MSKCC THERAPEUTIC/DIAGNOSTIC PROTOCOL

MSKCC THERAPEUTIC/DIAGNOSTIC PROTOCOL

Anti-GD2 3F8 Monoclonal Antibody and GM-CSF for High-Risk Neuroblastoma

Princ ipa l Investiga tor/Departme nt:	Xxxxx X. X ushner, MD	Pediatrics
Co-Princ ipa l Inve stigator(s)/Departme nt:	Nai-Kong X. Xxx xxx, MD, P hD	Pediatrics
Inve stigator(s)/Departme nt:	Elle n X. Xxxx, MD, P hD Xxxxxx x Xxxxx, MD Xxxxxx n X. Xxxx rts, MD Xxxxx Xxxxxxxx ya, P hD	Pediatrics Pediatrics Pediatrics Epidemiology a nd Biostatistics
Conse nting Professiona l(s)/Depa rtme nt:	Xxxx x X. Xxxx, MD, P hD Nai-Kong X. Xxx xxx, MD, PhD Xxxxx X. X xxxxxx, MD Xxxxxx x Xxxxx, MD Xxxxxx n X. Xxxx rts, MD	Pediatrics Pediatrics Pediatrics Pediatrics Pediatrics

Ple as e Note: A Conse nting Profe ssional mus t have comple tedthe xxxxxxx xx Human Subje cts Educatio n and Ce rtificatio n Prog ram.

Me moria l Sloa n-Kettering Ca nce r Center 0000 Xxxx Xxxxxx

New York, New York 10065

Table of Contents

1.0 PROTOCOL SUMMARY AND/OR SCHEMA 3

2.0 OBJECTIVES AND SCIENTIFIC AIMS 4

3.0 BACKGROUND AND RATIONALE 4

4.0 OVERVIEW OF STUDY DESIGN/INTERVENTION 7

4.1 Design 7

4.2 Intervention 7

5.0 THERAPEUTIC/DIAGNOSTIC AGENTS 8

6.0 CRITERIA FOR SUB JECT ELIGIBILITY 10

6.1 Subject Inclus ion Cr iteria 10

6.2 Subject Exclus ion Cr iteria 10

7.0 RECRUITMENT PLAN 10

8.0 PRETREATMENT EVALUATION 11

9.0 TREATMENT/INTERVENTION PLAN 11

10.0 EVALUATION DURING TREATMENT/INTERVENTION 12

11.0 TOXICITIES/SIDE EFFECTS 14

12.0 CRITERIA FOR THERAPEUTIC RESPONSE/OUTCOME ASSESSMENT 15

13.0 CRITERIA FOR REMOVAL FROM STUDY 15

14.0 BIOSTATISTICS 16

15.0 RESEARCH PARTICIPANT REGISTRATION AND RANDOMIZATION PROCEDURES16 15.1 Research Participant Registration 16

16.0 DATA MANAGEMENT ISSUES 16

16.1 Quality Assurance 16

16.2 Data and Safety Monitor ing 17

17.0 PROTECTION OF HUMAN SUBJECTS 17

17.1 Privacy 17

17.2 Serious Adverse Event (SAE) Reporting 18

17.2.1.......................................................................................................................................... 18

18.0 INFORMED CONSENT PROCEDURES 19

19.0 REFERENCES 19

20.0 APPENDICES 25

ACRON YM S and ABBREVIATIONS

ADCC, antibody- dependent celular cytotoxic ity AE, adverse event

ALT, alanine aminotransferase

ANC, absolute neutrophil count

ASCT, autologous stem-cel transplantation AST, aspartate aminotransferase

BM, bone marrow

BUN, blood urea nitrogen CBC, complete blood count CT, computed tomography CR, complete remission

CRDB, Clinical Research Database DSM, Data and Safety Monitor ing

DSMB, Data and Safety Monitoring Board DSMC, Data and Safety Monitoring Committee FDA, Food and Drug Administration

GM-CSF, granulocyte-macrophage colony-stimulating factor HAMA, human anti-mouse antibody

ICH, Internationa lConference of Harmonization IND, investigationa l new drug

IRB/PB, Institutiona lReview Board/Privacy Board iv, intravenous

MIBG, metaiodobenzylguanidine MoAb, xxxxx xxxx lantibody MRD, minima l residua l disease MRI, magnetic resonance imaging

MSKCC, Memoria l Xxxxx-Xxxxxxxxx Cancer Center NB, neuroblastoma

NCI, National Cancer Institute NIH, National Institutes of Health OS, overall surviva l

PD, progressive disease

PET, positron emiss ion tomography PI, Principal Investigator

PFS, progression-free surviva l

PPR, Protocol Participant Registration q, every

QA, quality assurance

RA, Research Authorization RSA, research study assistant SAE, serious adverse event sc, subcutaneous

VGPR, very good partia lremiss ion

1.0 PROTOCOL SUMMARY AND/OR SCHEMA

Efficacyof a nti-GD2 xxxxx xxxx l a ntibody (MoAb) therapy is prove n. This is a single-arm, ope n- labe l, open access study to provide the a nti- GD2 murine IgG3 MoAb 3F8 combined with

gra nuloc yte-ma crophage colony stimulating factor (GM-CSF) to patients with high-risk

ne uroblastoma (NB). This immunotherapy has shown efficacya ga inst minima l residual disease (MRD) in suc h patie nts. 3F8 is not yet lice nsed by the Food a nd Drug Administra tion (FDA) and commerc ia lly a va ilable but is listed on the FDA site as xxxxx n designa xxx.1a

2.0 OBJECTIVES AND SCIEN TIFIC AIM S

The objec tive of this ope n access protocol is to provide 3F8 to patie nts with high-risk NB. The treatme nt IND will collect additiona l usa ge, tolerability a nd safetydata.

