastim or oprelvekin; these hematopoietic growth factors may be administered according to ASCO, ASH, or institutional guidelines to treat an established cytopenia
- Erythropoietin, blood products, anti-emetics, steroids, and transfusions may be administered at the discretion of the Investigator based on established criteria.
untitled h1
7 efficacy assessments

7.1 **Tumor Assessments/Scans**
Tumor assessments/imaging studies must be obtained at baseline, at the end of cycle 2 and again after every other cycle (or every 8 weeks, whichever occurs first). The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up. All radiological images must be available for source verification. Images may be submitted for extramural review for final assessment of antitumor activity. Subjects who come off study should have a final end of study disease-specific assessment done when possible.
untitled h1
7.2 scan submission

All required study scans (CT's, MRI's, MIBG's and PET's) will be de-identified and sent to the NMTRC. All study required de-identified scans will be uploaded to HIPAA compliant database
(if available) or sent on disc to:
Alyssa VanderWerff NMTRC Clinical Program Coordinator 100 Michigan Avenue NE MC 272 Grand Rapids, MI 49503 Tel: (616) 267-0327 E-Mail: Alyssa.VanderWerff@ helendevoschildrens.org 7.3 **Response Criteria**
Overall response rate (ORR) in subjects with radiologically assessable disease will be determined by CT or MRI by cross-sectional imaging, MIBG/PET scans, and/or bone marrow assessment.  
Response Assessment: Each subject will be classified according to their "best response" for the purposes of analysis of treatment effect. Best response is determined from the sequence of the objective statuses described below.  
Response Criteria for Subjects with Solid Tumors: This study will use the (RECIST)
Response Evaluation Criteria measurements in Solid Tumor from the NCI (Therasse, et al, 2000)
modified for pediatrics.  
- Measurable disease: The presence of at least one lesion that can be accurately measured in at least one dimension with the longest diameter at least 10 mm
- Serial measurements of lesions are to be done with CT or MRI, using the same method of assessment is to be used to characterize each identified and reported lesion at baseline and during follow-up.  
- Quantification of Disease Burden The sum of the longest diameter (LD) for all target lesions will be calculated and reported as the disease measurement. For this evaluation, all scans will undergo central review.
- **Complete Response (CR):** Disappearance of all target lesions.  
- **Partial Response (PR):** At least a 30% decrease in the disease measurement, taking as reference the disease measurement done to confirm measurable disease at study entry.  
- **Stable Disease (SD):** Neither sufficient decrease to qualify for PR or sufficient increase to qualify for PD from study entry.  
- **Progressive Disease (PD):** At least a 20% increase in the sum of the longest diameters of all target lesions compared to study entry, the appearance of unequivocal new lesions, or laboratory evidence of clinical progression (e.g., spread to bone marrow or increasing catecholamines).
untitled h1
response criteria for subjects with

- Those subjects with morphologic evidence of neuroblastoma by routine H and E staining
(NSE staining only is not evaluable) will be evaluable to assess bone marrow response.  
- **Complete response:** No tumor cells detectable by routine morphology on two subsequent bilateral bone marrow aspirates and biopsies done at least three weeks apart after study entry.  
- **Progressive disease**: Tumor seen on morphology on two consecutive bone marrows done at least three weeks apart in subjects who had NO tumor in bone marrow at study entry. (Note:
Subject may be declared as progressive disease in bone marrow after only one diagnostic bone marrow at the discretion of the treating physician after discussion with the study chair.)
- **Stable disease**: Persistence of an amount of tumor in the bone marrow by morphology that does not meet criteria for either complete response or progressive disease.
untitled h1
response criteria for subjects with

- Subjects who have a positive MIBG or PET scan at the start of therapy will be evaluable for MIBG or PET response. All MIBG's and PET scans will be performed at the research institution and then centrally reviewed.  
- **Complete response** = complete resolution of all positive lesions - **Partial response** = resolution of at least one positive lesion, with persistence of other MIBG
positive lesions.  
- **Stable disease** = no change in scan in number of positive lesions (includes subjects who have same number of positive lesions but decreased intensity)
- **Progressive disease** = Development of new positive lesions
untitled h1
duration of response

