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Protocol DFMO Phase I/II
NMTRC 010B
A Phase I/II Trial of DFMO in Combination with Bortezomib in Patients with Relapsed or Refractory Neuroblastoma Version 2.1 April 28, 2015
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contact information

| Study Chair:      | Kathleen Neville, MD, MS Children's Mercy Director, Experimental Therapeutics in Pediatric Oncology  Program 2401 Gillham Rd. Kansas City, MO 64108 Tel: 816-234-3059 Fax: 816-855-1958 kaneville@cmh.edu                                             |
|-------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Study Vice Chair: | Giselle Sholler, MD  NMTRC Chair Director, Haworth Innovative Therapeutics Clinic Helen DeVos Children's Hospital 100 Michigan Avenue NE MC 272  Grand Rapids, MI 49503 Tel: (616) 267-0332 Fax: 616-391-2785 giselle.sholler@helendevoschildrens.org |
| Safety Officer:   | Stephen S. Roberts, MD Assistant Attending Physician, Neuroblastoma Program Memorial Sloan-Kettering Cancer Center 1275 York Avenue New York, NY 10065 Tel: 212-639-4034  robertss@mskcc.org                                                          |
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study committee study chairs

Kathleen Neville, MD, MS Children's Mercy Director, Experimental Therapeutics in Pediatric Oncology Program 2401 Gillham Rd. Kansas City, MO 64108 Tel: 816-234-3059 Fax: 816-855-1958 kaneville@cmh.edu Giselle Sholler, MD NMTRC Chair Director, Haworth Innovative Therapeutics Clinic Helen DeVos Children's Hospital 100 Michigan Avenue NE MC 272 Grand Rapids, MI 49503 Tel: (616) 267-0332 Fax: 616-391-2785 giselle.sholler@helendevoschildrens.org
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study committee members

André S. Bachmann, PhD Professor and Associate Chair for Research Department of Pediatrics and Human Development College of Human Medicine Michigan State University Cook-DeVos Center for Health Sciences 301 Michigan Street, NE, Office 514 Grand Rapids, Michigan 49503, USA 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 Phone office: 1-616-331-5982 E-mail: andre.bachmann@hc.msu.edu Tel: (616) 267-0335 Fax: 616-391-2785 E-Mail: Genevieve.bergendahl@helendevoschildrens.org Tel: (616) 267-0327 Fax: 616-391-2785 E-Mail: Alyssa.VanderWerff@helendevoschildrens.org Max Wicha, MD University of Michigan Distinguished Professor of Oncology Director, UMCCC 6302 CC 1500 E. Medical Center Drive Ann Arbor, MI 48109 Takamaru Ashikaga, PhD Professor and Director of Medical Biostatistics University of Vermont Burlington, VT 05405 William Ferguson, MD Pediatric Hematology-Oncology Cardinal Glennon Children's Medical Center Saint Louis University School of Medicine 1465 S. Grand Blvd. Saint Louis, MO 63104 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 Arnold Palmer Hospital for Children Pediatric Hematology/Oncology 50 W. Sturtevant St. Orlando, FL 32806 Jacqueline M 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 Tel: 1-734-936-1831 E-Mail: mwicha@umich.edu (P)1-802-656-2526 (F)1-802-656-3632 Takamaru.Ashikaga@uvm.edu Tel: (314) 577-5638 Fax: (314) 268-4081 Email: fergusws@slu.edu Tel: (858) 966-5811 Fax: (858) 966-8035 Email: wroberts@chsd.org Office: 321-841-8588 E-Mail: Don.Eslin@orlandohealth.com Office: 843-792-2957 Pager: 843-792-2123 Fax: 843-792-8912 E-Mail: kravekjm@musc.edu Javier Oesterheld, MD Levine Children's Hospital 1000 Blythe Blvd Charlotte, NC 28203 Phone-704-381-9900 Fax-704-381-9901 E-Mail: Javier.oesterheld@carolinashealthcare.org Phone: 616-391-2086 FAX: 616-391-8873 E-Mail: Deanna.Mitchell@devoschildrens.org Deanna Mitchell, MD Medical Director, Coagulation Disorders Program Assistant Professor, Pediatrics Michigan State University Helen DeVos Children's Hospital 100 Michigan NE
Grand Rapids, MI 49503 Nehal S. Parikh, MD Connecticut Children's Medical Center Pediatric Hematology/Oncology 282 Washington Street, Suite 2J Hartford, CT 06106 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 Office: 860-545-9630 Fax: 860-545-9622 E-Mail: nparikh@connecticutchildrens.org Office: 808-983-6000 E-mail: randalw@hawaii.edu Devang Pastakia, MD Vanderbilt-Ingram Cancer Center 397 Preston Research Building 2220 Pierce Ave Nashville, TN 37232 Mark Fluchel, MD Primary Children's Hospital Division of Pediatric Hematology/Oncology 100 North Mario Capecchi Drive, 4th Floor Salt Lake City, UT 84113 Phone: 615-875-6129 Email: devang.pastakia@Vanderbilt.Edu Phone: 801-662-4700 email: mark.fluchel@imail.org Valerie I. Brown, MD, PhD Associate Professor of Pediatrics Division of Pediatric Oncology/Hematology Clinical Director, Experimental Therapeutics Attending Physician, Hematopoietic Stem Cell Transplant Program Penn State Milton S. Hershey Medical Center and Children's Hospital 500 University Dr. P.O. Box 850, MC H085 Hershey, PA 17033-0850 Phone: 717-531-1042 Email: vbrown1@hmc.psu.edu Francis Eshun, MD Center for Cancer and Blood Disorders Phoenix Children's Hospital 1919 Thomas Road Phoenix Az 85016 feshun@phoenixchildrens.com
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research laboratories

Ping Zhao Pediatric Oncology Translational Research Laboratory (POTRL) Helen DeVos Children's Hospital Coopers Landing 1345 Monroe Ave, Suite 121 Grand Rapids, MI 49505 Ph: 616-486-8645 Ping.zhao@helendevoschildrens.org
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57binvestigator signature sheet

I have read the attached protocol and agree that it contains all the necessary details for performing the study. I will review copies of the protocol and of the preclinical and clinical information (Investigator's Brochure) on the test article, which was furnished to me by the Neuroblastoma and Medulloblastoma Translational Research Consortium (NMTRC), 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 NMTRC and of the IRB/IEC. I will submit the protocol modifications and/or any informed consent form (ICF) modifications to the NMTRC 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
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table of contents

| C CONTACT I NFORMATION .                                                                                                                                                  |                                                                                                                                                      |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------|
| STUDY COMMITTEE                                                                                                                                                           |                                                                                                                                                      |
| INVESTIGATOR SIGNATURE SHEET .                                                                                                                                            |                                                                                                                                                      |
| TABLE OF CONTENTS                                                                                                                                                         |                                                                                                                                                      |
| PROCEDURES AND ASSESSMENTS                                                                                                                                                |                                                                                                                                                      |
| 1.0                                                                                                                                                                       | P P ROTOCOL C ONCEPT .                                                                                                                               |
| 2.0                                                                                                                                                                       | S S TUDY O BJECTIVES .                                                                                                                               |
| 3.0                                                                                                                                                                       | S S TUDY D ESIGN .                                                                                                                                   |
| SUBJECT S ELECTION .                                                                                                                                                      |                                                                                                                                                      |
| 4.0                                                                                                                                                                       |                                                                                                                                                      |
| 5.0                                                                                                                                                                       | STUDY DRUG ADMINISTRATION ........................................................................................................................32 |
| 5.1 DFMO .                                                                                                                                                                |                                                                                                                                                      |
| 5.1.1 D ESCRIPTION , F ORMULATION , AND S TORAGE OF DFMO .............................................................................32                                  |                                                                                                                                                      |
| 5.1.2 DFMO D OSING : P HASE I                                                                                                                                             |                                                                                                                                                      |
| 5.1.3 DFMO DOSING: PHASE II                                                                                                                                               |                                                                                                                                                      |
| 5.1.4 A DMINISTRATION OF DFMO                                                                                                                                             |                                                                                                                                                      |
| 5.1.5 S UBJECTS U NABLE TO S WALLOW T ABLETS .........................................................................................36                                  |                                                                                                                                                      |
| 5.1.6 M ISSED D OSES                                                                                                                                                      |                                                                                                                                                      |
| 5.1.7 DFMO C LINICAL P HARMACOLOGY .............................................................................................................37                        |                                                                                                                                                      |
| 5.2 B OR TEZOMIB                                                                                                                                                          |                                                                                                                                                      |
| 5.2.1 DOSE CALCULATION AND ADMINISTRATION OF BORTEZOMIB- ALL PHASES ...................................39                                                                 |                                                                                                                                                      |
| 5.2.2 B ORTEZOMIB (V ELCADE ) ......................................................................................................................40                    |                                                                                                                                                      |
| 5.3 G UIDELINES FOR D OSE M ODIFICATIONS ...................................................................................................42                            |                                                                                                                                                      |
| 5.4 D OSE D EL A Y ....................................................................................................................................................42 |                                                                                                                                                      |
| 5.5 D EFINITION OF D OSE -L IMITING T OXICITY (DLT) .....................................................................................42                               |                                                                                                                                                      |
| 5.6 DFMO DOSE MODIFICATION ...........................................................................................................................43                  |                                                                                                                                                      |
| 5.7 B ORTEZOMIB D OSE M ODIFICATION ..................................................................................................................43                  |                                                                                                                                                      |
| 5.8 S TUDY D R U G A C C O U N T A B I L I T Y .                                                                                                                          |                                                                                                                                                      |
| 5.9 C ONCOMITANT M EDICATIONS AND T REATMENTS .......................................................................................46                                   |                                                                                                                                                      |
| 6.0                                                                                                                                                                       | STUDY PROCEDURES AND ASSESSMENTS ............................................................................................46                      |
| 6.1 E NROLLMENT OF S UBJECTS .................................................................................................................................46          |                                                                                                                                                      |
| 6.2 S CREENING                                                                                                                                                            |                                                                                                                                                      |
| 6.3 T REATMENT P HASE - C YCLE 1 .....................................................................................................................48                  |                                                                                                                                                      |
| 6.4 T REATMENT P HASE - C YCLES 2-6 ......................................................................................................................49              |                                                                                                                                                      |
| 6.5 O THER S CHEDULES :  .                                                                                                                                                |                                                                                                                                                      |
| 6.6 P ROTOCOL T REATMENT C OMPLETION                                                                                                                                      |                                                                                                                                                      |
| 6.7 S UBSEQUENT (M AINTENANCE ) C YCLES                                                                                                                                   |                                                                                                                                                      |
| 6.8 O FF -T HERAPY / 30 D AY F OLLOW -UP V ISIT                                                                                                                           |                                                                                                                                                      |
| 6.9 S URVIVAL F OLLOW - UP                                                                                                                                                |                                                                                                                                                      |  
| 7.0                                                                                                                                                                           | E FFICACY A SSESSMENTS                                                                                                                                          |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 7.1 T UMOR A SSESSMENTS /S CANS                                                                                                                                               |                                                                                                                                                                 |
| 7.2 S CAN S UBMISSION :  .                                                                                                                                                    |                                                                                                                                                                 |
| 7.3 R ESPONSE C RITERIA                                                                                                                                                       |                                                                                                                                                                 |
| A A DVERSE E VENT R EPORTING .                                                                                                                                                |                                                                                                                                                                 |
| 8.0                                                                                                                                                                           | 8.1 D EFINITIONS .                                                                                                                                              |
| 8.2 D OCUMENTING A DVERSE E VENTS                                                                                                                                             |                                                                                                                                                                 |
| 8.3 E XPEDITED R EPORTING OF A DVERSE E VENTS                                                                                                                                 |                                                                                                                                                                 |
| 8.4 G RADING AND R ELATEDNESS OF A DVERSE E VENTS                                                                                                                             |                                                                                                                                                                 |
| 8.4.1 G RADING OF S EVERITY OF AN A DVERSE E VENT                                                                                                                             |                                                                                                                                                                 |
| 8.5 G RADING OF H EMATOLOGIC A DVERSE E VENTS IN SPECIAL POPULATIONS                                                                                                          |                                                                                                                                                                 |
| 8.6 R ELATEDNESS TO S TUDY D R U G .                                                                                                                                          |                                                                                                                                                                 |
| 9.0                                                                                                                                                                           | SUBJECT W ITHDRA WAL AND T RIAL D ISCONTINUATION ............................................................................................61                 |
| 9.1 Criteria for Subject Off-Therapy ....................................................................................................................61                   |                                                                                                                                                                 |
| 9.2 Criteria for Subject Off-Study ...............................................................................................................................61          |                                                                                                                                                                 |
| 10.0                                                                                                                                                                          | DATA ANALYSIS                                                                                                                                                   |
| 10.1 DATA QUALITY ASSURANCE ..................................................................................................................................62              |                                                                                                                                                                 |
| 10.2 S TATISTICAL A NALYSIS ...............................................................................................................................................62 |                                                                                                                                                                 |
| ADMINISTRATIVE PROCEDURES ............................................................................................................................64                      |                                                                                                                                                                 |
| 11.0                                                                                                                                                                          | 1.1 P ATIENT I NFORMED C ONSENT .............................................................................................................................64 |
| 1.2 E THICAL C ONDUCT OF THE S TUDY AND IRB/IEC A PPROVAL ...............................................................................65                                   |                                                                                                                                                                 |
| 1.3 D ATA S AFETY M ONITORING B OARD (DSMB) ............................................................................................................65                    |                                                                                                                                                                 |
| 1.4 M ONITORING AND A UDITING ............................................................................................................................................65  |                                                                                                                                                                 |
| 1.5 P RE -S TUDY D OCUMENTATION                                                                                                                                               |                                                                                                                                                                 |
| 1.6 C ONFIDENTIALITY .                                                                                                                                                        |                                                                                                                                                                 |
| 1.7 S OURCE D OCUMENTS                                                                                                                                                        |                                                                                                                                                                 |
| 1.8 R ECORD R ETENTION .                                                                                                                                                      |                                                                                                                                                                 |
| 12.0                                                                                                                                                                          | BIOLOGICAL EVALUATION                                                                                                                                           |
| 12.1 B IOLOGICAL C ORRELATIVE S TUDIES S AMPLE I NSTRUCTIONS ............................................................................69                                   |                                                                                                                                                                 |
| 12.1.1 OPTIONAL URINE POLYAMINES COLLECTION:                                                                                                                                  |                                                                                                                                                                 |
| 12.1.2 O PTIONAL B LOOD C OLLECTION .........................................................................................................................70               |                                                                                                                                                                 |
| 12.1.3 O PTIONAL B ONE M ARROW C OLLECTION                                                                                                                                    |                                                                                                                                                                 |
| 12.1.4 O PTIONAL T UMOR S AMPLE C OLLECTION .........................................................................................................72                       |                                                                                                                                                                 |
| 12.2 S AMPLE S TORAGE AND D ESTRUCTION .                                                                                                                                      |                                                                                                                                                                 |
| A PPENDIX I : P ERFORMANCE S TATUS /S CORES                                                                                                                                   |                                                                                                                                                                 |
| APPENDIX II: L IST O F S TRONG A ND C LINICALLY R ELEVANT M ODERATE C YP3A4                                                                                                   |                                                                                                                                                                 |
| I NDUCERS A ND I NHIBITORS                                                                                                                                                    |                                                                                                                                                                 |  
| PROTOCOL SYNOPSIS PROTOCOL TITLE A Phase I/II Trial of DFMO in Combination with Bortezomib in  Patients with Relapsed or Refractory Neuroblastoma  PROTOCOL NUMBER NMTRC010B PHASE OF DEVELOPMENT Phase I/II OBJECTIVES Primary- Phase I  - To determine the safety and tolerability of DFMO in combination  with bortezomib at 3 dose levels of DFMO: 1500mg/m2 twice  daily, 2000mg/m2 twice daily, and 2500mg/m2 twice daily in subjects with relapsed or refractory neuroblastoma who receive  one full cycle of this dose. Primary- Phase I and II To evaluate the activity of DFMO in combination with bortezomib  in relapsed or refractory neuroblastoma based on:  Progression free survival (PFS)   Overall response rate (ORR) Secondary- Phase I and II - To determine the safety and tolerability of DFMO in combination  with bortezomib in pediatric subjects with refractory or recurrent  neuroblastoma. - For subjects followed with PET scan: comparison of changes in  PET activity and correlation with PFS  - Correlation of urinary polyamine levels with response and  progression of disease in neuroblastoma. - Biological Correlates to evaluate or explore minimally include: 1)  Urine: polyamine levels 2) Blood: Inflammatory markers (ESR,  CRP, IL-6), Biomarkers for Let 7 and SNPS, and explorative  biomarker analysis, 3) Bone Marrow/Tumor biopsies (if biopsied  per standard of care): research studies including Immunohistochemistry/Western Blot/qRT-PCR of bone marrow tumor cells or tumor to study Let-7, ODC, Lin28, MYCN, OKT4, BMI1, HMGA2, GLUT-4, p-AKT, NF-kB, SOCS3 will be  performed in an explorative biomarker analysis. Studies will  measure thymidine and polyamine levels. 4) In vitro research  studies of tumor cells collected above will include: Genomic  analysis of cells pre- and post- treatment (when applicable), cell  viability assays to determine subject IC50 and correlation of in  vitro response to in vivo response.  - To determine the dose effect of DFMO on biological correlates   |
|---|
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phase 1