3.0 BACK GROUND AND RATIONALE

3.1 Dise ase background: NB is the most common extracrania l solid tumor of childhood; 50-60% of patie nts prese nt with an unresectable prima ry tumor a nd metastases in bone marrow (BM).1 Inte nsive induction che motherapy a nd aggressive surgery ha ve improved re mission rates in xxxxx xxxxx nts2-4; results ha ve been less impressive in adolesce nts a nd adults in whom NB is espec ia lly c he moresistant.5,6 Realization of an effective xxxxxx xx for eradicating MRD ha s remained a formidable c halle nge. Post-surgica l use of loca l radiotherapy he lps control MRD in the primary site.7 Mye loabla tive therapy with a utologous ste m-ce ll tra nspla ntation (ASC T) has been the most common approac h for eradicating MRD in dista nt sites. The vita min A derivative 13-cis-retinoic ac id (isotretinoin) he lped to prolong relapse- free surviva l in a ra ndomized study.8 Immunotherapy using the a nti- GD2 c h14.18 MoAb plus GM-CSF and interle ukin-2 in a lterna ting c ycles improved outcome in a ra ndomized study.9 The se results ha ve made treatme nt with isotre tinoin a nd anti-GD2 MoAb sta ndard of care for high-risk NB.

3.2 Rationale for immunothe rapy : Various strategie s have undergone c linica l testing to induce or a ugme nt immune- media xxx attack aga inst ca nce x. Xxxx ver, few c linica l trials have used xxxxx xxxx l a ntibody-targeted immunothe rapy a ga inst solid tumors in childre n or adults, espec ia lly with antibody-depe nde nt ce llular c ytotoxic ity (ADCC) as t he princ ipa l unde rlying immune cytotoxic mec ha nism. Also, an antineoplastic role for gra nuloc ytes has received sca nt atte ntion.10 A treatme nt progra m combining 3F8 a nd GM-CSF thus represe nts an a ttempt to he lp fill a gap in the e merging fie ld of immunotherapy.

3.3 Rationale for 3F8 : 3F8 is a murine IgG3 MoAb tha t is well suited for targeted immunotherapy. The reasons are se xxxx x. First, the inte nsive c he mothe rapy required to produce the minima l disea se state optima l for immunotherapy in NB patie nts res ults in prolonged se vere lymphope nia.11 This setting is unfa vorable for active immunotherapy but a llows passive immunotherapy since the patie nt will be unable to reject alloge ne ic, xe noge ne ic, or ge netica lly- engineered a ntibodie s. Second, 3F8 recognizes the ga nglioside GD2.12 This targe t a ntige n is expressed at high density on NB (but ha s restric xxx distribution in xxxxx l huma n tissues), is not modulated from the cell surface whe n bound by antibodies, a nd is ge netica lly stable (unlike tumor- assoc ia xxx a ntige ns suc h as immunoglobulin idiotypes on lymphoma ce lls).13-15 Third, scintigraphy using 131I-3F8 confirms tha t 3F8 loca lizes se lec tive ly to GD2(+) tumor deposits in patie nts.16 The exce lle nt targeting pote ntia l of 3F8 was evidenced by its high tumor to non-tumor ratio, the high perce nt injected dose per gra m uptake, a nd the limited, if any, nonspecific liver a nd spleen

uptake.16,17 Fourth, 3F8 media tes destruction in vitro of GD2(+) huma n solid tumor ce lls by huma n comple me nt18 and by human lymphoc ytes, cultured monocyte s, and ne utrophils.19-21 Fina lly, the capac ity of 3F8 to activa te comple me nt on NB ce lls (which lack decay acce xxxx xxxx factor22) ra ises the possibility, not only of comple me nt- mediated lysis in patie nts, but also of the re lease of

comple me nt fragme nts that ma y e lic it an infla mma tory influx of gra nuloc ytes capable of lysing 3F8-

labe led tumor ce lls. F urthe rmore, the deposition of comple me nt fragme nts C3b a nd iC3b on NB ce lls may enha nce ADCC beca use the receptor for iC3b – variously ca lled Mac-1, CR3, CD11b/CD18, or άMβ2-inte grin – is a key ele me nt in a nti- GD2 MoAb- mediated tumor cell kill by ne utrophils,23,24 whic h are the most abunda nt circulating c lass of le ukoc yte.