Duration of response is defined as the period of time from when measurement criteria are met for complete response (CR) or partial response (PR), whichever is first recorded, until the first date that recurrent or progressive disease (PD) is objectively documented (taking as reference for PD
the smallest measurements recorded since the treatment started). The assessment of response will include the initial measurable targets and will be performed after the first and second cycle, then after every other cycle (or 8 weeks, whichever occurs first). Serial results of bone marrow aspirates, biopsies and urinary catecholamines will be reviewed for responding subjects to confirm response or lack of progression.
untitled h1
clinical response

A subject will be defined as having a clinical response if they have stable disease or better and a decrease in their tumor markers by ≥ 50%. Clinical response will also be defined as a subject that has a complete clearing of a previously positive bone marrow while on study.
untitled h1
progression free survival

Time to progression, defined as the period from the first day of administration of study drug until the criteria for progression are met taking as reference the screening measurements, will be assessed.
untitled h1
8 sample size justification feasibility

Since this is primarily a feasibility trial, the definition of feasibility for this study will include:
"Enrollment onto study, RNA expression profile completed, DNA Mutation Panel completed, genomic analysis and report generation, tumor board held with treatment decision, treatment review completed and start of treatment by 21 days post biopsy/surgical resection date, and then completion of 1 cycle of therapy."
Our preliminary results for 5 subjects indicates that we have a sample collection success rate of 5/5 (100%) subjects, a genetic report profile success rate of 5/5 (100%) within the 7-10 day time window as well as a treatment agreement success rate of 5/5 (100%) within the 5 day time window. The average time (+/-SD) for completion of the genetic profile was 6.6 days (+/-1.7 days) with a median of 7 days. The average time (+/-SD) for time to Treatment was 3.2 days (+/- 0.8 days) with a median of 3 days. Data preliminary to date for each of these three feasibility markers are very consistent with our anticipated performance standards.  
Our initial feasibility trial continues to show that it is feasible and safe to use RNA expression analysis for tumor boards creating treatment decisions for patients without curative options. This study will ask the feasibility of adding a DNA mutation panel and expansion to other noncurative pediatric cancers as well.  
Assumptions: The feasibility outcome is a binary measure for each of the three clinical strata with a lower success null value of 50% which would require reconsideration of the full process and an upper success value of 75% for the alternative which would be deemed worthy of further consideration for an efficacy trial at the next phase. A binomial distribution was used for the testing process with a combination of Type I error levels (10%) and Power (75%). The basic design overall is a MiniMax approach which minimizes the overall sample size. **The MiniMax**
design with an interim look will require an overall sample size of n = 16. Each of the three clinical strata will use a two stage decision rule.
untitled h1
the two-stage stopping rule for

Stage 1: Stop and accept the null hypothesis if the observed feasibility rate is less than or equal to 5/9. Otherwise, continue to stage 2. The probability of stopping for futility is 0.746 when Ho is true and 0.166 when Ha is true.  
Stage 2: Stop and accept the null hypothesis if the observed feasibility rate is less than or equal to 10/16. Otherwise, stop and reject the null hypothesis.
untitled h1
9 laboratory evaluations 9.1 specimens

Required samples will be collected at the start of study. Recommended/Secondary
(volunteer/optional) samples will also be collected at the start of study. Additionally, optional bone marrow samples will be collected every 6-8 weeks per Study Procedure Table.
untitled h1
9.2 tumor collection and correlative