| STUDY DESIGN   | NMTRC010B is an open label, multicenter, study to evaluate the  efficacy of DFMO in combination with Bortezomib in patients with  relapsed or refractory neuroblastoma. Phase 1   |
|----------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
This portion of the study uses a standard 3+3 design in which cohorts of 3 patients will receive oral DFMO at a starting dose of 1500 mg/m2 BID of a 21-day cycle. Subjects that complete one cycle will be considered as having completed Phase I. The dose escalation scheme for subsequent cohorts and modifications for dose limiting toxicities (DLT) are detailed in the protocol. The MTD of single agent therapy will be defined as the dose level below which DLTs are seen in ≥ two of six subjects dosed.
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cycle 1 through 6 dfmo

Phase I: Subjects will receive oral DFMO twice daily at their assigned dose level on each day of this 21-day cycle.  
| DFMO Dose   | Dose           |
|-------------|----------------|
| Level 1     | 1500 mg/m2 BID |
| 2           | 2000 mg/m2 BID |
| 3           | 2500 mg/m2 BID |  
Phase II: The highest tolerated DFMO dose from the Phase I dose escalation will be used for the Phase II portion of the study. Subjects will receive oral DFMO twice daily on each day of this 21-day cycle. All Phases: Bortezomib will be given at 1.3mg/m2/dose IV Push on Days 1, 4, and 8 of each 21-day cycle
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phase i subjects

The Phase I subjects on this study will be the first pediatric subjects to receive DFMO in combination with Bortezomib. If a Dose Limiting Toxicity (DLT) is seen in 2/6 subjects, this portion of the study will allow for a -1 dose level reduction (1000mg/m2/dose). If the DLT is seen in 2/6 subjects at the -1 dose level, a further reduction to dose level -2 (500mg/m2/dose) will be allowed. Enrollment will be held at the end of this phase of the study until all subjects within the cohort have completed one cycle and a safety analysis has been completed by the study committee and Data Safety and Monitoring Board (DSMB). The study committee will then decide whether the study can continue to the next cohort and subsequently to the Phase II portion of the trial.  
| Phase I: At least three and up to six subjects per cohort, for an  estimated three cohorts (based on safety) may be enrolled for a  projected total of 12 to 24 subjects.  Phase II: A total of 32-38 relapsed/refractory neuroblastoma subjects  will be enrolled to ensure that there will be 32 subjects evaluable for  efficacy. Phase 1 subjects assigned to the final dosing level that complete at least 2 cycles and re-evaluation will be eligible to  continue onto the Phase II portion of the study and will be included  in the total enrollment numbers. Response Response will be evaluated at the end of cycles 2, 4, and 6 with: - CT and/or MRI- same as baseline - MIBG or PET scan- same as baseline - Bone Marrow- if positive at study entry Maintenance Cycles Subjects that complete 6 cycles will be considered as having  completed the protocol defined therapy, however they may continue  on cycles of DFMO (at their current dose level) and bortezomib for  as long as they are tolerating it well and not having progression.  Subjects in maintenance cycles will have response evaluations done  as per standard of care.   |    |
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| ELIGIBILITY                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   | Inclusion Criteria 1. Age: ≤ 21 years at the time of diagnosis. 2. Diagnosis: Histologic verification at either the time of original  diagnosis or relapse of neuroblastoma. 3. Disease Status: For the purposes of this study, aggressive multidrug chemotherapy is defined as chemotherapy including 2 or more agents that must include an alkylating agent and a platinumcontaining compound. Patients must have ONE of the following: 1) Any episode of recurrent disease following completion of aggressive multi-drug frontline therapy. 2) Any episode of progressive disease during aggressive multi-drug frontline therapy. 3) Primary resistant/refractory disease detected at the  conclusion of at least 4 cycles of aggressive multidrug induction chemotherapy on or according to a high-risk neuroblastoma protocol (examples include Children's  Oncology Group trials: A3973, ANBL0532, ANBL09P1,  etc.).    |  
4. Measurable or evaluable disease, including at least one of the following: Measureable tumor by CT or MRI; or A positive MIBG or PET; or Positive bone marrow biopsy/aspirate.  
5. Current disease state must be one for which there is currently no known curative therapy or no additional therapies proven to prolong survival with an acceptable quality of life.  
6. A negative serum or urine pregnancy test is required for female subjects of child bearing potential (onset of menses or ≥13 years of age).  
7. Organ Function Requirements a. Subjects must have adequate liver function as defined by:
- AST and ALT <5x upper limit of normal
- Serum bilirubin must be ≤ 2.0 mg/dl b. Subjects must have adequate Bone Marrow function defined as:
For patients without bone marrow involvement:
- Peripheral absolute neutrophil count (ANC) ≥
750/µL - Platelet count ≥ 50,000/µL (transfusion independent, defined as not receiving platelet transfusions within a 7 day period prior to enrollment. Exception: Patients that are platelet dependent due to previous extensive treatment- e.g. - MIBG therapy).  
- Hemoglobin ≥ 8.0 g/dL (may receive RBC
transfusions)
Patients known to have bone marrow involvement with neuroblastoma are eligible provided that minimum ANC and platelet count criteria are met but are not evaluable for hematological toxicity. Patients that do not have bone marrow involvement but that are transfusion dependent at study entry are eligible provided that minimum ANC and platelet count criteria are met but are not evaluable for platelet toxicity.  
| -                             | Serum creatinine based on age/gender as follows: Maximum Serum Creatinine (mg/dL) Male Female   |     |
|-------------------------------|-------------------------------------------------------------------------------------------------|-----|
| 1 month to < 6 months         | 0.4                                                                                             | 0.4 |
| 6 months to < 1 year          | 0.5                                                                                             | 05  |
| 1 to < 2 years                | 0.6                                                                                             | 0.6 |
| Age                           | 0.8                                                                                             | 0.8 |
| 2 to < 6 year 6 to < 10 years | 1                                                                                               | 1   |
| 10 to < 13 years              | 1.2                                                                                             | 1.2 |
| 13 to < 16 years              | 1.5                                                                                             | 1.4 |
| ≥ 16 years                    | 1.7                                                                                             | 1.4 |
| 8.                            | Informed Consent: All subjects and/or legal guardians must sign                                 |     |  
6 to < 10 years 1 1 10 to < 13 years 1.2 1.2 13 to < 16 years 1.5 1.4 ≥ 16 years 1.7 1.4 8. Informed Consent: All subjects and/or legal guardians must sign informed written consent. Assent, when appropriate, will be obtained according to institutional guidelines
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exclusion criteria 9. lansky score