3.4 Rationale for GM -CSF: GM-CSF has the pote ntia l for a mplifying 3F8 a ntitumor activity in patie nts via effects on gra nuloc yte s a nd tissue -based macropha ges. Reasons for combining GM-CSF with 3F8 include the following. F irst, gra nuloc yte production is only tra nsie ntly suppressed with che motherapy a nd GM-CSF increases numbers of c ircula ting neutrophils a nd eosinophils, does not affect comple me nt le ve ls, a nd is well tole rated compared to other cytokines suc h as interleukin-

2.2 5 Second, gra nuloc ytes from patie nts receiving c he motherapy and from xxxxx l volunte ers are effective in mo unting ADCC a ga inst NB ce lls via non-oxida tive mec ha nisms, a nd GM-CSF enha nces this cytotoxicity.24,26-30 Third, eosinophilic infiltra tion of some ca ncers has fa vorable prognostic significa nce, a nd eosinophils e xhibit pote nt a ntitumor activity in animal mode ls.31,32 Fourth, activated monoc ytes- mac ropha ges effic ie ntly pha goc ytose N B ce lls, a nd e xposure in vitro or in vivo to GM- CSF primes monoc ytes- macropha ge s for grea ter a ntineoplastic cytotoxic ity.33-38 Fifth, GM-CSF enha nce s the prolife ration, ma turation, a nd function of a ntige n-prese nting ce lls, inc luding antige n processing a nd prese nta tion by macropha ges a nd de ndritic ce lls25,39,40 - effects that might promote induction, or antitumor activity, of an idiotypic network. 41-44 Fina lly, GM-CSF is not a growth factor for NB ce lls in vitro.45

3.5 GM -CSF-me diatedactivationof ne utrophils : Neutrophils are the predomina nt c lass of circula ting le ukoc ytes; c he motherapy only tra nsie ntly decreases the ir numbers a nd large ly spares the ir c ytotoxic capabilities. The se features are adva nta geous for immune-based attack a ga inst cance r.10 We and others ha ve shown tha t Mac-1 (CD11b/CD18), FcRII, a nd Fc RIII are required for optima l ADCC in a syste m using huma n ne utrophils as effectors, huma n N B ce lls as targe ts, a nd c linica lly active a nti- GD2 MoAbs as mediators.23,24 Effic ie nt tumor-ce ll kill occ urs despite the large size of the targets (whic h precludes a role for pha goc ytosis) a nd the ir relative resista nce to reac t ive oxyge n species.29 GM-CSF enha nceme nt of ADCC in this syste m corre la tes with upre gulation of Mac-1 and with incre ased e xoc ytosis of azurophil (primary) gra nules whic h conta in cathepsin G a nd defe nsins capable of lysing N B ce lls.30 To eluc idate the c ytotoxic mec hanisms of anti-GD2 MoAb- media xxx ADCC, we used blocking MoAbs, ne utrophils de void of Mac-1 (from donors with le ukoc yte adhesion defic ie nc y), a nd ne utrophils that do not generate reactive oxyge n species (from donors with c hronic gra nulomatous disea se),23 while othe rs used blocking MoAbs, e lec tron microscopy, a nd ne utrophils from hea lthy donors as well as from patie nts.24 Activa tion of ne utrophils by GM-CSF is correlated with improved anti-NB effects of treatme nt with a nti-GD2 MoAb.46

3.6 Clinical e xpe rience with 3F8 plus GM -CSF: The optima l method of using GM- CSF with the aim of enha ncing ADCC in patie nts with solid tumors has only rece ntly been eluc xxx xxx. In the past, we c hose to administe r GM-CSF by a 2- hr iv infusion (followed by a 1-hr interval before starting 3F8 infusion) because GM-CSF disappea rs rapidly (<2-3 hr) from the blood with that schedule.47,48 We wished to a void the prolonged (>12 hr) bioa ctive le ve ls in blood assoc ia xxx with the

subc uta neous (sc) route; our concern was tha t high serum le ve ls of GM-CSF might impede gra nuloc yte trafficking into tissues.49 Xxxx ve r, for circulating tumor ce lls in BM or blood, this conce rn is dwarfed by the more pote nt antitumor effects fromprolonged c ytokine e xposure following subc uta neous injection.25

The 03-077 study of 3F8+sc GM-CSF immunothe rapy plus isotretinoin e nrolled patie nts in 1st comple te/ve ry good partia l re mission (CR/VGP R) (n=85), patie nts with primary refractory NB in BM (n=79), a nd patie nts in >2 CR/VGP R (n=101). Sa lie nt findings included:

1) Ac ute toxicitie s were ma na geable which a llowed outpa tie nt treatme nt. This finding sta nds in xxxxx contrast with studie s using other anti-GD2 MoAbs either alone or in combination with interle ukin-2, where treatme nt is inpatie nt, often in an intensive care unit, with complic ations suc h as capillary leak syndrome.9

2) For patie nts in 1st CR/VGPR, prior mye loabla tive therapy + ASCT had no impac t on 5- year progression-free surviva l (PFS) 62% or xxx xxxx surviva l (OS) 81%.50 This supports the welcome possibility of a voiding the risk of significa nt ac ute a nd long-term toxic ities of mye loabla tive therapy.

3) For patie nts with prima ry refractory NB, CR rate in BM by histology was 87% a nd CR rate of metaiodobe nzylgua nidine (MIBG) scans was 38%. These results vividly doc ument the anti-NB activity of a nti- GD2 MoAb immunotherapy, i.e., the possibility of overcoming histologica lly- and/or radiologica lly- visible c he moresista nt NB in BM using an a nti- GD2 MoAb regime n tha t has ma na geable toxic ity a nd is administe red outpatie nt.