Viable, fresh tumor >0.2grams from tumor biopsy should be placed in neurobasal tissue culture media using sterile technique for, cell line and xenograft generation. Viable, fresh tumor >0.2grams will also be placed in kit for RNA for CRL laboratory and >0.2grams will be fresh frozen to be shipped to the Neuroblastoma Translational Research Laboratory for RNA
analysis and to TGen for RNA and DNA sequencing analysis along with 4-6ml blood sample in a PAXgene tube. Samples should be labeled with subjects study number. If excess tissue is available, then snap frozen tumor tissue approximately 5mm in size should be wrapped in foil, snap frozen in liquid nitrogen and stored at -80ºC. Tissue samples will be coded. Fixed tissue remaining from diagnostic evaluation may be used. Bone marrow samples will also be sent to the Neuroblastoma Translational Research Laboratory for tumor cell isolation and culture and Spectrum Health to be sorted by flow cytometry using the immunophenotyping six-color, ganglioside GD2, analysis using monoclonal antibodies to membrane antigen expression of ganglioside GD2FITC dye, CD81FITCdye, the absence of CD45PerCPdyeleukocytic antigen, and the presence of CD56APC NCAM antigen, CD9, and/or CD34. Cells or tumor samples will have RNA
isolated and run on an Affymetrix U133+ Array chip. Data will be analyzed for comparison of bone marrow tumor and solid tumor expression profiles.  
At the Neuroblastoma Translational Research Laboratory tumor cells will be grown to 70%
confluency in neurobasal media with EGF and FGF. Cell lines will be maintained in culture for biology studies. These will include determining the growth curves and responsiveness of cells derived from these tumors to a variety of agents identified on the report *in vitro* (both the chosen regimen as well as alternate options). Cells will be injected into the inguinal fat pad of NOD
SCID mice for generation of subject xenografts. These mice will be used for correlative drug testing experiments based on predictive models. Evaluation of mechanism of action of new drug combinations will be evaluated in mice models through immunohistochemical staining and western blotting of tumor preps. These models will allow us the opportunity to evaluate alternate drug combinations that might have been superior to the chosen regimen.
untitled h1
9.3 detailed procedure for sending

At screening, subjects will have biopsy samples sent. The main sample for study report will be referred to as the "Primary Sample." The "Primary Sample" will be a current solid tumor biopsy or core bone marrow biopsy (with >75% tumor).  
If a solid tumor sample cannot be obtained and the subject's bone marrow is <75% tumor then they will be ineligible for study. In addition to the "Primary Sample", if subjects have signed the additional consent for optional sample collection, they will also have these secondary samples sent as follows.
untitled h1
9.3.1 sample procurement and shipping

Label all tubes with subject's unique identifier, date/time of sample collection, and contents (i.e. solid tumor, bone marrow, blood).
untitled h1
9.3.1.1 send the following required

Required- **Solid Tumor or Bone Marrow Core Biopsy** (only if solid tumor is not available)- You will be provided a kit for tumor collection. This kit will include instructions, collection tubes containing RNA later (place tumor biopsy here within 20 minutes of harvest), and shipping instructions. Follow collection instructions (also listed in Appendix II) and ship overnight at ambient temperature to:
Clinical Reference Laboratory Attn: Molecular 11711 W. 83rd Terrace Lenexa, KS 66214 Phone: 913-492-3652
untitled h1
2. sample 2- required tumor

Add tumor or needle core biopsy to cryo-vial, snap freeze and ship on at least 1kg of dry ice to:
Spectrum Health Molecular Diagnostics 145 Michigan St Ne Ste 6201 Lemmen-Holton Cancer Pavilion Grand Rapids, Michigan 49503-2566 Ph: 616-486-6264 9.3.1.2 **Send the following** Recommended secondary tumor **samples** along with appropriate forms listed **in order of priority** (please work your way down the list of tumor samples until you either run out of available sample or have completed all samples. Once this occurs move on to Bone Marrow samples:
You will be provided a kit for tumor collection (fresh snap frozen and in culture media). This kit will include instructions, collection tools, shipping box, and shipping instructions. Follow collection instructions and ship as directed overnight.
untitled h1
3. sample 3

With sterile technique and within 20 minutes add tumor sample or needle core biopsy to T25 flask containing cell growth media (provided), seal with parafilm, and overnight at ambient temperature to:
untitled h1
ping zhao

Neuroblastoma Translational Research Laboratory Coopers Landing 1345 Monroe Ave Grand Rapids, MI 49503 Ping.zhao@helendevoschildrens.org
untitled h1
4. sample 4

Add tumor or needle core biopsy to cryo-vial, snap freeze and ship on at least 1kg of dry ice to:
Ping Zhao Neuroblastoma Translational Research Laboratory Coopers Landing 1345 Monroe Ave Grand Rapids, MI 49503 Ping.zhao@helendevoschildrens.org
untitled h1
5. sample 5

Add tumor or needle core biopsy to cryo-vial, snap freeze and ship on at least 1kg of dry ice to:
Ping Zhao Neuroblastoma Translational Research Laboratory Coopers Landing 1345 Monroe Ave Grand Rapids, MI 49503 Ping.zhao@helendevoschildrens.org
untitled h1
6. sample 6