10. BSA (m2) of <0.25 11. Prior Therapy- Patients must have fully recovered from the acute toxic effects of all prior anti- cancer chemotherapy and be within the following timelines:
a. Myelosuppressive chemotherapy: Must not have received within 2 weeks of enrollment onto this study (6 weeks if prior nitrosourea). b. Hematopoietic growth factors: At least 5 days since the completion of therapy with a growth factor. c. Biologic (anti-neoplastic agent): At least 7 days since the completion of therapy with a biologic agent. For agents that have known adverse events occurring beyond 7 days after administration, this period must be extended beyond the time during which adverse events are known to occur. The duration of this interval must be discussed with the Study Chair.  
d. Immunotherapy: At least 6 weeks since the completion of any type of immunotherapy, e.g. tumor vaccines. e. Monoclonal antibodies: At least 7 days or 3 half-lives, whichever is longer, must have elapsed since prior treatment with a monoclonal antibody. f. XRT: At least 14 days since the last treatment except for radiation delivered with palliative intent to a non-target site. g. Stem Cell Transplant or Rescue: No evidence of active graft vs. host disease and ≥ 2 months must have elapsed since transplant.  
12. Investigational Drugs: Subjects who have received another investigational drug within the last 14 days are excluded from participation.  
13. Infection: Subjects who have an uncontrolled infection are not eligible until the infection is judged to be well controlled in the  
| opinion of the investigator.                                                                                                                                                                                                                                                                                                                                                                                                 |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 14. Subjects who, in the opinion of the investigator, may not be able  to comply with the safety monitoring requirements of the study,  or in whom compliance is likely to be suboptimal, should be  excluded.  Additional criteria: Subjects willing to participate in the correlative biologic studies will  sign an additional consent form to provide bone marrow, blood,  urine, and tumor (if available) for analysis. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |
| ESTIMATED NUMBER OF  SUBJECTS/GEOGRAPHIC  REGIONS                                                                                                                                                                                                                                                                                                                                                                            | A projected total of 12 to 24 subjects for the Phase I and 32- 38  subjects for the Phase II will be enrolled.  A total of 38-62 subjects will be enrolled to complete the dose  escalation and achieve 32 evaluable subjects for the Phase II. Up to 16 sites in the United States will participate.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |
| LENGTH OF STUDY                                                                                                                                                                                                                                                                                                                                                                                                              | Accrual to this study is estimated to be 2 years, with follow up for  five years after last subject completion.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |
| INVESTIGATIONAL PRODUCT DOSE/ROUTE/REGIMEN                                                                                                                                                                                                                                                                                                                                                                                   | DFMO is an oral agent that will be administered twice daily on every day of each 21-day cycle in combination with bortezomib given IV on Days 1, 4, and 8 of each 21-day cycle.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |
| STUDY ASSESSMENTS                                                                                                                                                                                                                                                                                                                                                                                                            | Refer to Table of Assessments for timing of study procedures.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              |
| CRITERIA FOR EVALUATION                                                                                                                                                                                                                                                                                                                                                                                                      | Safety Measures:  - Safety analysis will be conducted on all subjects who have  received at least one dose of study drug, and will include the  frequency and grade of all adverse events. Response Measures:  - Overall Response Rate (ORR)  To determine the overall response rate (ORR) by the presence  of radiologically assessable disease by cross-sectional imaging  and in MIBG or PET scans. Response will be defined by  criteria in section 9.0; or disappearance of abnormal MIBG or  PET foci.  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 the second cycle, then after  every other cycle. Serial results of bone marrow aspirates, and |  
| urinary catecholamines will be followed to confirm clinical  response in subjects with stable disease.   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        |
|----------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| -                                                                                                        | A subject will be defined as a responder if CR/PR is observed at  any time during the treatment.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
| -                                                                                                        | Time to progression, defined as the period from the start of the  treatment until the criteria for progression are met.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                |
| -                                                                                                        | Survival will be recorded at all times during the study, and  follow-up will be every three months for one year and then  annually for five years.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     |
| STATISTICS/SAMPLE SIZE                                                                                   | Statistics:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            |
| ESTIMATE                                                                                                 | All baseline subject characteristics will be summarized in a tabular  format. Safety data will be described for all subjects receiving at  least one dose of DFMO. Safety data will include all grade 2 or  greater adverse events and reporting of DLT's and MTD. The  proportion of subjects experiencing adverse events, serious adverse  events, and treatment delays will be summarized for each dosing  cohort. Response data will be summarized as the proportion of subjects  experiencing progressive disease, stable disease, partial responses or  complete responses summarized in tabular format and supplemented  with exact confidence intervals. Progression free survival and  duration of any responses will also be summarized based on the  Kaplan-Meier approach.  Sample Size: Phase I: A 3+3 design with 3 dose levels will be used. At least  three and up to six subjects per cohort, for an estimated three cohorts  (based on safety) may be enrolled for a projected total of 12 to 24  subjects.  Phase II: Primary endpoint of ORR, a sample size of n=32 (6 from  Phase I MTD) will be required to test whether treatment with  DFMO can result in an ORR of 10% (Reference Value set at ORR  <= 1%) The sample size to detect this difference uses a two-stage  test with 5% Type I error level and 80% power. Examination of the secondary endpoint of PFS will require a sample  size of n = 23 subjects to test whether treatment with DFMO can  prolong PFS by 48 days (from 42 days based on historical data in  this subject population to 90 days). The sample size to detect this  difference uses a two-stage test with 5% Type I error level and 80%  power. |  
| Procedures and Assessments                                                  |         |                                                                                                                                                                   |                               |                                                     |       |       |       |     |     |    |    |
|-----------------------------------------------------------------------------|---------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------|-----------------------------------------------------|-------|-------|-------|-----|-----|----|----|
| Screen                                                                      | Cycle 1 | Cycles 2 through 6                                                                                                                                                | Subsequent  Cycles Follow  Up | a Optional  additional  informed  consent  required |       |       |       |     |     |    |    |
| Day 1                                                                       | Day     | Day                                                                                                                                                               | Day                           | Day 16- 21                                          | Day 1 | Day 4 | Day 8 | Day | Day |    |    |
| 4                                                                           | 8       | 15                                                                                                                                                                | 15                            | 16-21                                               |       |       |       |     |     |    |    |
| Informed consent                                                            | X       |                                                                                                                                                                   |                               |                                                     |       |       |       |     |     |    |    |
| Medical & surgical history, demographics  histologic evidence of malignancy | X       | X                                                                                                                                                                 |                               |                                                     |       |       |       |     |     |    |    |
| Prior therapy for malignancy                                                | X       | b At the end  of cycle 2, 4,  and 6, then  every other  cycle                                                                                                     |                               |                                                     |       |       |       |     |     |    |    |
| Tumor Tissue (Pharmacogenomics)a                                            | X       |                                                                                                                                                                   |                               |                                                     |       |       |       |     |     |    |    |
| Interval Medical History & Physical Exam                                    | X       | X                                                                                                                                                                 | X                             | X                                                   | X     | X     | X     | X   | X   |    |    |
| Vital signs including O2 saturation                                         | X       | X                                                                                                                                                                 | X                             | X                                                   | X     | X     | X     | X   | X   | X  | X  |
| ECOG or Lansky play status                                                  | X       | X                                                                                                                                                                 | X                             | X                                                   |       |       |       |     |     |    |    |
| CBC and differential                                                        | X       | X                                                                                                                                                                 | X                             | X                                                   | X     | X     | X     | X   |     |    |    |
| Serum chemistries                                                           | X       | X                                                                                                                                                                 | X                             | X                                                   | X     | X     | X     |     |     |    |    |
| CRP, ESR, IL-6 (IL-6 is recommended)                                        | X       | X                                                                                                                                                                 |                               |                                                     |       |       |       |     |     |    |    |
| Pregnancy test (urine)                                                      | X       | X                                                                                                                                                                 | X                             |                                                     |       |       |       |     |     |    |    |
| Urinary VMA/HVA                                                             | X       | X                                                                                                                                                                 | X                             | X                                                   |       |       |       |     |     |    |    |
| BSA calculation                                                             | X       | X                                                                                                                                                                 | X                             |                                                     |       |       |       |     |     |    |    |
| Administration of Bortezomib                                                | X       | X                                                                                                                                                                 | X                             | X                                                   | X     | X     | X     |     |     |    |    |
| Administration of DFMO                                                      | X       |                                                                                                                                                                  |                              |                                                    |      |      |      |    |    |   |   |
| Dispense (and collect) drug dosing diary                                    | X       | X                                                                                                                                                                 |                               |                                                     |       |       |       |     |     |    |    |
| AE monitoring                                                               | X       | X                                                                                                                                                                 | X                             | X                                                   | X     | X     | X     | X   | X   | X  |    |
| Concomitant medications                                                     | X       | X                                                                                                                                                                 | X                             | X                                                   | X     | X     | X     | X   | X   | X  | X  |
| CT or MRI                                                                   | X       | Xb                                                                                                                                                                | Xb                            | Xb                                                  |       |       |       |     |     |    |    |
| MIBG or PET scan                                                            | X       | Xb                                                                                                                                                                | Xb                            | Xb                                                  |       |       |       |     |     |    |    |
| Bone Marrow aspirate and biopsy ^ (if + at enrollment)                      | X       | Xb                                                                                                                                                                | Xb                            | Xb                                                  |       |       |       |     |     |    |    |
| Audiogram                                                                   | X       | Xc                                                                                                                                                                | Xc                            | Xc                                                  |       |       |       |     |     |    |    |
| Urine for biological correlatesa                                            | X       | X                                                                                                                                                                 | X                             |                                                     |       |       |       |     |     |    |    |
| Blood for biological correlatesa                                            | X       | X                                                                                                                                                                 | X                             |                                                     |       |       |       |     |     |    |    |
| Survival                                                                    | X       | c At the end  of cycle 2 and  6, then yearly ^Standard of  Care Bone  Marrow.  (Separate  consent  required for  additional  research  samples per  section 12.0) |                               |                                                     |       |       |       |     |     |    |    |
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1.0 protocol concept

High risk Neuroblastoma (NB) remains a challenge in pediatric oncology, accounting for 15% of all pediatric cancer deaths. While most patients are able to attain remission, approximately 50% will relapse. Once relapsed, there is currently no curative treatment for these children, and for these children the 5-year survival rate is <10%. As such, new therapeutic approaches are needed to treat these children. These more aggressive forms of NB respond poorly to hormonal and chemotherapeutic approaches, and therefore, there is a great need for antineoplastic agents with novel mechanisms of action. The MYCN protein up-regulates ornithine decarboxylase *(ODC*), a gene encoding for the ODC enzyme that is pivotal in polyamine biosynthesis. High polyamine content and elevated ODC activities are commonly found in NB as well as many other tumors, and therefore, suppression of polyamines in cancer cells is an effective means to reduce tumor cell proliferation. We have shown ODC inhibition reverses the LIN28/Let7 pathway, an important pathway in cell differentiation and regulation of glycolytic metabolism. In studying this pathway, it was found to be regulated as well by the NFkB pathway. We have therefore studied the combination of DFMO with Bortezomib and showing synergy of these medications in neuroblastoma in vitro and in vivo. This study will address this concept in children with relapsed or refractory neuroblastoma.
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background and preliminary data 1.1

Neuroblastoma (NB) is a tumor of the autonomous nervous system originating from the adrenal medulla and autonomous ganglia in the chest and abdomen. After leukemia and brain tumors, NB is the third most frequent malignant tumor of childhood. The incidence in the United States is approximately one in 7,000 children (Ater, Gardner et al. 1998). Therapy for NB is very intense, especially in advanced stages of the disease with widespread metastases to liver, bone, lymph nodes, and bone marrow. Current therapy includes chemotherapy, radiation, and high dose chemotherapy with subsequent bone marrow transplantation followed by differentiating therapy. More recently, immunotherapy has been added using monoclonal antibodies to the GD2 glycolipid antigen that is heavily expressed by NB cells (Bosslet, Mennel et al. 1989; Cheung, Kushner et al. 1998). Over the last 30 years, significant therapeutic progress has been made with an increase in the five-year relative survival rate from approximately 25% to 55%. However, almost 50% of patients are estimated to die of their tumor, and over the past decade improvement in the five-year survival rate of NB patients has been slow (Harras 1996). NB has a particularly poor prognosis in patients older than 2 years at diagnosis, advanced stage disease and/or disease characterized by *MYCN* gene amplification (Seeger, Brodeur et al. 1985; Brodeur 2003). These more aggressive forms of NB respond poorly to chemotherapeutic approaches, and therefore, there is great need for a better understanding of the cellular regulation of *MYCN*-amplified NB tumors in an effort to search for alternative molecular drug targets. Although a role for the MYCN oncoprotein has been established in NB pathogenesis, the mechanism by which MYCN contributes to both the development of this disease and its poor prognosis is still unclear. The MYCN oncoprotein functions as a transcriptional regulator (Ben-Yosef, Yanuka et al. 1998) and thus may influence tumorigenesis and patient survival by regulating the expression of key genes involved in the NB malignant phenotype. MYCN regulates the expression of genes that encode ornithine decarboxylase (ODC), the multi-drug resistance-associated protein 1 (MRP1), and MDM2 (Slack, Chen et al. 2005). ODC is the rate-limiting enzyme in the production of polyamines (Marton and Pegg 1995). Although polyamines, and therefore ODC, are essential for normal cell proliferation, increased ODC activity can induce cellular transformation *in vitro* (Auvinen, Paasinen et al. 1992), and high ODC levels are associated with a variety of tumors, including those of the brain and prostate (Mohan, Challa et al. 1999; Ernestus, Rohn et al. 2001). Several studies have identified ODC as a target gene for the MYCN oncoprotein (Lutz, Stohr et al. 1996; Ben-Yosef, Yanuka et al. 1998; Lu, Pearson et al. 2003), and it is possible that ODC, and therefore polyamines, play a significant role in NB tumorigenesis.
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role of polyamines in cancer

The identification of novel inhibitors of enzymes involved in polyamine biosynthesis with antitumor activities has recently revived interest in polyamine homeostasis and in designing strategies of cancer chemotherapy (Mamont, Duchesne et al. 1978; Porter, Regenass et al. 1992; Seiler, Atanassov et al. 1998). Selective pharmacological interference with the synthesis of natural polyamines results in tumor cell growth inhibition under both in vitro and in vivo conditions (Mamont, Duchesne et al. 1978; McCann and Pegg 1992). Although the precise mechanism by which polyamines contribute to cell proliferation is not well known, it has been suggested that it may be a result of their ability to bind DNA and affect gene expression by bringing about structural changes in chromatin, thereby stimulating cell growth (Marton and Morris 1987). Furthermore, the dramatic increases in the activity of ODC in certain tumor cells have been linked to G1-S transition (Fuller, Gerner et al. 1977; Kahana and Nathans 1984; Kaczmarek, Calabretta et al. 1987). As mentioned above, an apparent molecular basis for this derives from the fact that ODC is among those genes, which can be regulated by c-Myc and MYCN (Bello-Fernandez, Packham et al. 1993; Pena, Reddy et al. 1993; Wagner, Meyers et al. 1993; Lutz, Stohr et al. 1996; Lu, Pearson et al. 2003), both of which regulate entry into and exit from the cell cycle. Because cell growth is absolutely dependent on polyamines, interference with polyamine biosynthesis has long been considered a promising therapeutic approach against proliferative diseases, including various malignancies (Heby and Persson 1990; Auvinen, Paasinen et al. 1992; McCann and Pegg 1992). α-difluoromethylornithine (DFMO or eflornithine), a suicide substrate inhibitor of ODC (Metcalf, Bey et al. 1978; Poulin, Lu et al. 1992), has been the prototype tool for the study of therapeutic effectiveness of polyamine depletion in experimental tumors (McCann and Pegg 1992; Meyskens and Gerner 1999) (Fig. 1).  
  