4) Patie nts treated in >2 CR/VGPR achie ved 48- month PFS 33% a nd OS 53%, with no adve rse impac t if patie nts had received anti-GD2 MoAb a nd isotre tinoin as part of initial (pre-re lapse) therapy a nd with only 2/35 very long-term relapse- free survivors having received ASCT as part of xxxxx xx. These results are noteworthy, give n tha t studies show <5% long-term OS after relapse51-55 and a nti- GD2 MoAb, isotretinoin, a nd ASCT are sta nda rd treatme nts for newly-dia gnosed patie nts.56 Inde ed, the results support a c ha nge in mind-set: no longe r should relapse of high-risk NB be considered “invariably xxxxx l”,57 but rather a cura tive xxxxxx xx should be applied.

5) Positive MRD after 2 c yc le s of 3F8+scGM-CSF was a significa ntly adverse indepe nde nt prognostic factor for all groups of patie nts.50,58,59 This finding should be taken into account whe n determining subseque nt therapy in a given patient.

6) Corre lative studie s highlight the a nti- neoplastic pote nc y of mye loid effectors whe reas lymphoc ytes are the effectors usua lly he ra lded in ca ncer immunothe rapy.

7) GM-CSF by sc route is associated with be tter PFS and OS tha n by iv route,60 as used in the predecessor study of 3F8+ivGM-CSF. 61

3.7 Is otre tinoin (13 -cis-retinoic acid) was shown in a ra ndomized na tiona l study to decrease the risk of relapse in patie nts treated in complete re mission.8 This age nt has subseque ntly become sta ndard of care for N B patie nts in complete re mission. It will be used in this protocol after patie nts are evalua xxx for response (a nd for toxic ity) to 3F8/GM-CSF.

3.8 Inte rpre tatio ns and implicatio ns The ease of administra tion of the treatme nts (a subc uta neous injection, whic h is give n at home, a nd a 30- minute intra ve nous infusion in the

outpatie nt c linic), plus the ir tra nsie nt ac ute side-effects, are compatible with wide spread usa ge of this regimen be yond our hospita l – a strong positive factor in furthering product de ve lopme nt.

In addition to the outpatie nt treatme nt with a nti- GD2 MoAb, the Memorial S loa n-Kette ring Cancer Center (MSKCC) ne urobla stoma treatme nt progra m remains unique in that ASC T is not included a nd the immunothe rapy is a va ilable for patie nts in >2nd C R/VGP R. Results to date, as summa rized in section 3.6, are highly e ncouraging a nd ma y have implications world-wide.

4.0 OVERVIEW OFSTUD Y DESIGN/IN TERVENTION

4.1 Des ign

This treatme nt use s 3F8/GM-CSF and isotre tinoin for: Group 1 patie nts are in 1st CR/VGP R; Group 2 patie nts are in a ≥2nd CR/VGP R; a nd Group 3 patie nts ha ve prima ry refractory NB in BM. All patie nts will receive 3F8/GM-CSF through 24 months.

4.2 Inte rve ntion

Road Map/Sc he ma for Gro up 1 (1st CR/VGP R) a nd Gro up 2 (≥ 2 nd CR/VGPR) patie

nts: Cyc le 1 3F8 (iv) + GM-CSF subcuta neous (sc) (1 wk) 2-4-wk inte rva l

Cyc le 2 3F8 (iv) + GM-CSF (sc) (1 wk)

2-4-wk inte rva l* – oral isotretinoin x14 da ys Cyc le 3 3F8 (iv) + GM-CSF (sc) (1 wk)

2-4-wk inte rva l – oral isotretinoin x14 da ys Cyc le 4 3F8 (iv) + GM-CSF (sc) (1 wk)

6-8-wk inte rva l – oral isotretinoin x14 da ys on, 14 da ys off, 14 da ys on

Cyc le 5 3F8 (iv) + GM-CSF (sc) (1 wk)

6-8-wk inte rva l – oral isotretinoin x14 da ys on, 14 da ys off, 14 da ys on (6th cyc le) Cyc le 6 3F8 (iv) + GM-CSF (sc) (1 wk)

6-8-wk inte rva l

Cyc le 7 3F8 (iv) + GM-CSF (sc) (1 wk)

Continue with 6-8-wk inte rva ls through 24 months from 1st dose of 3F8.

* assessme nt of BM status by sta ndard histology

Road Map/Sc hema for Gro up 3 patients (BM positive): The break between end of a cyc le of 3F8/GM-CSF and start of ne xt c yc le is approximate ly 2-to-4-weeks through 4 c yc les after achie veme nt of CR in BM; subseque nt breaks are ~6-8 weeks. Please see roadmap be low for a patie nt achie ving CR in BM after c yc le 1.