Add tumor or needle core biopsy to RNA Later and ship overnight at ambient temperature to:
Ping Zhao Neuroblastoma Translational Research Laboratory Coopers Landing 1345 Monroe Ave Grand Rapids, MI 49503 Ping.zhao@helendevoschildrens.org
untitled h1
7. sample 7

Add tumor or needle core biopsy to cryo-vial, snap freeze and ship on at least 1kg of dry ice to:
Ping Zhao Neuroblastoma Translational Research Laboratory Coopers Landing 1345 Monroe Ave Grand Rapids, MI 49503 Ping.zhao@helendevoschildrens.org 9.3.1.3 **For all Tumor Types: Send the following** Recommended secondary blood samples
untitled h1
listed in order of priority

Blood- Send 4-6 ml of blood in tubes (tubes and instructions will be provided).
untitled h1
1. sample 8

Send blood in EDTA tube (ambient) to:
DORRANCE CLINICAL LABORATORY at TGen 445 N 5th Street, Suite 400 Phoenix, AZ 85004 Phone: 602-343-8796 Lab Fax: 602-343-8545
untitled h1
2. sample 9 send pax

Ping Zhao Neuroblastoma Translational Research Laboratory Coopers Landing 1345 Monroe Ave Grand Rapids, MI 49503 Ping.zhao@helendevoschildrens.org
untitled h1
9.3.1.4 for neuroblastoma subjects or

Only- Send the following Recommended secondary bone marrow samples listed in order of priority (along with appropriate forms):
Bone Marrow Aspirate- Send 4cc of bilateral bone marrow in green top (sodium heparin) tube(s) priority overnight at room temperature to:
untitled h1
1. sample 10

Send bone marrow aspirate ambient to:
Pam Kidd, MD Spectrum Health Flow Cytometry Lab 145 Michigan St Ne Ste 6201 Lemmen-Holton Cancer Pavilion Grand Rapids, Michigan 49503-2566 Ph: 616-486- 6270 2. **Sample 11:**
Send bone marrow aspirate ambient to:
(Please alert site 2-3 days prior to procedure. Contact site to confirm shipment as well.)
Ping Zhao Neuroblastoma Translational Research Laboratory Coopers Landing 1345 Monroe Ave Grand Rapids, MI 49503 Ping.zhao@helendevoschildrens.org Notes: If all samples are collected, you will be collecting 11 samples and mailing 6 packages. The samples above are listed in decreasing priority (1-11) * Sample 1 to CRL (ambient)
** Sample 2 to Spectrum Health Molecular Diagnostics (frozen)
***Sample 3, 6, 9, & 11 to VIA (ambient). ****Samples 4, 5 & 7 to VIA (frozen). ***** Sample 8 to TGen (ambient) ****** Sample 10 to Spectrum Health Flow (ambient) ******* Keep all collection tubes at 4 degrees until ready to use. Please contact Ping Zhao if you have any trouble with the preparation or packaging of the samples. Ping.zhao@helendevoschildrens.org
untitled h1
9.3.2 for neuroblastoma subjects or

Send the following **Recommended** samples to **Spectrum Health** (Priority if limited samples- **First):**
1. **Bone Marrow Aspirate**- Send 2cc of bilateral bone marrow in green top (sodium heparin) tube(s) priority overnight at room temperature to:
Flow Cytometry Lab of Spectrum Health Attn: Pam Kidd, MD
Lemmen-Holton Cancer Pavilion 145 Michigan Street NE Suite 6201 Grand Rapids, MI 49503 Ph: 616-486- 6270 Send the following Recommended samples (along with appropriate forms) to the Neuroblastoma Translational Research Laboratory (Priority if limited samplesSecond)
2. **Bone Marrow Aspirate-** Send 4cc of bilateral bone marrow in green top (sodium heparin) tube(s) priority overnight at room temperature to:
untitled h1
ping zhao

Neuroblastoma Translational Research Laboratory Coopers Landing 1345 Monroe Ave Grand Rapids, MI 49503 Ping.zhao@helendevoschildrens.org
untitled h1
9.4 correlative biology studies