  
DFMO inhibits cell growth of many cancer cells including NB and induces cell differentiation (Chapman 1980; Melino, Farrace et al. 1988). These processes are accompanied by an apparent depletion of putrescine (Put) and spermidine (Spd) pools (Pegg 1988; Heby and Persson 1990; McCann and Pegg 1992). DFMO has also been shown to induce apoptosis and inhibit metastasis in a human gastric cancer model (Takahashi, Mai et al. 2000).
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role of polyamines in nb

The fluctuation in the levels of intracellular polyamines such as Put, Spd, and spermine (Spm) has been observed in association with cell differentiation (Heby 1981; Tabor and Tabor 1984; Pegg 1986), and inhibition of ODC by DFMO and reduction in polyamine pools stimulates various cancer cells including NB cells to differentiate (Chen, Nau et al. 1983; Melino, Farrace et al. 1988; Melino, Piacentini et al. 1991). DFMO treatment of NB cells can change the triangular NB morphology by inducing a different phenotype; one which resembles elongated fibroblastlike cells without typical neuritic processes. By comparison, treatment with retinoic acid (RA)
induces neural differentiation of NB cells as indicated by the outgrowth of definite neurites (Melino, Farrace et al. 1988; Melino, Piacentini et al. 1991; Wainwright, Lasorella et al. 2001). The role of polyamines in cell differentiation has been studied for many years, and yet the precise role of polyamines at the cellular and molecular level is still not well understood and may play a key role in tumor regression.
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effect of polyamine inhibitor dfmo

Encouraging data by two groups (Hogarty, Norris et al. 2008; Rounbehler, Li et al. 2009) recently emerged and confirmed the effect of DFMO *in vivo* using the *TH-MYCN* NB mouse model, and DFMO in combination with cisplatin and cyclophosphamide increased the tumor-free survival of TH-MYCN homozygous mice (Fig. 2) (Hogarty, Norris et al. 2008). Additional studies have revealed that DFMO combined with SAM486A act synergistically and result in a significantly reduced tumor burden in TH-MYCN mice (56).
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relevance of odc in patients

Further evidence of the importance of ODC in NB tumorigenesis is available from our recent studies with human NB tumors. We analyzed the expression levels of ODC mRNA from 88 NB
patients and found significant correlations between ODC expression and the overall survival probability. High levels of ODC were predictive of low survival probability and vice versa (Fig.  
3A). Most surprisingly, ODC was also predictive in tumors without MYCN amplification (Fig.  
3B), thus suggesting that ODC also plays a role in NB tumorigenesis independent of MYCN amplification (57). These findings were independently confirmed by two other groups (Hogarty, Norris et al. 2008; Rounbehler, Li et al. 2009).
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combination of dfmo and bortezomib

Metabolism.  
Overexpression of LIN28 correlates with poor outcome in neuroblastoma (NB) [1]. The LIN28/Let-7 axis affects many cellular processes including cell differentiation and glycolytic metabolism [2-4]. The LIN28/Let-7 axis also affects MYCN, which is often overexpressed in high risk NB [5, 6]. Studies in colon cancer have shown that ornithine decarboxylase (ODC) inhibition decreases LIN28 levels, thereby reversing the LIN28/Let-7 axis [7]. Our preliminary studies demonstrate that therapy targeting ODC reverses the LIN28B/Let-7 axis in NB and decreases MYCN protein expression (Figure 4). Our studies show that cells overexpressing LIN28B also have greater sensitivity to difluoromethylornithine (DFMO) treatment, which inhibits ODC and decreases cellular polyamines [8].  
Many cancer cells are reprogramed to produce ATP primarily via glycolytic metabolism as opposed to oxidative phosphorylation, a phenomenon termed the "Warburg effect" [9-13]. This phenomenon is well documented in NB [14]. Our studies showed a decrease in ATP/cell with DFMO treatment in NB, indicating a decrease in glycolytic metabolic activity (data not shown). Patient observations in our Phase I study with DFMO and *in vivo* xenograft studies using PET scans further validated the findings that DFMO treatment reduces glycolytic metabolism in NB (Figure 2). Recent studies have shown that the LIN28/Let-7 and metabolism pathways are important in cancer stem cell (CSC) regulation. Our collaborator, Dr. Max Wicha, has demonstrated the importance of these pathways in breast CSCs. His studies show that elevated glycolytic metabolism is a characteristic of CSCs [15] and suggests that the LIN28/Let-7 axis is of critical importance in CSCs [16]. Additionally, his research has demonstrated the importance of SOCS3 and the NF-kB/IL-6 inflammatory loop in CSCs [17]. The NF-kB/IL-6 inflammatory loop is linked to the LIN28/Let-7 axis [3]; therefore, to further inhibit this pathway, we studied bortezomib (Velcade). Bortezomib inactivates NF-kB through proteasome inhibition. NF-kB binds and enhances expression at the LIN28 promoter [3], therefore inhibition by bortezomib reduces LIN28. Downstream effects of this include inhibition of the insulin receptor and GLUT-4 and decrease in glycolytic metabolism [2]. Proteosome inhibition also results in an increase in SOCS3 which in turn inhibits the JAK/STAT pathway and decreases IL-6 [17] (Figure 3). Our preliminary studies show synergy between DFMO and bortezomib in NB.  
Figure 5: DFMO treatment reduces glycolytic metabolism in  
  
  
NB. A) Patient PET scans before and after one cycle of DFMO treatment. B) Average SUVmax (measure of metabolism) was lower in mice treated with DFMO compared to untreated. C) PET scans of one untreated and one DFMO-treated mouse at baseline, day 19, and day 32 of treatment.  
This combination was tested in *in vitro* and in vivo studies and shown to be well tolerated and effective together. A mice xenograft model treated with the combination with DFMO and bortezomib resulted in a decrease in tumor growth (Figure 5).
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1.2 clinical work summary of

NTC01059071 was a multi-center, investigator initiated dose escalation study that was completed in 2013. The following dose levels of DFMO (CPP-1X) were investigated: 500 mg/m2, 750 mg/m2, 1000 mg/m2 and 1500 mg/m2. Cycles were 21 days, with CPP-1X given days 1-21 for all cycles and for cycles 2 and greater, etoposide is given on days 1-14 of each cycle. Subjects must complete the first 2 cycles of treatment (cycle 1 CPP-1X alone, cycle 2 CPP-1X with 50 mg/m2/dose etoposide) in order to be included in the dose limiting toxicity assessment. Subjects who did not complete 2 cycles were replaced in the cohort. Subjects are considered to have completed the study if they have received 5 cycles of treatment. They may continue treatment if there are no safety concerns, there is no disease progression, and/or there is an indication of clinical benefit. At the close of the study 21 subjects had received at least one dose of DFMO, with 3 evaluable subjects at 500 mg/m2, 3 evaluable subjects at 750 mg/m2, 3 evaluable subjects at 1000 mg/m2, and 6 evaluable subjects at 1500 mg/m2. All subjects were multiple relapsed/refractory, with 14 males and 7 females, mean age 9, range (1-17), and 60% White/Caucasian. There were no DLT's reported during this study and no serious adverse events. The most prevalent adverse events were low hemoglobin, neutropenia and thrombocytopenia. The majority of the events were considered unrelated to treatment. Nine subjects completed at least 5 cycles of therapy therefore completing study defined protocol therapy. Time on study ranged from 1 to 43 cycles, with 4 subjects on therapy 1 year or more.
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summary of phase ii neuroblastoma

NCT01586260 is a multi-center, open label, single agent study that was opened in 2012 for children with neuroblastoma that is in remission. The dose level of DFMO being investigated is:
1000 mg/m2 twice daily. Cycles are 28 days, with DFMO given days 1-28 for all cycles. Subjects are considered to have completed the study if they have received 27 cycles of treatment (2 years) without a remission. To date 51 subjects had received at least one dose of DFMO, with 44 subjects remaining on study at this time. All subjects had neuroblastoma that is in remission, with 33 males and 18 females, mean age 9, range (1-17), and 69% White/Caucasian. There have been no related SAE's reported during this study to date.
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summary of dosing and schedule

Bortezomib (Velcade®, IND \#58443) is an FDA approved proteasomal Inhibitor for the treatment of multiple myeloma and mantle cell lymphoma. Pre-clinical studies have demonstrated anti-tumor activity of bortezomib in pediatric cancers, including leukemia, nonHodgkin lymphoma, neuroblastoma, rhabdomyosarcoma, and Ewing's sarcoma. Bortezomib has been studied in one phase 1 and three phase 2 Children's Oncology Group (COG) studies and is currently under investigation in a phase 3 study. The Phase 1 study was combined with Vorinostat and the MTD for bortezomib was 1.3mg/m2 IV on D1, 4, 8 and 11 of a 21 day cycle. On this trial, one child developed grade 4 neuropathy late in treatment. Due to this, the phase 2 trials have used bortezomib at either 1.2 mg/m2 or 1.3mg/m2 on D1, 4 and 8 of a 21 day cycle. The one trial using the 1.2mg/m2 combined bortezomib with more myelosuppressive therapy (Ifosfamide/Vinorelbine). The remaining COG trails, including the current phase 3 trial, have use the dose of Bortezomib at 1.3mg/m2 IV on D1, 4 and 8 of a 21 day cycle. This dose and schedule of Bortezomib is chosen for this study as the standard of care dose in pediatrics.
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summary

In summary, our studies show that DFMO reverses the LIN28/Let-7 pathway, reverses MYCN overexpression and decreases cellular ATP and PET activity in NB cells. These pathways, in addition to the proteasome pathway, have been shown to be important in CSCs but have yet to be linked to CSCs in NB. Given the high rate of relapse in NB, targeting of the CSC is important to prevent relapse. We believe that our preliminary study lays the framework for an investigation of the effects of ODC-Lin 28/Let-7 inhibition in NB using DFMO and bortezomib in children with relapsed/refractory neuroblastoma.
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2.0 study objectives 2.1 primary

2.1.1 To determine the safety and tolerability of DFMO in combination with bortezomib at 3 dose levels of DFMO: 1500mg/m2 twice daily, 2000mg/m2 twice daily, and 2500mg/m2 twice daily in subjects with relapsed or refractory neuroblastoma who receive one full cycle of this dose.
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2.2 primary- phase i and

2.2.1 To evaluate the activity of DFMO as a single agent and in combination with bortezomib in relapsed or refractory neuroblastoma based on:
Progression free survival (PFS) Overall response rate (ORR)
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2.3 secondary objectives

2.3.1 To determine the safety and tolerability of DFMO in combination with bortezomib in pediatric subjects with refractory or recurrent neuroblastoma.  
2.3.2 For subjects followed with PET scan: comparison of changes in PET
activity and correlation with PFS
2.3.3 Correlation of urinary polyamine levels with response and progression of disease in neuroblastoma.  
2.3.4 Biological Correlates to evaluate or explore minimally include: 1) Urine:
polyamine levels 2) Blood: Inflammatory markers, Biomarkers for Let 7 and SNPS, and explorative biomarker analysis, 3) Bone Marrow/Tumor biopsies (if biopsied per standard of care): research studies including Immunohistochemistry/Western Blot/qRT-PCR of bone marrow tumor cells or tumor to study Let-7, ODC, Lin28, MYCN, OKT-4, BMI1, HMGA2, GLUT-4, p-AKT, NF-kB, SOCS3 will be performed in an explorative biomarker analysis. Studies will measure thymidine and polyamine levels. 4) In vitro research studies of tumor cells collected above will include: Genomic analysis of cells pre- and post- treatment (when applicable), cell viability assays to determine subject IC50 and correlation of in vitro response to in vivo response.  
2.3.5 To determine the dose effect of DFMO on biological correlates
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3.0 study design

NMTRC010B is an open label, multicenter, study to evaluate the efficacy of DFMO in combination with bortezomib in subjects with relapsed or refractory neuroblastoma.
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3.1 cycles 1-6 3.1.1 dfmo-

Subjects will receive six (6) cycles of oral DFMO twice daily at their assigned dose level on each day of this 21-day cycle.  
| DFMO Dose   | Dose           |
|-------------|----------------|
| Level 1     | 1500 mg/m2 BID |
| 2           | 2000 mg/m2 BID |
| 3           | 2500 mg/m2 BID |  
DFMO is provided as 250mg tablets.
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3.1.2 dfmo- phase ii

The highest tolerated DFMO dose from the Phase I dose escalation will be used for the Phase II portion of the study. Subjects will receive oral DFMO twice daily on each day of this 21day cycle.  
DFMO is provided as 250mg tablets.
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3.1.3 bortezomib

Bortezomib will be given at 1.3mg/m2/dose IV Push on Days 1, 4, and 8 of each 21-day cycle The Phase I subjects on this study will be the first pediatric subjects to receive DFMO in combination with Bortezomib. If a Dose Limiting Toxicity (DLT) is seen in 2/6 subjects, this portion of the study will allow for a -1 dose level reduction (1000mg/m2/dose). If the DLT is seen in 2/6 subjects at the -1 dose level, a further reduction to dose level -2 (500mg/m2/dose) will be allowed. Enrollment will be held at the end of this phase of the study until all subjects within the cohort have completed one cycle and a safety analysis has been completed by the study committee and Data Safety and Monitoring Board (DSMB). The study committee will then decide whether the study can continue to the next cohort and subsequently to the Phase II portion of the trial.  
3.2 Response **Evaluations**
At the times indicated in Section 6.0 and the Table of Procedures and Assessments, scans will be obtained to evaluate response for subjects enrolled in this study. Response will be assessed according to criteria outlined in Section 7.0 to evaluate the potential benefit of DFMO in this subject population.  
3.3 **Biological Studies**
Subjects will have an opportunity to participate in additional correlative biological studies on a voluntary basis collecting urine, blood, bone marrow and tumor (if a standard of care sample is being collected). These samples will be sent to the POTRL at Helen DeVos Children's Hospital and University of Arizona for studies described in Section 12. The studies will evaluate the effect of DFMO and bortezomib on neuroblastoma. These studies will include evaluation of the ODC-Lin28/Let-7 pathway, glycolytic metabolism and inflammation. These biologic studies may be able to contribute to our knowledge of molecular profiles or biomarkers of response to therapy to help guide future treatment studies.
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4.0 subject selection 4.1 inclusion