Cyc le 1 3F8 (iv) + GM-CSF (sc) (1 wk) 2-4-wk inte rva l* – BM ne ga tive

Cyc le 2 3F8 (iv) + GM-CSF (sc) (1 wk)

2-4-wk inte rva l* – oral isotretinoin x14 da ys

Cyc le 3 3F8 (iv) + GM-CSF (sc) (1 wk)

2-4-wk inte rva l – oral isotretinoin x14 da ys Cyc le 4 3F8 (iv) + GM-CSF (sc) (1 wk)

2-4-wk inte rva l – oral isotretinoin x14 da ys Cyc le 5 3F8 (iv) + GM-CSF (sc) (1 wk)

6-8-wk inte rva l – oral isotretinoin x14 da ys Cyc le 6 3F8 (iv) + GM-CSF (sc) (1 wk)

6-8-wk inte rva l – oral isotretinoin x14 da ys on, 14 da ys off, 14 da ys on (6th cyc le) Cyc le 7 3F8 (iv) + GM-CSF (sc) (1 wk)

6-8-wk inte rva l

Continue with 6-8-wk inte rva ls through 24 months from 1st dose of 3F8.

* assessme nt of BM response by sta nda rd histology

The treatme nt sc hedule ma y require minor adjustme nt as c linica lly indica xxx or due to c irc umsta nce (e.g., due to Pediatric Day Hospita l closure for holida ys or due to inc leme nt weathe r). Patie nts can complete the missed day of 3F8 the following week, so the y receive a full 5-day cyc le. GM-CSF will be continued through the 3F8 make-up day at the 500 mc g/m2 dose.

5.0 THERAPEUTIC/DIAGNOSTIC AGENTS

5.1 3F8 xxxxx xxxx l a ntibody (IND number of BB-IND-8449)

m3F8 is an investigationa l new drug de ve loped at MSKCC and ma nufac tured by a qua lified CMO in complia nce with GMP guida nce appropria te for phase I c xxxxxx x xxxx ls. The final produc t specifications, a na lytical test me thods a nd stora ge conditions/stability are described in the CMC section of this IND. Brie fly, m3F8 is purified by column chromatography followed by viral filtra tion a nd fina le sterile filtra tion. The fina l produc t is tested to assure tha t it is free of aggre gates, nuc leic ac id, murine viruses, bacteria, fungi, mycoplasma a nd pyroge ns. m3F8 is also tested for a ntibody specificity. For iv administration, 3F8 should be diluted into 10 ml 5% huma n serum a lbumin a nd millipore (0.2 µm) filtered before use. Route of Administra tion: iv infusion.

5.2 Sargra mostim (YEAST-DERIVED HUMAN RECOMBINANT GRANULOCYTE- MACROPHAGE COLONY STIMULATING FACTOR; GM-CSF)

5.2.1 Source and Pharmacology: Yeast derived recombina nt huma n GM-CSF.

5.2.2 Supplier: LEUKINE (sargra mostim) is distributed by Ge nzyme Corpora tion, Cambridge, MA a nd ma nufac tured by Ba yer HealthCare P ha rmace utica ls, Seattle, WA.

5.2.3 Formulation and Stability: Sargra mostim is a va ilable as a sterile, white, preserva tive- free, 250 mc g single use via l of lyophilized powder or a 500 mc g/1 mL injectable solution.

The 250 mcg via ls require aseptic reconstitution with 0.5 mL Sterile Water for Injection, USP (without preserva tive ). During reconstitution, the Sterile Water for Injec tion, USP should be directed at the side of the via l and the xxxxx nts ge ntly swirled to a void foa ming

during dissolution. Avoid e xce ssive or vigorous agitation; do not shake. The reconstituted sargra mostim solutions are clear, colorless, isotonic with a pH of 7.4 ± 0.3, a nd conta in 500 mc g/mL of sargra mostim. The single-use 250 mc g via ls contain no a ntibacteria l preserva tive, and the refore should be administered as soon as possible, and within 6 hours following reconstitution. The y are inte nded for single use only a nd should not be re-entered or re used. Do not sa ve a ny unused portion for later administration. Do not use be yond the e xpira tion date printed on the via l.

Via xx xxxxx ining 500 mc g/1 mL of sargra mostim are already in solution and are multiple dose vials. For subcuta neous administration, further dilution is not required. Liquid sargra mostim may be stored for up to 20 da ys at 2-8°C once the vial has been entered. Disca rd a ny remaining solution after 20 da ys.

Store sargra mostim powder, or reconstituted solutions, under refrigeration at 2-8°C (36-46°F); do not free ze or shake.

Aseptic tec hnique should he employed in the preparation of all sargra mostim solutions. To assure correct conce ntration following reconstitution, care should be e xerc ised to eliminate any air bubbles from the needle hub of the syringe used to prepare t he dilue nt. Parenteral products should be inspec xxx visually for particula te matter a nd discolora tion prior to administration whe ne ver solution a nd conta iner permit.