At the Neuroblastoma Translational Research Laboratory tumor cells (from either biopsy or bone marrow samples) will be grown to 70% confluency in neurobasal media with EGF and FGF. Cell lines will be maintained in culture for these and future biology studies. These will include studies such as determining the growth curves and responsiveness of cells derived from these tumors to a variety of agents identified on the report in vitro (both the chosen regimen as well as alternate options). Cells will be injected into the inguinal fat pad of NOD SCID mice for generation of subject xenografts. These mice will be used for correlative drug testing experiments based on predictive models. Evaluation of mechanism of action of new drug combinations will be evaluated in mice models through immunohistochemical staining, western blotting, and RNA expression profiling of tumor preps. These models will allow us the opportunity validate predictive modeling in a laboratory model.  
.  
Samples will be sent to the Translational Genomics Institute (TGen) for DNA and RNA sequencing. Isolation of DNA and RNA from subject samples will be performed for use in genomic studies. Nucleic acids will be extracted from blood, bone Marrow or tumor tissues. Analysis for mutations or Single Nucleotide Variants (SNVs) in tumors, using germ line DNA as control, will be conducted using blood and fresh or frozen tumor tissue. Comparative Genomics Hybridization, whole genome sequencing of tumor and germline DNA, messenger RNA sequencing and expression analysis and epigenentic studies including whole methylome analysis using next generation sequencing may be performed.  
Bone marrow samples will also be sent to Spectrum Health to be sorted by flow cytometry using the immunophenotyping six-color, ganglioside GD2, analysis using monoclonal antibodies to membrane antigen expression of ganglioside GD2FITC dye, CD81FITCdye, the absence of CD45PerCPdyeleukocytic antigen, and the presence of CD56APC NCAM antigen, CD9, and/or CD34. Cells or tumor samples will have RNA
isolated and run on an Affymetrix U133+ Array chip. Data will be analyzed for comparison of bone marrow tumor and solid tumor expression profiles.
untitled h1
9.5 storage

Blood and tumor samples collected for any studies performed in this protocol, and any other components from the processed cells, may be stored indefinitely to research scientific questions related to cancer and/or study drugs. The subject retains the right to have the sample material destroyed at any time by contacting the principal investigator.
9.6
9.5 storage

9.7 **Plan for Communication of Collaborating Sites**
Communication between centers will be critical in this trial. Prior to consent of the subject, the NMTRC research coordinator will be contacted (via e-mail). If a spot is available at the time, the potential subject will undergo consent and completion of all required screening procedures and certification of all inclusion and exclusion criteria by the Investigator. If the subject fits all enrollment criteria, the site will again contact the coordinator at the NMTRC who will notify the Clinical Reference Laboratory, Spectrum Health, Neuroblastoma Translational Research Laboratory , TGen, and Intervention Insights in preparation for collection and processing of tissue sample. The research coordinator at NMTRC will organize the convening of members of the tumor board for discussion of the current case once the full report is available. In addition, a study enrollment form will be faxed to the coordinator at NMTRC, a unique subject identifier will be assigned and the enrollment form will be sent back to the participating site. The NMTRC will contact all sites via e-mail if the study enrollment is on hold or closed at any time.  
Subject samples will be collected at each institution and follow the flow chart above. Once the reports are generated they will be sent to NMTRC/Dr. Sholler. The Principal Investigator enrolling the patient will prepare a tumor board presentation. The presentation and reports will be sent to all tumor board members by the NMTRC coordinator. There will be a meeting of the tumor board within 4 days of receiving the report to decide on the treatment plan recommendation. The recommendation will be reviewed in depth for safety by the Pharmacist. The treatment plan and memo will be discussed with the subject and their parents and upon their signed agreement the treatment will begin within 1 week of the discussion with parents.  
Each site will also participate in a 1 hour monthly video or teleconference meeting to review study progress and subjects currently enrolled. There will also be a yearly face-face study committee meeting hosted by a member institution.
9.6
10 adverse event reporting 10.1

An *adverse event* is any untoward medical occurrence in a subject or clinical investigation subject administered a pharmaceutical product and which does not necessarily have to have a causal relationship with this treatment. An adverse event can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding, for example), symptom, or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product.  
An untoward medical event which occurs outside the period of follow-up as defined in the protocol will not be considered an adverse event unless related to study drug. Worsening of a medical condition for which the efficacy of the study drug is being evaluated will not be considered an adverse event.
9.6
10.1.2 unexpected adverse event