1. Age: ≤ 21 years at the time of diagnosis. 2. Diagnosis: Histologic verification at either the time of original diagnosis or relapse of neuroblastoma.  
3. Disease Status: For the purposes of this study, aggressive multidrug chemotherapy is defined as chemotherapy including 2 or more agents that must include an alkylating agent and a platinumcontaining compound. Patients must have ONE of the following:
1) Any episode of recurrent disease following completion of aggressive multi-drug frontline therapy.  
2) Any episode of progressive disease during aggressive multi-drug frontline therapy.  
3) Primary resistant/refractory disease detected at the conclusion of at least 4 cycles of aggressive multidrug induction chemotherapy on or according to a high-risk neuroblastoma protocol (examples include Children's Oncology Group trials: A3973, ANBL0532, ANBL09P1, etc.).  
4. Measurable or evaluable disease, including at least one of the following: Measureable tumor by CT or MRI; or A positive MIBG or PET scan; or Positive bone marrow biopsy/aspirate.  
5. Current disease state must be one for which there is currently no known curative therapy or no additional therapies proven to prolong survival with an acceptable quality of life.  
6. A negative serum or urine pregnancy test is required for female subjects of child bearing potential (onset of menses or ≥13 years of age).  
7. Organ Function Requirements:
a. Subjects must have adequate liver function as defined by:
- AST and ALT <5x upper limit of normal - Serum bilirubin must be ≤ 2.0 mg/dl b. Subjects must have adequate Bone Marrow function defined as:
For patients without bone marrow involvement:
- Peripheral absolute neutrophil count (ANC) ≥ 750/µL
- Platelet count ≥ 50,000/µL (transfusion independent, defined as not receiving platelet transfusions within a 7 day period prior to enrollment. Exception: Patients that are platelet dependent due to previous extensive treatment- e.g. - MIBG therapy).  
- Hemoglobin ≥ 8.0 g/dL (may receive RBC transfusions)
Patients known to have bone marrow involvement with neuroblastoma are eligible provided that minimum ANC and platelet count criteria are met but are not evaluable for hematological toxicity. Patients that do not have bone marrow involvement but that are transfusion dependent at study entry are eligible provided that minimum ANC and platelet count criteria are met but are not evaluable for platelet toxicity.  
c. Subjects must have adequate renal function defined as:
Serum creatinine based on age/gender as follows:  
| Maximum Serum Creatinine  (mg/dL) Male Female   |     |     |
|-------------------------------------------------|-----|-----|
| 1 month to < 6  months                          | 0.4 | 0.4 |
| 6 months to < 1 year                            | 0.5 | 05  |
| 1 to < 2 years                                  | 0.6 | 0.6 |
| 2 to < 6 year                                   | 0.8 | 0.8 |
| 6 to < 10 years                                 | 1   | 1   |
| 10 to < 13 years                                | 1.2 | 1.2 |
| 13 to < 16 years                                | 1.5 | 1.4 |
| ≥ 16 years                                      | 1.7 | 1.4 |
| Age                                             |     |     |  
8. Informed Consent: All subjects and/or legal guardians must sign informed written consent.  
Assent, when appropriate, will be obtained according to institutional guidelines.
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4.2 exclusion criteria 1. lansky

2. BSA (m2) of <0.25 3. Prior Therapy- Patients must have fully recovered from the acute toxic effects of all prior anti- cancer chemotherapy and be within the following timelines:
a. Myelosuppressive chemotherapy: Must not have received within 2 weeks of enrollment onto this study (6 weeks if prior nitrosourea). b. Hematopoietic growth factors: At least 5 days since the completion of therapy with a growth factor. c. Biologic (anti-neoplastic agent): At least 7 days since the completion of therapy with a biologic agent. For agents that have known adverse events occurring beyond 7 days after administration, this period must be extended beyond the time during which adverse events are known to occur. The duration of this interval must be discussed with the Study Chair. d. Immunotherapy: At least 6 weeks since the completion of any type of immunotherapy, e.g. tumor vaccines. e. Monoclonal antibodies: At least 7 days or 3 half-lives, whichever is longer, must have elapsed since prior treatment with a monoclonal antibody. f. XRT: At least 14 days since the last treatment except for radiation delivered with palliative intent to a non-target site. g. Stem Cell Transplant or Rescue: No evidence of active graft vs. host disease and ≥ 2 months must have elapsed since transplant.  
4. Investigational Drugs: Subjects who have received another investigational drug within the last 14 days are excluded from participation.  
5. Infection: Subjects who have an uncontrolled infection are not eligible until the infection is judged to be well controlled in the opinion of the investigator.  
6. Subjects who, in the opinion of the investigator, may not be able to comply with the safety monitoring requirements of the study, or in whom compliance is likely to be suboptimal, should be excluded.
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additional criteria

Subjects willing to participate in the correlative biologic studies will sign an additional consent form to provide bone marrow, blood, urine and tumor (if available) for analysis.
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5.0 study drug administration 5.1

DFMO (eflornithine hydrochloride is an inhibitor of ornithine decarboxylase (ODC) designated chemically as 2-(difluoromethyl)-DL-ornithine monohydrochloride monohydrate. The dosage form to be used in this study are provided as a convex tablet containing 250 mg of eflornithine HCl, monohydrate. The tablets are packaged and sealed in opaque white HDPE bottles, and each bottle contains 100 tablets. The DMFO tablets are supplied by KC Pharma.  
The tablets are to be stored at room temperature (20-250C).
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5.1.2 dfmo dosing phase i

Subjects will be given oral DFMO twice daily at their assigned dose level on each day of this 21day cycle. Upon enrollment subjects will be assigned a DFMO dose level by NMTRC staff.  
Dosing will be per Dose level charts below. Doses are rounded to the nearest whole tablet.  
The dose of DFMO will be recalculated at the beginning of each cycle based on BSA using a weight and height obtained up to 5 days prior to the cycle start date.  
| DFMO Dose   | Dose           |
|-------------|----------------|
| Level 1     | 1500 mg/m2 BID |
| 2           | 2000 mg/m2 BID |
| 3           | 2500 mg/m2 BID |  
| BSA (m2)      | Tablets to be Dispensed for          | Total Tablets   | Actual                 |
|---------------|--------------------------------------|-----------------|------------------------|
| each dose     | Per Day                              | Mg/m2           |                        |
| >1.5          | Nine (9) tablets orally twice a day  | Eighteen (18)   | 1500 and               |
| down per dose |                                      |                 |                        |
| 1.3 to 1.5    | Eight (8) tablets orally twice a day | Sixteen (16)    | 1539 to 1333  per dose |
| 1.1 to 1.2    | Seven (7) tablets orally twice a day | Fourteen (14)   | 1458 to 1591  per dose |
| 1.0           | Six (6) tablets orally twice a day   | Twelve (12)     | 1500 per dose          |
| 0.8 to 0.9    | Five (5) tablets orally twice a day  | Ten (5)         | 1389 to 1563  per dose |
| 0.6 to 0.7    | Four (4) tablets orally twice a day  | Eight (8)       | 1429 to 1667  per dose |
| 0.5           | Three (3) tablets orally twice a day | Six (6)         | 1500 per dose          |
| 0.3 to 0.4    | Two (2) tablets orally twice a day   | Four (4)        | 1250 to 1667 per dose  |
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dfmo dose table for dose

| BSA (m2)    | Tablets to be Dispensed for          | Total Tablets   | Actual                |
|-------------|--------------------------------------|-----------------|-----------------------|
| each dose   | Per Day                              | Mg/m2           |                       |
| 1.5 to >1.5 | Twelve (12) tablets orally twice a   | Twenty-four     | 2000 and              |
| day         | (24)                                 | down per dose   |                       |
| 1.4         | Eleven (11) tablets orally twice a   | Twenty-two (22) | 1964 per dose         |
| day         |                                      |                 |                       |
| 1.2 to 1.3  | Ten (10) tablets orally twice a day  | Twenty (20)     | 1923 to 2083 per dose |
| 1.1         | Nine (9) tablets orally twice a day  | Eighteen (18)   | 2046 per dose         |
| 1.0         | Eight (8) tablets orally twice a day | Sixteen (16)    | 2000 per dose         |
| 0.9         | Seven (7) tablets orally twice a day | Fourteen (14)   | 1944 per dose         |
| 0.7 to 0.8  | Six (6) tablets orally twice a day   | Twelve (12)     | 1875 to 2143 per dose |
| 0.6         | Five (5) tablets orally twice a day  | Ten (10)        | 2083 per dose         |
| 0.5         | Four (4) tablets orally twice a day  | Eight (8)       | 2000 per dose         |
| 0.4         | Three (3) tablets orally twice a day | Six (6)         | 1875 per dose         |
| 0.3         | Two (2) tablets orally twice a day   | Four (4)        | 1667 per dose         |  
| BSA (m2)      | Tablets to be Dispensed for each         | Total Tablets      | Actual        |
|---------------|------------------------------------------|--------------------|---------------|
| dose          | Per Day                                  | Mg/m2              |               |
| 1.5 to >1.5   | Fifteen (15) tablets orally twice a day  | Thirty (30)        | 2500 and      |
| down per dose |                                          |                    |               |
| 1.4           | Fourteen (14) tablets orally twice a day | Twenty-eight  (28) | 2500 per dose |
| 1.3           | Thirteen (13) tablets orally twice a day | Twenty-six (26)    | 2500 per dose |
| 1.2           | Twelve (12) tablets orally twice a day   | Twenty-four (24)   | 2500 per dose |
| 1.1           | Eleven (11) tablets orally twice a day   | Twenty-two (22)    | 2500 per dose |
| 1.0           | Ten (10) tablets orally twice a day      | Twenty (20)        | 2500 per dose |
| 0.9           | Nine (9) tablets orally twice a day      | Eighteen (18)      | 2500 per dose |
| 0.8           | Eight (8) tablets orally twice a day     | Sixteen (16)       | 2500 per dose |
| 0.7           | Seven (7) tablets orally twice a day     | Fourteen (14)      | 2500 per dose |
| 0.6           | Six (6) tablets orally twice a day       | Twelve (12)        | 2500 per dose |
| 0.5           | Five (5) tablets orally twice a day      | Ten (10)           | 2500 per dose |
| 0.4           | Four (4) tablets orally twice a day      | Eight (8)          | 2500 per dose |
| 0.3           | Three (3) tablets orally twice a day     | Six (6)            | 2500 per dose |
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dfmo dose table for dose

The highest tolerated DFMO dose from the Phase I dose escalation will be used for the Phase II portion of the study. Subjects will receive oral DFMO twice daily on each day of this 21-day cycle per dosing chart above for appropriate dose level. Doses are rounded to the nearest whole tablet.
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5.1.4 administration of dfmo

Treatment will be administered on an outpatient basis unless hospitalization is required for another reason.  
Each entire dose of DFMO tablets should be swallowed. Subjects will be advised (but not required) to maintain a low Polyamine diet during the duration of this study. A handout will be provided to subjects with foods they should avoid while on this study. Subjects will be given a 'patient instruction sheet' for home administration. Subjects will be given a 'dosing diary' to keep track of doses given at home.
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5.1.5 subjects unable to swallow

For subjects unable to swallow tablets, DFMO tablets may be chewed or crushed in a teaspoon and administered with a small amount of liquid. Note; tablets may not completely dissolve. Recommended liquids for mixing (to cover taste)- Lemonade, Apple, Cranberry, Grape, or Pineapple
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dfmo may not be mixed

Crushed DFMO tablets may also be mixed in 1-2 tablespoons of chocolate syrup, applesauce, or other food. The DFMO tablets will not dissolve, and this is acceptable. This technique is simply to mask the flavor.
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5.1.6 missed doses

If a subject vomits or misses a dose they should skip the missed dose and continue the drug at the next dose. Do not make up missed doses.
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5.1.7 dfmo clinical pharmacology

DFMO (Eflornithine hydrochloride) is a member of the following drug classes: 1) inhibitor of ornithine decarboxylase (ODC), 2) hirsutism (excess hair growth) retardant, and 3)
antiprotozoals. Eflornithine is FDA approved as a cream for treatment of female hirsutism, and in intravenous form for treatment of trypanosomiasis. The oral tablet form is not available outside of the clinical trial setting in the U.S., and the formulation used in this trial is similar to that used in the Phase III colon adenoma clinical trial in combination with sulindac (Meyskens et al., 2008). Contraindications: Prior hypersensitivity to eflornithine.
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common side effects

Overall, the most frequently reported AEs in NCI, DCP-sponsored chemoprevention studies were diarrhea (9.0%), headache (7.5%), nausea (6.5%), hearing loss (5.6%), tinnitus (4.3%) and asthenia (4.7%). Other common toxicities (each accounting for 2 to 3% of all AEs reported) were epigastric pain, flatulence, dyspepsia, anemia, dizziness and skin rash. Less common toxicities (each accounting for 1 to 2% of all AEs reported) were: stomatitis, rhinitis, insomnia, infections, vomiting, vasodilation, dry mouth, constipation, dry skin, menstrual disorders, pharyngitis, emotional lability, pruritis, myalgia, and pain (miscellaneous). The most significant adverse effects associated with clinical administration of DFMO in chemoprevention trials are loss of hearing acuity and tinnitus. These effects have generally been found to be reversible when DFMO treatment is stopped. Patients who receive a cumulative oral dose of DFMO below 150 g/m2 experience minimal ototoxicity.  
Infrequent side effects: Hearing loss/change by audiometry testing has been reported in 8.4%
of patients on high dose eflornithine. Rash and alopecia have been reported in 3% of patients. Anorexia and abdominal pain have been reported in 2% of patients treated with eflornithine. Rare but serious side effects include dizziness (1%), headaches (2%), and seizures (8%), have been reported in patients on intravenous eflornithine. Myelosuppression (including leukopenia, [37%], anemia [55%], and thrombocytopenia [14%]) has been reported at high intravenous doses.  
Pregnancy and Lactation: Pregnancy category C. It is unknown if eflornithine crosses the placenta. Case reports in humans along with animal studies (mice, rats) indicate potential for fetotoxicity. Experiments in rodents indicate that eflornithine blocks yolk sac formation and trophoblast differentiation, affecting processes such as vasculogenesis and steriodgnesis (LopezGarcia, *et al.*, 2008). The World Health Organization has not determined a breast feeding rating for eflornithine due to insufficient data. The Thompson lactation rating is that infant risk cannot be ruled out. No studies investigating the safety of lactation after eflornithine administration have been conducted, nor are there data to determine drug levels in breast milk after drug administration.  
| Toxicity for DFMO Potential Risk: Likely Happens to 10-30 patients out  of every 100                                   | Rare                                                                                                                                                                                                              |                  |
|------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------|
| Happens to fewer than 3 patients out of every 100                                                                      |                                                                                                                                                                                                                   |                  |
| -                                                                                                                      | Fewer red and white  blood cells  o a low number of red  blood cells can make  you feel tired and  weak and may  require transfusion. o a low number of  white blood cells can  make it easier to get  infections |                  |
| -                                                                                                                      | Decrease in the number  of platelets made in the  bone marrow                                                                                                                                                     | Less Likely      |
| Happens to 3-10 patients out of  every 100 - Nausea  - Hearing Loss - Ringing in ears - Diarrhea - Headache - Weakness | -                                                                                                                                                                                                                 | Loss of appetite |
| -                                                                                                                      | Abdominal Pain                                                                                                                                                                                                    |                  |
| -                                                                                                                      | Flatulence (gas)                                                                                                                                                                                                  |                  |
| -                                                                                                                      | Dizziness                                                                                                                                                                                                         |                  |
| - •                                                                                                                    | Skin Rash                                                                                                                                                                                                         |                  |
| -                                                                                                                      | Seizures                                                                                                                                                                                                          |                  |
| -                                                                                                                      | Sores in the mouth                                                                                                                                                                                                |                  |
| -                                                                                                                      | Runny nose                                                                                                                                                                                                        |                  |
| -                                                                                                                      | Difficulty sleeping                                                                                                                                                                                               |                  |
| -                                                                                                                      | Infections                                                                                                                                                                                                        |                  |
| -                                                                                                                      | Dry mouth                                                                                                                                                                                                         |                  |
| -                                                                                                                      | Constipation                                                                                                                                                                                                      |                  |
| -                                                                                                                      | Dry skin                                                                                                                                                                                                          |                  |
| -                                                                                                                      | Menstrual disorders                                                                                                                                                                                               |                  |
| -                                                                                                                      | Sore throat                                                                                                                                                                                                       |                  |
| -                                                                                                                      | Vomiting                                                                                                                                                                                                          |                  |
| -                                                                                                                      | Vasodilation (the  relaxation of blood  vessels possibly causing  low blood pressure)                                                                                                                             |                  |
| -                                                                                                                      | Emotional ups and downs                                                                                                                                                                                           |                  |
| -                                                                                                                      | Itchiness                                                                                                                                                                                                         |                  |
| -                                                                                                                      | Body aches                                                                                                                                                                                                        |                  |
| -                                                                                                                      | Pain                                                                                                                                                                                                              |                  |  
Four particular areas of concern have been identified with regard to the safety of subjects participating in this study and an attempt to address each of them is outlined below: The main considerations are: thrombocytopenia, hearing loss, gastrointestinal and non-G.I. side effects.
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a) dfmo specific adverse events