5.2.4 Route of Administration: Subcuta neous

5.3 13-cis-Re tinoic Acid (NDC #0000-0000-00,0004-0169, 0004-0156-01-

ISOTRETINOIN, ACCUTANE)

5.3.1 Source and Pharma cology: The e xact mec ha nism of retinoic acid- induced matura tion of tumor ce lls is not known. In ne uroblastoma cell lines it has been shown to down regulate MYCN RNA and protein e xpression, a nd suc h down re xxxx tion correlates with the ability of isotre tinoin to induce tumor cell growth arrest. Rece nt studie s using ge ne tra nsfection direc tly implica te down re gulation of MYCN expre ssion by isotretinoin as a key eve nt in achie ving susta ined arrest of ne uroblastoma cell prolife ration. Retinoic acid also appears to enhance xxxxx l hematopoietic differe ntiation by increasing the responsive ness of mye loid a nd erythroid proge nitor ce lls to the action of mye loid colony stimulating activity and erythropoie tin, respective ly. Me tabo lism: Retinoic ac id is 99.9% bound in plasma (almost entire ly to albumin) a nd has a ha lf- life of 10-20 hours. The major me tabolite is 4-

oxoisotretinoin, a nd e xcre tion is in the urine a nd feces. A single oral dose of 100mg/m2

isotretinoin will produce peak plasma leve ls of 1-2mM. The mea n peaktime as 3.2 hours after 80mg ora lly, with a termina l t½ of 10 to 20 hours. Administering 160 mg/m2/day to childre n after ASCT ha s beenshown to achie ve 13-c is-retinoic acid le ve ls of 5 to 7 micromula r.

5.3.2 Formulation a nd Stability: Isotretinoin, whic h is the 13-cis xxxxx r of retinoic ac id, will be used. This is a ye llow--orange crysta lline powder with a molec ula r weight of

300.44. Isotretinoin is sensitive to light a nd oxyge n, a nd so it should not be re moved from the capsule for longe r than one hour prior to administration to the patie nt a nd it should be kept in subdued light as much as possible.

5.3.3 Guide lines for Administration: Take orally with fat-conta ining food or milk to enha nce absorption. Administra tion of the entire capsule is to be encouraged a nd sma ll childre n can be trained to swallow them using similar sized ca ndy.

5.3.4 Supplier: Isotretinoin is a va ilable commerc ia lly under the trade na me ACCUTANE (Roc he Xxxxxxxx xxxx) in 10mg, 20mg, a nd 40mg soft ge latin capsules. See package insert for further information.

6.0 CRITERIA FOR SUBJECT ELIGIBILITY

6.1 Subje ct Inclus ion Xxxxx xxx

• Dia gnosis of NB as defined by internationa l criteria,62 i.e., histopa thology (confirmed by the MSKCC Departme nt of Pathology) or BM metastases plus high urine catec hola mine le ve ls.

• High-risk NB, as defined by risk-re la xxx treatme nt guide line s a nd the Interna tiona l NB

Staging System, i.e., sta ge 4 with (any age ) or without (>18 months) MYCN- a mplifica tion,63 or M YCN-amplified NB othe r tha n sta ge 1.64,65

• Patie nts are in CR/VGP R or ha ve primary refractory NB in BM – i.e., NB resista nt to

sta ndard therapy, as evide nced by persiste nce of NB in BM by histology or MIBG scan, but all othe r findings in sca ns show VGP R.

• Childre n a nd adults are e ligible.

• Signed informed conse nt indicating aware ness of the sc heduling a nd side effects, as well as testing require me nts, of this progra m.

6.2 Subje ct Exclus ion Xxxxx xxx

• Existing se ve re major orga n dysfunction, i.e., re na l, cardiac, hepa tic, ne urologic, pulmonary, or gastrointestina l toxic ity > grade 3, e xcept for grade 3 he matologic toxic ity.

• Progressive dise ase (PD) (section 12).

• History of a lle rgy to mouse proteins.

• Active life-threa tening infection.

• Huma n anti- mouse antibody (HAMA) tite r >1000 Elisa units/ml.

• Pregna nt women

• Inability to comply with protocol require me nts.

7.0 RECRUITM ENT PLAN

Patie nts will be offered partic ipation in this ope n access study by the ir atte nding physic ian in the Departme nt of Ped iatrics, MSKCC. No patie nt will be ide ntified by cha rt review or direct adve rtising. The a tte nding physic xxx will be responsible for e xpla ining the study, obta ining written informed consent, a nd registering the patie nt on study. Patie nts will ma inly be

children a nd adole sce nts beca use of the na ture of N B (90% of patie nts are <6 yea rs old at diagnosis). Patie nts of both se xes a nd all ethnic backgrounds are e ligible for this study.

8.0 PRETREATM ENT EVALUATION

Pre-treatme nt e va luations should be completed within 30 da ys of start of treatme nt.

8.1 Complete history a nd physica l e xa mination.

8.2 Complete blood count (C BC), serum creatinine, blood urea nitroge n, serum aspartate a xxxxxxx nsferase, serum a la nine a xxxxxxx nsfe xxxx, a nd serum total bilirubin

8.3 BM aspirates from xxxx xxxxx a nte rior a nd bilate ral posterior iliac crests, a nd biopsies from a ny two separate sites. The spec ime ns are studied by sta ndard histoche mica l methods for the prese nce of tumor ce lls.

8.4 Computed tomography (CT) or ma gnetic resona nce xxx xxxx (MRI) of prima ry tumor site, plus othe r specific or suspe cted sites of tumor.

8.5 Scintigraphic studies (MIBG sca n66 and/or positron e mission tomography [PET] scan.67).

8.6 Serum for a na lysis of HAMA, if applicable (i.e., for patie nts previously treated with murine or chime ric a ntibody). If the patie nt was previously treated with an antibody, HAMA results sinc e last a ntibody treatme nt can be used.