An *unexpected adverse event* is one for which the nature or severity of the event is not consistent with the applicable product information as outlined in package insert filed with the FDA.  
10.1.3 **Serious Adverse Event**
A *serious adverse event* is any untoward medical occurrence that:
- Results in death
- Is life-threatening (an event in which the subject was at risk of death at the time of the event; it does not refer to an event which hypothetically might have caused death if it were more severe)
- Requires in-patient hospitalization or prolongation of existing hospitalization - Results in persistent or significant disability/incapacity - Results in a congenital anomaly or birth defect - Other important medical events that may not be immediately life-threatening or result in death or hospitalization but may jeopardize the subject or may require intervention to prevent one of the other outcomes listed above. Examples of such events are intensive treatment in an emergency room for allergic bronchospasm; blood dyscrasias or convulsions that do not result in hospitalization; or development of drug dependency or drug abuse.
The term "severe" is often used to describe the intensity (severity) of an event; the event itself may be of relatively minor medical significance (such as a severe headache). This is not the same as "serious", which is based on subject/event outcome or action criteria usually associated with events that pose a threat to a subject's life or functioning.
9.6
10.1.4 documenting adverse events

The Investigator should elicit information regarding the occurrence of adverse events through open-ended questioning of the subject, physical examination and review of laboratory results. All adverse events, whether serious or not, will be described in the source documents and Grade 2 or higher (per CTCAE 4.0) adverse events that occur while on study should be captured on the adverse event case report form. All Baseline AE's will be captured on a separate baseline Adverse event case report form. All Grade 2 or higher new events, as well as those that worsen in intensity or frequency relative to baseline, which occur after administration of study drug through the period of protocol-specified follow-up, must be captured.  
Information to be reported in the description of each adverse event includes:  
- A medical diagnosis of the event (if a medical diagnosis cannot be determined, a description of each sign or symptom characterizing the event should be recorded)
- The date of onset of the event and relatedness to treatment regimen. - The date of resolution of the event and whether the event is serious or not - Action taken; drug treatment required; non-drug treatment required; hospitalization or prolongation of hospitalization required; diagnostic procedure performed; subject discontinued from the study
- Outcome: complete recovery or return to baseline; unknown/lost to follow-up; adverse event persisting; subject died (notify the NMTRC immediately)
Adverse events, regardless of suspected cause, will be collected for 30 days following the last treatment, suspected study drug-related toxicity at the 30 day follow-up visit must continue to be followed until resolution to baseline or ≤ Grade 2 or stabilization of the event.
9.6
10.1.5 expedited reporting of serious

All fatal or life–threatening adverse events must be reported to the NMTRC immediately by telephone, fax, or e-mail within 24 hours of knowledge of the event. If full information is not known, additional follow-up by the Investigator will be required. All other serious adverse events that are unexpected (not listed in the relevant appendices) which occur any time after the subject has been consented up to 30 days after the last dose of treatment, and are possibly probably, or definitely related to the research must be reported to the study chair and appropriate regulatory authorities (local IRB and FDA if required) within 7 days of notification of the event. All Grade 3 and 4 (CTCAE), events that are not in the relevant appendices and that are possibly, probably, or definitely related to the research, but are not included in the SAE category above should also be reported to the study chair and appropriate regulatory authorities within 7 days of notification of the event. The Investigator must report all serious adverse events reported to regulatory authorities in an expedited manner to the local IRB or IEC. All serious adverse events must be followed until resolution or stabilization. Unanticipated problems involving risk to subjects or others, serious adverse events related to participation in the study, and all volunteer deaths related to participation in the study should be promptly reported to the NMTRC.
9.6
10.1.6 grading and relatedness of

Each adverse event (Grade 2 or higher) will be graded for severity per the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE V 4.0), and these criteria must be used in grading the severity of adverse events. The criteria can be found at:
http://ctep.cancer.gov/reporting/ctc.html.  
Grading of Severity of an Adverse Event Not Listed in Published Criteria:
For those adverse events which are not listed as part of the NCI CTCAE V 4.0, the same grading system should be used, where:
- **Mild** corresponds to an event not resulting in disability or incapacity and which resolves without intervention
- **Moderate** corresponds to an event not resulting in disability or incapacity but which requires intervention
- **Severe** corresponds to an event resulting in temporary disability or incapacity and which requires intervention
- **Life-threatening** corresponds to an event in which the subject was at risk of death at the time of the event
- **Fatal** corresponds to an event that results in the death of the subject