Although hearing loss has been a problem at higher doses (see below), clinical changes in hearing have been uncommon (one of 123 subjects in the phase IIb trial) and reversible in the doses proposed for this trial (62). An extended analysis of these observations is in press (65). There was no statistically significant shifts in distortion product otoacoustic emission levels. For auditory pure tone thresholds, there was a subtle 2-3 dB decrease in hearing sensitivity for the two higher DFMO doses (0.2 and 0.4 gm/M2/day), but only for the two lowest frequencies at 250 and 500 HZ. However in two phase I trials using lower doses of DFMO, done by other investigators, no audiometric changes were seen after approximately 6 months of DFMO therapy at 0.50Gm/m2/d (total dose 90Gm) although some changes were seen at higher doses (79).  
Hearing loss may occur in association with DFMO administration at high doses. In a metaanalysis of previous studies, (64), it was reported that less than 10% of the subjects who received cumulative doses below 150 Gm/m2 developed a demonstrable hearing deficit, while hearing losses were observed in up to 75% of subjects who received cumulative doses above 250 Gm/m2.  
This side effect was thought to be totally reversible upon drug discontinuation. Some study participants taking DFMO at doses similar to those used in this study have experienced mild decreases in hearing soft sounds. These changes have been uncommon and usually subclinical (that is, noticeable on special hearing tests called audiograms, but not in normal conversation or daily activities). Although an affected participant's level of hearing usually returns to its usual state when drug is stopped, in a small percentage of cases (occurring in less than 5% of participants taking the drug) effects have persisted even after drug was stopped. These changes are most,likely not reversible in all subjects. However, in a recently completed phase IIb trial of DFMO audiologic changes that were clinically significant were not detected, even in the highest dose group (0.4Gm/m2/day) (extended analysis, 65). It is probable that at low doses of DFMO
ongoing recovery of inner ear polyamines occurs and clinical hearing loss will be rarely, if ever, seen. Since hearing loss is usually totally reversible after drug discontinuation, this approach appears safe and cost effective. (2) Thrombocytopenia (low platelets) Thrombocytopenia has been reported predominantly in studies using doses of DFMO
(>3Gm/m2/day) and primarily in cancer patients who had previously undergone chemotherapy or patients with compromised bone marrow. Other side effects ascribed to DFMO have been rare and, to date, seen only at high doses. (3) Other Skin rash, anemia, and neutropenia have also been seen with DFMO administration (4) GI Abdominal pain, loss of appetite, diarrhea have been reported.
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5.2 bortezomib 5.2.1 dose calculation

Bortezomib will be given at 1.3mg/m2/dose IV Push on Days 1, 4, and 8 of each 21-day cycle. Doses should be rounded per institutional standard. The dose of bortezomib will be recalculated at the beginning of each cycle based on BSA using a weight and height obtained up to 5 days prior to cycle start date.
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5.2.2 bortezomib (velcade)

Formulation and Stability: Bortezomib is a commercially available product and will not be supplied for this study. Bortezomib should be stored and dispensed per institutional standards and product insert.  
Source and Pharmacology: Bortezomib (PS - 341) is a reversible inhibitor of the chymotrypsin -like activity of the 26S proteasome (a multicatalytic protease present in all eukaryotic cells). The 26S proteasome is a large protein complex that degrades proteins that have been conjugated to ubiquitin. The ubiquitin-proteasome pathway plays an essential role in regulating the intracellular concentration of specific proteins, and constitutes the major mechanism for intracellular protein degradation (80%). Those intracellular proteins which maintain homeostasis within cells include numerous regulatory proteins involved in cellular integrity, such as cellcycle control, cellular apoptosis, transcription factor activation, and tumor growth via ATPdependent processes. Inhibition of the 26S proteasome prevents this targeted proteolysis, which can affect multiple signaling cascades within the cell. This disruption of normal homeostatic mechanisms can lead to cell death. The binding of bortezomib to human plasma proteins averages 83% over a concentration range of 100 to 1000 ng/mL. The mean elimination halflife of bortezomib after multiple dosing ranged from 40 to 193 hours after the 1 mg/m 2 dose and 76 to 108 hours after the 1.3mg/m2 dose. In vitro studies with human liver microsomes and human cDNA-expressed cytochrome P450 isozymes indicate that bortezomib is primarily oxidatively metabolized via cytochrome P450 enzymes 3A4, 2C19, and 1A2. Bortezomib metabolism by CYP 2D6 and 2C9 enzymes is minor. The major metabolic pathway is deboronation to form 2 deboronated metabolites that subsequently undergo hydroxylation to several metabolites. Deboronated bortezomib metabolites are inactive as 26S proteasome inhibitors. Bortezomib is metabolized by multiple cytochrome P450 (CYP) enzymes, with CYP3A4 being the primary contributor, and with only minor contributions of CYPs 2C19, 1A2, 2C9 and 2D6. It is a weak inhibitor of CYP2C19 and does not inhibit CYP1A2, 2C9, 2D6, and 3A4 at clinically relevant concentrations (IC50 > 30 μM). Co-administration of strong CYP3A4 inducers should be discussed with pharmacy prior to use throughout therapy with bortezomib (refer to Appendix II for a list of strong inducers). Co-administration of strong and clinically relevant moderate CYP3A4 inhibitors should be avoided until 72 hours beyond administration of Day 8 bortezomib for each course (exception: administration of prophylactic fluconazole may begin ≥ 24 hours after the Day 8 bortezomib dose). Similarly, patients receiving strong and clinically relevant moderate inhibitors should discontinue use of these agents 72 hours prior to start of Day 1 bortezomib in all courses (refer to Appendix II for a list of strong and clinically relevant moderate inhibitors). Additional inducers or inhibitors of CYP450 enzymes can be found at http://medicine.iupui.edu/clinpharm/ddis. In vitro and in vivo studies showed that green tea compounds, ascorbic acid (vitamin C) and other antioxidants, have the potential to significantly inhibit the activity of bortezomib. To avoid the risk of any possible interaction it is recommended that green tea containing products and any supplemental products containing vitamin C, flavonoids or other antioxidants (eg, vitamins, herbal supplements) be discontinued from at least 24 hours prior to the initiation of bortezomib through 72 hours after the last bortezomib dose. Injectable multivitamins used as a component of parenteral nutrition should also be avoided during this time period to minimize the risk of direct vitamin C inactivation of bortezomib. In addition, it is recommended that the total dietary intake of vitamin C not exceed the RDA for age. Normally balanced diets are acceptable; supplementation with high doses of vitamin C or injectable vitamin C should be avoided.
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bortezomib risks

| Risks and side effects related to Bortezomib include those which are: Less Likely Rare  Happens to 3-10 patients out of every 100 Happens to fewer than  3 patients out of every  100 - A feeling of  weakness and/or  tiredness - Nausea and/or  vomiting - Constipation or  diarrhea - Loss of appetite - Fever - Fewer red blood cells  and platelets in the  blood o a low number of  red blood cells  can make you feel  tired and weak o a low number of  platelets causes  you to bruise and  bleed more easily - Fluid retention and  build-up in the arms  and legs leading to  swelling and an  increase in weight - Infection - Nerve damage that  may cause pain,  burning, numbness,  and tingling in the  hands and feet and  may affect the ability  to perform tasks that  require fine  movements - Nerve damage that  may cause muscle  weakness or  paralysis in the  hands and feet and  may affect the ability  to perform tasks that  require fine  movements Likely Happens to 10-30  patients out of every  100 - Low blood pressure  - Dizziness  - Fainting - Difficulty sleeping or falling asleep - Chills including shaking chills - Skin rash with the presence of macules (flat  discolored area) and papules (raised bump) - Excessive loss of water from the body - Acid or upset stomach (heartburn) - Pain which may be in the abdomen (belly),  back, bone, head, joints, arms and legs,  muscles, and nerves - Headache or head pain - Cough - Shortness of breath - Fewer white blood cells in the blood o a low number of white blood cells can  make it easier to get infections - Low numbers of white blood cells called  lymphocytes that may last a long time and  make it easier to get infections which may be  life threatening - A stoppage (or blockage) of the intestine  which may require treatment - Inflammation and/or sores in the mouth  and/or throat that may make swallowing  difficult and are painful (painful mouth sores) - Bleeding in the gut that may show in the  stools - Nose bleed - Fever with a low white blood cell count which  could indicate infection and may require  hospitalization and treatment with antibiotics - Infection caused by hard to treat bugs  including bacteria, virus, and fungus - Muscle weakness of the whole body - Anxiety - Blurred vision - Fluid build-up in the lungs that can make you  feel short of breath - A hole in the  intestines which  would cause leakage  into the abdomen  (belly) with pain and  infection - Damage to the brain  associated with high  blood pressure  which may lead to  difficulty thinking,  carrying out normal  tasks, headache,  seizures  (convulsions),  difficulty seeing,  blindness, or other  visual changes,  which if caught early  can be reversed - Severe damage to  the brain tissue  which could lead to  difficulty carrying out  normal daily tasks or  could lead to coma - Severe kidney  damage (which may  be permanent)   |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
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5.3 guidelines for dose modifications

Toxicities and dose modifications will be monitored in all cycles. Adjustments to the doses of study drug will be based upon toxicity, graded according to the NCI Common Toxicity Criteria (CTC), Version 4.0, if these were normal at baseline. Events that are not described in the NCI criteria will be assigned grades according to the criteria provided in Section 8.0. Criteria for determining the relatedness of clinical adverse events to treatment (Section 8.5) should be utilized to determine the relationship of adverse events to the treatment.  
5.4 **Dose Delay**
Subjects without bone marrow metastases must have an ANC > 500/μl before starting Day 1 of each cycle. Subjects experiencing any related toxicity specified in 5.5 or any intolerable toxicity on a scheduled treatment day will have their dose of study drug held per Sections 5.6 and 5.7. If toxicity is encountered on Day 1 of any cycle, the day of resolution and administration of the dose will be considered Day 1 of that cycle. Treatment may be delayed no more than 14 days to allow recovery from related toxicity.
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5.5 definition of dose-limiting toxicity

DLT will be defined as any of the following events that are possibly, probably or definitely attributable to DFMO or bortezomib. Dose limiting hematologic and non-hematologic toxicities are defined differently.
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5.5.1 non-hematologic dose-limiting toxicity

5.5.1.1 Any Grade 3 or Grade 4 non-hematological toxicity attributable to the investigational drug with the specific exclusion of:
Grade 3 nausea, vomiting, diarrhea unless > 3 days despite optimal treatment with antiemetics ALT/AST elevation > 10x ULN that returns to Grade ≤ 2 or baseline prior to the time for the next treatment cycle. Note: For the purposes of this study the ULN for ALT/AST is defined as 50 U/L.  
Direct Bilirubin level > 1.5x - 3x upper limit of normal Grade 3 fever Grade 3 infection Grade 3 hypophosphatemia, hypokalemia, hypocalcemia or hypomagnesemia responsive to oral supplementation.  
Grade 2 allergic reactions that results in discontinuation of study drug 5.5.1.2 A peripheral neuropathy that results in a discontinuation of the bortezomib dose (See Section 5.7).  
5.5.1.3 Non-hematological toxicity that causes a delay of >14 days between treatment cycles.
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5.5.2 hematological dose limiting toxicity

Hematological dose limiting toxicity is defined as:
Grade 4 neutropenia for > 7 days Grade 4 thrombocytopenia for > 14 days (excluding special populations defined in 8.4.2)
Myelosuppression that causes a delay of >14 days between treatment cycles.
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any suspected or confirmed dose-limiting