8.7 Pregna ncytest, if applicable

9.0 TREATMENT/INTERVENTION PLAN

9.1 Schedule : The total dosa ge of 3F8 per cycle is 100 mg/m2, administered at 20 mg/m2/day a nd infused iv over ~1.5 hr or le ss (0.5 hr is c ustomary), with a na lgesics a nd antihista mines used as needed for e xpected side -effects. 3F8 is started after GM-CSF administration. GM-CSF is dosed at 250 mc g/m2/day from day –5 to day +1 (Wednesda y to Tuesda y is c ustomary), a nd is 500 mc g/m2/day thereafter (i.e., on da ys +2 to +4; Xxxxxxxx y to Xxxxx y), as in the predecessor protocol. 61 Patie nts come off study for PD or a life- threatening grade 4 toxic ity from 3F8; othe rwise, patients will continue treatme nt through 24 months. It is e xpec xxx that patie nts will receive ~4-to-10 cycles. Patie nts may receive local radiation the rapy.

For Group 1 and 2 patie nts (enrolled respectively on study in 1st or >2nd CR/VGP R,

i. e., with no evide nce of disease), the break between end of a c yc le a nd start of ne xt cyc le is approxima tely 2-to-4 weeks through 4 c yc le s; subsequent breaks are ~6-8 weeks. Isotretinoin is started after c yc le 2 of 3F8/GM-CSF. It is only to be started after c yc le 1 if HAMA deve lops a nd precludes time ly administration of c yc le 2. Road map/sc hema is in section 4.2.

For Group 3 patie nts (e nrolled on study for treatme nt of prima ry refractory disea se), the break between e nd of a cyc le of 3F8/GM-CSF and start of ne xt c yc le is approxima tely 2-

to-4-weeks through 4 cyc les after achie ve ment of CR in BM; subseque nt breaks are ~ 6-8 weeks. Isotretinoin is started after c yc le 2 of 3F8/GM-CSF. It is only to be started after cyc le 1 if HAMA deve lops a nd preclude s time ly administra tion of cyc le 2. Road ma p/sc he ma is in section 4.2.

Patie nts who deve lop early HAMA, whic h precludes time ly treatme nts with 3F8/GM- CSF, are e ligible to receive low-dose ma inte na nc e regime ns suc h as irinoteca n a lone,68 te mozolomide a lone,69 irinoteca n-te mozolomide,70 or cyc lophospha mide-topoteca n.71 These patie nts can also receive a nti- HAMA a ge nts such as rituxima b a nd c yc lophospha mide. The y resume treatment with 3F8/GM-CSF if HAMA becomes ne gative.

9.2 3F8/GM-CSF treatme nt sc hedule (one c yc le).

Da ys -5 to -1: GM-CSF 250 mc g/m2/da y, subc uta neously. Da ys 0 a nd +1: GM-CSF 250 mc g/m2/da y, subc uta neously.

3F8 20 mg/m2/day by iv infusion ove r ~1.5 hr or le ss (usua lly 0.5 hr).

Da ys +2 to +4: GM-CSF 500 mc g/m2/da y, subc uta neously.

3F8 20 mg/m2/day by iv infusion ove r ~1.5 hr or le ss (usua lly 0.5 hr).

Note: The da ily GM-CSF is not administered if the absolute ne utrophil count (ANC) is >20,000/ µl. The last doc ume nted ANC count will be used to determine whe ther GM-CSF will be administered or not. If HAMA de velops, cyc les are deferred until HAMA titer decreases to <1000 Elisa units/ml. Emla cream (lidoca ine 2.5% a nd priloca ine 2.5%) can be used to pre ve nt pain from GM-CSF shots.

9.3 Isotretinoin is administered at 160 mg/m2/d, divided into two doses, x14 days. This treatme nt can be repeated after a minimum rest period of 14 days, for a total of 6 c yc les. I t is not taken on the same da ys as 3F8. All patients receive a minimum of 2 c ycles of 3F8/GM- CSF before starting isotretinoin (only to be started after cyc le 1 if HAMA de ve lops a nd precludes timely administra tion of c yc le 2). All patie nts take a total of 6 c yc le s of isotretinoin, but those patie nts who ha ve persiste nce of disease (but no progressive disease) can take this age nt until BM a nd MIBG show CR. Dose reductions due to e xpec xxx side effects of isotretinoin (e.g., he adac hes, dry skin, etc.) are a llowed.

10.0 EVALUATION DURING TREATMENT/IN TERVENTION

Note : If for any re as onte sts cannot be co mple tedon day 0, the y will be comple tedon day 1.

10.1 CBC on da ys 0 a nd +3 (Xxxxx xx a nd Thursda ys). If ANC is >10,000/ µl, CBC is repeated the ne xt da y.

10.2 C3 and CH50 on day 0 before a nd approximately 3 hours after 3F8 in c yc le 1

10.3 HAMA is c hecked before eachcyc le of 3F8 (Dr. C he ung, Research Lab).

10.4 Serum creatinine, blood urea nitroge n, serum aspartate a xxxxxxx nsfe xxxx, serum a la nine aminotra nsferase, a nd serum total bilirubin on days 0 a nd +4 of eachc ycle.