5.6 **DFMO Dose Modification**
Subjects experiencing any dose limiting toxicity (DLT) specified above (Section 5.5) attributable to DFMO or any other intolerable toxicity will have their dose of DFMO held until toxicities have reverted to ≤ Grade 2 toxicity. Upon resolution of the toxicity, subjects will receive a -1 dose reduction of DFMO to one step down on the dosing table in section 5.1.2 Example- If the subject is currently taking 3 tabs twice a day they will step down to the weight range/dose directly below the one they are at and take 2 tabs twice a day. Subjects that are currently only taking one tablet per dose BID will be dose reduced to one tablet per day (QD). Subjects will be allowed to dose reduce for subsequent toxicities one more time to a -2 dose level. At that point if they experience another dose reducing toxicity (as defined here) they will be required to go off protocol therapy.  
If there is no resolution of above toxicity attributable to DFMO to ≤Grade 2 by 14 days, DFMO
should be discontinued, and subjects should be discontinued from the study. .  
For subjects entering this trial with bone marrow disease and for subjects entering this trial with platelet transfusion dependency, modifications will not be made based on platelet count.
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5.7 bortezomib dose modification

Subjects experiencing any dose limiting toxicity (DLT) specified above (Section 5.5) attributable to bortezomib or any other intolerable toxicity will have their dose of bortezomib held until toxicities have reverted to ≤ Grade 2 toxicity. Upon resolution of the toxicity, subjects will have bortezomib restarted per recommendations below.
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bortezomib-related hepatotoxicity

Subjects with mild hepatic impairment do not require dose adjustment of bortezomib. Subjects with severe hepatic impairment should receive bortezomib at modified doses as outlined below:  
| Direct Bilirubin Level         | Bortezomib Dose Modification                                                                             |                                                                     |
|--------------------------------|----------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------|
| Moderate*                      | > 1.5x - 3x upper limit of normal                                                                        | Reduce bortezomib to 0.7 mg/m2 for all doses in the                 |
| * Will not be considered a DLT | cycle in which hepatotoxicity is present. Consider  dose escalation to 1 mg/m2 if transaminitis resolves |                                                                     |
| Severe                         | > 3x upper limit of normal                                                                               | and is not possibly, probably or definitely related to  bortezomib. |  
-No adjustment necessary for elevated SGOT (ALT).
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bortezomib-related peripheral neuropathy

Subjects with bortezomib-related neurotoxicity will remain on protocol therapy but have bortezomib dose de-escalated or held as specified below. Peripheral neuropathy will be closely monitored during each course of treatment and toxicities graded using CTCAE v 4.0. Peripheral sensory neuropathy grading should be based on the maximum toxicity occurring during the previous course. All dose modifications should be based on the worst preceding toxicity. Bortezomib dose will be decreased for sensory peripheral neuropathy as follows:  
| Severity of peripheral                  | Bortezomib modification                                                                                                                                                                                       |
|-----------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| sensory neuropathy Grade 1 without pain | None                                                                                                                                                                                                          |
| Grade 1 with pain or                    | Decrease bortezomib to the next lower dose level.                                                                                                                                                             |
| Grade 2 without pain*                   | For patients receiving 1.0 mg/m2/day bortezomib  decrease to 0.7 mg/m2/day. * Will not be considered a DLT                                                                                                    |
| Grade 2 with pain or                    | Hold bortezomib treatment until symptoms have                                                                                                                                                                 |
| Grade 3*                                | resolved to < Grade 1. When toxicity resolves,  reinitiate bortezomib at the next lower dose level. For patients receiving 1.0 mg/m2/day bortezomib, decrease to 0.7 mg/m2/day. *Will not be considered a DLT |
| Grade 4*                                | Discontinue bortezomib *DLT                                                                                                                                                                                   |  
If Grade 3 peripheral sensory neuropathy persists for > 2 weeks, bortezomib should be discontinued. Subjects who discontinue bortezomib will be off protocol therapy.  
If Grade 3 peripheral sensory neuropathy recurs despite bortezomib dose reduction to 1 mg/m 2, the subject should discontinue bortezomib. Subjects who discontinue bortezomib will be off protocol therapy.  
Pulmonary Toxicity There have been rare reports of acute diffuse infiltrative pulmonary disease of unknown etiology such as pneumonitis, interstitial pneumonia, lung infiltration and acute respiratory distress syndrome (ARDS) in subjects receiving bortezomib. For this reason, pulmonary toxicity will be monitored carefully during the study. **Bortezomib should be held in subjects with ARDS that**
may be bortezomib related.
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monitoring

Pulmonary function will be monitored by pulse oximetry. Measurement of pulse oximetry should be documented within 12 hours prior to each dose of bortezomib. A chest radiograph should be performed with the development of respiratory symptoms. In the absence of identifiable causes of respiratory stress, all subjects should demonstrate oxygen saturation on room air of ≥ 94% at sea level (> 90% at high altitude) prior to each bortezomib dose. Pulmonary toxicity due to bortezomib is frequently delayed and can occur up to 3 weeks after the final dose of bortezomib is administered. Pulmonary symptoms such as increased respiratory rate may indicate toxicity related to bortezomib and pulse oximetry should be checked for persistent, unexplained hyperpnea. Respiratory symptoms such as cough, hypoxia and dyspnea should be aggressively evaluated.
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dose reductions and discontinuation of

Bortezomib will be dose reduced to 1 mg/m2 for any resolving Grade 3 pulmonary toxicity (excluding hiccups or voice changes/laryngitis), including hypoxia that is possibly, probably or definitely related to bortezomib. Subjects that experience a Grade 4 pulmonary toxicity possibly, probably or definitely related to bortezomib (excluding hiccups and voice changes/laryngitis) will discontinue bortezomib. Subjects who discontinue bortezomib will be removed from protocol therapy. Subjects re-experiencing the same bortezomib-related qualifying Grade 3 pulmonary toxicity following a single bortezomib dose reduction will be removed from protocol therapy.
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dose reduction for other bortezomib-associated

Bortezomib will be dose reduced to 1 mg/m2 for non-hematological severe toxicities (other than those described above) that are possibly, probably or definitely related to bortezomib. A severe toxicity is defined as any Grade 3 or 4 toxicity as described in Section 5.5. For subjects entering this trial with stable platelets between 50,000-100,000/ μl (those with poor bone marrow recovery after previous treatment) study drug should be held if platelets are Grade 3 as defined in Section 8.4.2. For subjects entering this trial with platelet transfusion dependent, modifications will not be made based on platelet count. Bortezomib doses may not be re-escalated in subjects with dose limiting toxicity. If a specific bortezomib-related toxicity recurs despite one dose reduction, subject will discontinue study.
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5.8 study drug accountability

Study drug must only be used for subjects enrolled in the trial. The investigational site staff must maintain a careful inventory of study drug. Drugs will be distributed to investigational site staff and subjects using FDA guidelines for distribution of investigational agents. Study drug use will be recorded on a Study Drug Inventory Form. This form will contain the following information:
- Subject number and initials for each subject receiving study drug - Date and quantity of DFMO received by the site - Date and quantity of DFMO dispensed - Date and quantity of DFMO returned At each monitoring visit, the clinical monitor will reconcile with the actual inventory of study drug at each site.
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5.9 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
- Any radiotherapy, including systemically administered radioisotopes, unless administered with palliative intent; another site of disease must be available for response evaluation.  
Erythropoietin, blood products, anti-emetics, steroids, and transfusions may be administered at the discretion of the Investigator based on established criteria
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6.0 study procedures and assessments

Prior to consent of the subject, the research coordinator at the lead site will be contacted (via email). This coordinator will then reply with study space availability. If the subject fits all enrollment criteria, the site will again contact the coordinator at the lead site for official enrollment confirmation and unique subject identifier and dose level assignment. In addition, a study enrollment form will be faxed to the coordinator at the lead site. This determination will be made based on subject data (subject qualifies for study) and current subjects enrolled in trial (i.e. how many subjects are currently enrolled). A subject may NOT be enrolled on trial until official approval from the lead site is received. All subjects (or subjects' legal representatives) must provide written informed consent before any study specific assessments may be performed.
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6.2 screening

The Investigator is responsible for keeping a record of all subjects screened for entry into the study and subsequently excluded.  
The following screening procedures must be performed within 21 days **prior to the first dose of** study drug. Studies must be done *after* **last previous treatment for malignancy:**
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 will also be obtained; 2. CT or MRI of measurable disease sites; 3. MIBG or PET scan; 4. Audiogram; 5. Bilateral bone marrow aspirate and biopsy; 6. Additional Optional Bone Marrow-for subjects with additional informed consent bone marrow samples for biological correlatives per section 12.0).
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the following screening procedures must

1. 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 2. Demographics; 3. Vital signs, including temperature, pulse rate, blood pressure, and O2 saturation; 4. BSA calculation (from body weight and height); 5. ECOG Performance status/Lansky Play status (Appendix I); 6. CBC with differential; 7. Serum electrolytes, blood urea nitrogen (BUN), creatinine, bilirubin, LDH, ALT, AST
and ferritin; 8. C-reactive protein (CRP) and Erythrocyte sedimentation rate (ESR) and IL-6 level (IL-6 recommended).  
9. Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid (HVA); 10. Serum or Urine pregnancy test for female subjects of child bearing potential (onset of menses or ≥13 years of age);
11. Concomitant medications/therapies; 12. 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 lead site 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 and drug dose assigned. The lead site will act as the central coordinating body. The subject may not start on study until the lead site has provided official approval of enrollment.
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6.3 treatment phase - cycle

The first cycle will be 21 days in duration. The following procedures must be completed:
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cycle 1 day 1 (procedures

1. Physical examination (including body weight), including documentation of an update of all previous abnormalities, any new abnormalities, and a detailed neurological exam 2. Vital signs, including temperature, pulse rate, blood pressure, and O2 saturation; 3. Review and recording of concomitant medications; 4. Monitoring and documentation of all AEs and review of concurrent illnesses 5. Optional: Urine for Biological Correlates per section 12.0 (additional consent required); 6. Optional: Blood for Biological Correlates per section 12.0 (additional consent required); 7. Dispense drug dosing diary; 8. DFMO dosing begins; 9. Bortezomib administration.
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cycle 1 day 4 (+/-

Subjects will return to the clinic on Cycle 1 Day 4 for Bortezomib and evaluations. The following evaluations will be conducted:
1. History and Physical exam; 2. Vital signs, including temperature, pulse rate, blood pressure, and O2 saturation; 3. Monitoring and documentation of all AEs and review of concurrent illnesses; 4. Review and recording of concomitant medications; 5. DFMO dosing continues daily; 6. Bortezomib administration.
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cycle 1 day 8 (+/-

Subjects will return to the clinic on Cycle 1 Day 8 for evaluations. The following evaluations will be conducted:
1. History and Physical exam; 2. Vital signs, including temperature, pulse rate, blood pressure, and O2 saturation; 3. CBC with differential; 4. Serum electrolytes, BUN, creatinine, bilirubin, ALT, AST; and LDH; 5. Monitoring and documentation of all AEs and review of concurrent illnesses; 6. Review and recording of concomitant medications; 7. DFMO dosing continues daily; 8. Bortezomib administration.
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cycle 1 day 15 (+/-

Subjects will return to the clinic on Cycle 1 Day 15 for evaluations. The following evaluations will be conducted:
1. History and Physical exam; 2. Vital signs, including temperature, pulse rate, blood pressure, and O2 saturation; 3. CBC with differential; 4. Serum electrolytes, BUN, creatinine, bilirubin, ALT, AST; and LDH; 5. Review and recording of concomitant medications; 6. Monitoring and documentation of all AEs and review of concurrent illnesses; 7. DFMO dosing continues daily; 8. Optional: Urine for Biological Correlates per section 12.0 (additional consent required);
9. Optional: Blood for Biological Correlates per section 12.0 (additional consent required).
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6.4 treatment phase - cycles

All Cycles will be 21 days in duration. The following procedures must be completed:
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cycle 2-6 day 1 (+/-

1. History and Physical examination (including body weight), including documentation of an update of all previous abnormalities, any new abnormalities, and a detailed neurological exam (same day);
2. Vital signs, including temperature, pulse rate, blood pressure, and O2 saturation (same day);
3. Review and recording of concomitant medications; 4. Monitoring and documentation of all AEs and review of concurrent illnesses (same day); 5. BSA calculation (from body weight and height); 6. ECOG Performance status/Lansky Play status (Appendix I); 7. CBC with differential; 8. Serum electrolytes, blood urea nitrogen (BUN), creatinine, bilirubin, LDH, ALT, and AST;
9. C-reactive protein (CRP) and Erythrocyte sedimentation rate (ESR), IL-6 level (IL-6 recommended);
10. Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid (HVA); 11. Optional: Urine for Biological Correlates per section 12.0 (additional consent required); 12. Optional: Blood for Biological Correlates per section 12.0 (additional consent required); 13. Collection of previous cycle drug dosing diary and dispensing of new drug dosing diary; 14. Serum or Urine pregnancy test for female subjects of child bearing potential (onset of menses or ≥13 years of age);
15. DFMO dosing continues daily; 16. Bortezomib administration.
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cycle 2-6 day 4 (+/-

Subjects will return to the clinic on Cycle 1 Day 4 for Bortezomib and evaluations. The following evaluations will be conducted:
1. Vital signs, including temperature, pulse rate, blood pressure, and O2 saturation; 2. Monitoring and documentation of all AEs and review of concurrent illnesses; 3. Review and recording of concomitant medications; 4. DFMO dosing continues daily; 5. Bortezomib administration.
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cycle 2-6 day 8 (+/-

Subjects will return to the clinic on Cycle 1 Day 8 for evaluations. The following evaluations will be conducted:
1. History and Physical exam; 2. Vital signs, including temperature, pulse rate, blood pressure, and O2 saturation; 3. CBC with differential; 4. Monitoring and documentation of all AEs and review of concurrent illnesses; 5. Review and recording of concomitant medications; 6. DFMO dosing continues daily; 7. Bortezomib administration.
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cycle 2-6 day 15 (+/-

Subjects will return to the clinic on Cycle 1 Day 15 for evaluations. The following evaluations will be conducted:
1. History and Physical exam; 2. Vital signs, including temperature, pulse rate, blood pressure, and O2 saturation; 3. CBC with differential; 4. Serum electrolytes, BUN, creatinine, bilirubin, ALT, AST; 5. Review and recording of concomitant medications; 6. Monitoring and documentation of all AEs and review of concurrent illnesses. 7. DFMO dosing continues daily.
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end of cycles 2 4

1. MIBG or PET scan (use same as baseline) 2. CT/MRI (use same radiologic method as baseline); 3. Bone marrow biopsy and aspirate if positive at study entry (If the subject has signed informed consent for the use of tissue in the correlative biologic study please send additional samples per section 12.0);
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end of cycles 2 6

1. Audiogram (Audiogram should also be done at any time point for any suspected hearing loss)  
6.5 **Other Schedules:**
1. Additional imaging or assessments may be done if clinically indicated. Type of imaging, type of assessment and timing should be recorded as well as reason for imaging and/or assessment.  
2. Survival will be monitored on an ongoing basis during the study, then every 3 months from the time the subject is off-treatment for a period of 1 year, then yearly for up to five years.
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6.6 protocol treatment completion

Subjects who receive 6 total 21-day treatment cycles will be considered as having completed the protocol. Additional treatment cycles may be delivered in a maintenance setting if there are no safety concerns and there is no disease progression. Maintenance monitoring will be conducted as described for Subsequent Cycles (Section 6.7).
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6.7 subsequent (maintenance) cycles (for

Drug administration will be according to guidelines with premedication as specified. The following evaluations will be performed on the days of each cycle as indicated. Evaluations will be performed prior to dosing unless otherwise indicated. All Cycles will be 21 days in duration. The following procedures must be completed:
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day 1 (+/- 3 day

The following procedures may be performed up to 5 days prior to starting treatment/Day 1 unless otherwise indicated:  
1. History and Physical examination (including body weight), including documentation of an update of all previous abnormalities, any new abnormalities, and a detailed neurological exam (same day);
2. Vital signs, including temperature, pulse rate, blood pressure, and O2 saturation (same day);
3. Review and recording of concomitant medications; 4. Monitoring and documentation of all AEs and review of concurrent illnesses
(same day);
5. BSA calculation (from body weight and height); 6. ECOG Performance status/Lansky Play status (Appendix I); 7. CBC with differential; 8. Serum electrolytes, blood urea nitrogen (BUN), creatinine, bilirubin, LDH, ALT, and AST;
9. Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid (HVA); 10. Serum or Urine pregnancy test for female subjects of child bearing potential (onset of menses or ≥13 years of age).  
11. DFMO dosing continues daily; 12. Bortezomib administration.
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day 4 (+/- 1 day

Subjects will return to the clinic on Cycle 1 Day 4 for Bortezomib and evaluations. The following evaluations will be conducted:  
1. Vital signs, including temperature, pulse rate, blood pressure, and O2 saturation; 2. Monitoring and documentation of all AEs and review of concurrent illnesses; 3. Review and recording of concomitant medications; 4. DFMO dosing continues daily; 5. Bortezomib administration.
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cycle day 8 (+/- 1

Subjects will return to the clinic on Cycle 1 Day 8 for evaluations. The following evaluations will be conducted:
1. History and physical exam; 2. Vital signs, including temperature, pulse rate, blood pressure, and O2 saturation; 3. CBC with differential; 4. Monitoring and documentation of all AEs and review of concurrent illnesses; 5. Review and recording of concomitant medications; 6. DFMO dosing continues daily; 7. Bortezomib administration.
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6.8 off-therapy / 30 day

Subjects will return to the clinic 30 (+7) days after the last dose of DFMO, and the following evaluations will be conducted:
1. History and Physical examination (including body weight), including documentation of an update of all previous abnormalities, any new abnormalities, and a detailed neurological exam; 2. ECOG Performance status/Lansky Play status (Appendix I); 3. Vital signs, including temperature, pulse rate, blood pressure, and O2 saturation; 4. CBC with differential; 5. Serum electrolytes, BUN, creatinine, bilirubin, LDH, ALT, AST; 6. Urine for Vanillylmandelic Acid (VMA) & Homovanillic Acid (HVA); 7. Review and recording of concomitant medications; 8. Monitoring of AEs and review of concurrent illnesses; 9. Collect previous cycles drug dosing diaries (if early withdrawal); 10. MRI/CT; MIBG or PET scan; if not already obtained within the previous two **cycles.**
11. Bone marrow biopsy and aspirate (if positive at study entry); if not already **obtained**
within the previous two **cycles;**
12. Audiogram (if clinically indicated). Any subject with a suspected study drug-related toxicity at the follow-up visit must be followed until the related event(s) has resolved to baseline or ≤ grade 2. 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. Subjects that have started a new anti-cancer treatment since going off DFMO will be censored from any further new AE collection at the date of starting the new therapy but will continue survival follow up.
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6.9 survival follow-up

Subjects will be followed for long term survival by contact with parent or treating institution to confirm survival at the following time points (time from last dose of study drug):  
− 3 months
− 6 months
− 9 months − 1 year
− Then Yearly after (up to 5 years total)
Follow up will continue for five (5) years or until subject death or subject is lost to follow up (per definition in section 9.0). The follow-up results including date of any progression of disease, survival status or date of death will be recorded on the appropriate page of the CRF, as well as in the subject's source documentation.
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7.0efficacy assessments 7.1 tumor assessments/scans

Tumor assessments/imaging studies must be obtained at baseline and at the end of the second and fourth cycles, and again after every other cycle. 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 for any reason should have a final end of study disease-specific assessment done.
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7.2 scan submission

All required study scans (CT's, MRI's, and MIBG's) will be reviewed by central radiology. All study required scans should be sent on disc. Scans will be sent as a batch at the end of each subject's therapy or sooner if any concern for progression 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
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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, by MIBG or 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 status described below. Response Criteria for Subjects with Solid Tumors: This study will use the (RECIST) Response Evaluation Criteria in Solid Tumor (version 1.1) from the NCI. Key points are that a maximum of 5 target lesions are identified and that changes in the *largest* diameter (unidimensional measurement) of the tumor lesions are used in the RECIST v1.1 criteria.  
- 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 (CT scan slice thickness no greater than 5 mm). The investigator will identify up to 5 measurable lesions to be followed for response. Previously irradiated lesions must demonstrate clear evidence of progression to be considered measurable.  
- Measurable Disease Response will be assessed at end of Cycle 2 and then at least every other cycle
- 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.  
- **Complete Response (CR):** Disappearance of all target and non-target lesions. - **Very Good Partial Response (VGPR):** Greater than 90% decrease of the disease measurement for CT/MRI lesions, taking as reference the disease measurement done to confirm measurable disease at study entry. Non-target CT/MRI lesions stable to smaller in size.  
- **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 enrollment.  
No new lesions or progression of any non-target measurable lesion.  
- **Stable Disease (SD):** Neither sufficient decrease to qualify for PR or sufficient increase to qualify for PD taking as reference the smallest disease measurement since the treatment started.  
- **Progressive Disease (PD):** At least a 20% increase in the sum of the disease measurements for measurable lesions, taking as reference the smallest disease measurement recorded since the start of treatment (nadir), or the appearance of one or more new lesions.  
Response Criteria for Subjects with Bone Marrow Disease: - 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 consecutive bilateral bone marrow aspirates and biopsies done at least three weeks apart after study entry.  
- **Progressive disease**: Tumor seen on morphology on two consecutive bilateral bone marrow aspirates and biopsies 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 any amount of tumor in the bone marrow by morphology that does not meet criteria for either complete response or progressive disease.
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response criteria for subjects with

- Subjects who have a positive MIBG or PET scan at the start of therapy will be evaluable for MIBG/PET response. The use of I123 for MIBG imaging is recommended for all scans. If this radioisotope is unavailable at the treating institution, the use of the same radioisotope for all MIBG scans for an individual subject is strongly encouraged.  
- **Complete response** = complete resolution of all MIBG/PET positive lesions - **Partial response** = resolution of at least one MIBG/PET positive lesion, with persistence of other MIBG/PET positive lesions.  
- **Stable disease** = no change in MIBG/PET scan in number of positive lesions (includes subjects who have same number of positive lesions but decreased intensity)
- **Progressive disease** = Development of new MIBG/PET positive lesions The intensity of MIBG/PET uptake is not to be considered in the above institutional evaluation.
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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. Serial results of bone marrow aspirates, biopsies and urinary catecholamines will be reviewed for responding subjects to confirm response or lack of progression.
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8.0 adverse event reporting

Common Terminology Criteria for Adverse Events (CTCAE)
This study will utilize the version 4.0 of the CTCAE of the National Cancer Institute (NCI) for toxicity and performance reporting. A copy of the CTCAE version 4.0 can be downloaded from the NCI website at: http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.
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8.1 definitions 8.1.1 adverse event

An *adverse event* is any untoward medical occurrence in a patient 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.
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8.1.2 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, e.g., the investigator's brochure.
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8.1.3 serious adverse event

A *serious adverse event* is any untoward medical occurrence that at any dose:
- 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 - Is 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.
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8.2 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 Grade 2 or higher (per CTCAE version 4.0) adverse events, whether serious or not, will be described in the source documents and the adverse event page of the case report form. All new events (Grade 2 or higher), 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 - 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 lead site immediately, and complete the Serious Adverse Event page and the Final Visit section of the case report form)
Adverse events, regardless of suspected cause, will be collected for 30 days following the last treatment or until a new treatment is started. 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.
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8.3 expedited reporting of adverse

All fatal, life–threatening, or dose limiting **adverse events must be reported to the lead site**
immediately by telephone, fax, or e-mail within 24 hours **of discovery** as well as to the appropriate regulatory authorities (local IRB). The lead site, NMTRC, will then report directly to the safety officer, FDA if required, and KC Pharma as appropriate. If full information is not known, additional follow-up by the Investigator will be required.  
All other serious adverse events must be reported to the lead site by telephone, fax, or email within 5 days **of discovery** as well as to the appropriate regulatory authorities (local IRB).  
The lead site will then report directly to the safety officer, FDA if required, and KC Pharma as appropriate. 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.
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nmtrc reporting contact

Please report all expedited reports to:
Alyssa VanderWerff and Genevieve Bergendahl, RN NMTRC Tel: (616) 267-0327 E-Mail: Alyssa.VanderWerff@helendevoschildrens.org E-Mail: Genevieve.Bergendahl@helendevoschildrens.org
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8.4 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
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8.5 grading of hematologic adverse

Subjects who are platelet transfusion dependent at study entry will not have grading of platelet levels. Subjects who have bone marrow involvement at study entry will not have grading of any hematologic levels.
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8.6 relatedness to study drug

The Investigator must attempt to determine if an adverse event is in some way related to the use of the study drug and define an attribution category. This relationship should be described as follows:  
| RELATIONSHIP                                                                                                                                                                                                                                                                                                                                                                               | ATTRIBUTION                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              | DESCRIPTION                                                                                                                                                                                                                                                                                                                                                                                                                          |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| The AE is clearly NOT related to the intervention. The event is clearly due to causes distinct from the use of the  study drug, such as a documented pre-existing condition, the  effect of a concomitant medication, or a new condition which,  based on the pathophysiology of the condition, and the  pharmacology of the study drug, would be unrelated to the use  of the study drug. |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                      |
| Unrelated                                                                                                                                                                                                                                                                                                                                                                                  |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                      |
| Unrelated to  investigational                                                                                                                                                                                                                                                                                                                                                              |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                      |
| agent/intervention                                                                                                                                                                                                                                                                                                                                                                         | The AE is doubtfully related to the intervention. Adverse event does not have temporal relationship to  intervention, could readily have been produced by the  subject's clinical state, could have been due to environmental  or other interventions, does not follow known pattern of  response to intervention, does not reappear or worsen with  reintroduction of intervention.                                                                                                                     |                                                                                                                                                                                                                                                                                                                                                                                                                                      |
| Unlikely                                                                                                                                                                                                                                                                                                                                                                                   | The AE may be related to the intervention. The event follows a reasonable temporal sequence from  administration of the study drug and the event follows a  known response pattern to the study drug BUT the event could  have been produced by an intercurrent medical condition  which, based on the pathophysiology of the condition, and the  pharmacology of the study drug, would be unlikely related to  the use of the study drug OR the event could be the effect of a  concomitant medication. |                                                                                                                                                                                                                                                                                                                                                                                                                                      |
| Possible                                                                                                                                                                                                                                                                                                                                                                                   | The AE is likely related to the intervention. The event follows a reasonable temporal sequence from  administration of the study drug, the event follows a known  response pattern to the study drug AND the event cannot have  been reasonably explained by an intercurrent medical  condition OR the event cannot be the effect of a concomitant  medication.                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                      |
| Related to                                                                                                                                                                                                                                                                                                                                                                                 |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                      |
| investigational                                                                                                                                                                                                                                                                                                                                                                            |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                      |
| agent/intervention                                                                                                                                                                                                                                                                                                                                                                         | Probable                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 | The AE is clearly related to the intervention. The event follows a reasonable temporal sequence from  administration of the study drug, the event follows a known  response pattern to the study drug and based on the known  pharmacology of the study drug, the event is clearly related to  the effect of the study drug. The adverse event improves upon  discontinuation of the study drug and reappears upon repeat  exposure. |
| Definite                                                                                                                                                                                                                                                                                                                                                                                   |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          |                                                                                                                                                                                                                                                                                                                                                                                                                                      |