10.5 BM studies (section 8.4) at e nd of c yc le 2 a nd subseque ntly, BM studies are repeated in conjunc tion with MIBG or PET scan(section 10.7) through 2 ye ars while on study in patie nts with history of BM or cortica l bone involve me nt, but are repeated ~e very 6 months in other patie nts (i.e., patie nts who are sta ge 2 or 3, or were sta ge 4 by virtue of metastases indista nt lymph nodes).

10.6 CT a nd/or MRI of prima ry site approxima tely every 3 months through 2 years while on study.

10.7 MIBG and/or PET scanapproxima tely e ve ry 3 months through 2 years while on study.

10.8 Pregna ncyscreen (fema le s of child-bearing a ge ) before starting isotre tinoin.

Table 4: Evaluatio ns on Pro tocol
Te s ts	Pre -tre atme nt	During Tre atme nt	During Follo wup
Complete history a nd physica l	✓	-	-
Complete blood count	✓	On Day 0 (Xxxxx y) a nd 3 (Thursda y) of each cyc le. If ANC >10,000/µl, CBC is repeated the ne xt da y. CBC is not repeated after Day 4.	-
C3 & CH50	-	Before a nd approximate ly 3 hours after 3F8 on Day 0 (Xxxxx y) of cyc le 1.	-
Live r a nd Re na l function tests (ALT, AST, BUN and creatinine)	✓	On Day 0 (Xxxxx y) a nd 4 (Xxxxx y) of eachcyc le.	-
Pregna ncytest, if applicable	✓	Prior to starting c yc le 1 of isotretinoin	-
Blood for HAMA	✓, if applic able	Before eachc xx xx	-
Xxxx ma rrow studies	✓	After c yc le 2, and subseque ntly with MIBG or PET through 2 yea rs for patie nts with history of BM or cortica l bone involve ment. For patie nts who are stage 2 or 3, or stage 4	Approximate ly q 3 months for 2 years from first treatme nt or until patie nt is off study, whic he ve r is earlier a nd thereafter as c linica lly indica xxx

		by virtue of me tastases in dista nt lymph nodes: approximate ly e very 6 months while on study
CT a nd/or MRI	✓, of primary tumor site, plus other specific or suspected sites of tumor	~Q3 months of primary site	Approximate ly q 3 months for 2 years from first treatme nt or until patie nt is off study, whic he ve r is earlier a nd thereafter as c linica lly indica xxx
MIBG a nd/or PET scan	✓	~Q3 months	Approximate ly q 3 months for 2 years from first treatme nt or until patie nt is off study, whic he ve r is earlier a nd thereafter as c linica lly indica xxx

11.0 TOXICITIES /SIDE EFFECTS

11.1 Toxic ities are graded by the Common Toxic ity Criteria (Version4.0) deve loped by the Nationa l Ca ncer Institute (NCI) of the USA.

11.2 3F8: Re ve rsible side-effects inc lude pain, paresthesia, hyperte nsion, hypote nsion, tachycardia, urtica ria, fe ver, na usea, e me sis, a nd rarely, diarrhea, serum sickne ss, hypona tre mia, somnole nce a nd posterior re versible e nc xxxx xxxx thy syndrome . Other pote ntia l side-effects that may occ ur are bronc hospa sm, anaphylaxis, periphe ral ne uropathy, impa ired accommoda tion of the e ye, a nd poor reactivity of pupils to light.

11.3 GM-CSF: Common side-e ffects include bone pain, flushing, local reaction at site

of injection, le ukope nia shortly after injec tion, a nd decrease in platelet count. Rare side-e ffects (predomina ntly in adults) inc lude a llergic reactions, weight ga in, ple ural or xxxxx xxxxx l effusion, pericardial embolism, thrombosis, a nd difficulty breathing after first injection.

11.4 Isotretinoin: Dry skin, c he ilitis, dry eye s, hype rcalce mia, pse udotumor cerebri, headac hes, hepatotoxic ity, teratoge nic effect on fetus, depressio n, suic idal ideation.

12.0 CRITERIA FOR THERAPEUTIC RESPONSE/OUTCOM E ASSESSM ENT

12.1 Response duration is ca lc ulated from first day of treatment with 3F8.

12.2 All patie nts are considered a treatme nt failure under this protocol if PD de ve lops.

12.3 Disea se status is defined by the Internationa l Ne uroblastoma Response Criteria,62

supple me nted by results of MIBG scans.

▪ Complete response /re mission (CR): no evide nce of dise ase.

▪ Very good partial response /re mission (VGPR): >90% decrease in all disease para me ters, including prima ry tumor, except bone scan unc ha nged or improved; bone marrow must be free of disease ; MIBG scan ne gative in osteomedulla ry sites.

▪ Partia l response/re mission: >50% decrease in all disease parame ters, e xc ept bone scan unc ha nged or improved; no more tha n 1 positive bone ma rrow site ; MIBG scan improved in all lesions.

▪ Mixed response : >50% decrease in >1 but not all disease markers, MIBG scan improved in some but not all sites.

▪ Stable disease: <50% decrease in all tumor ma rkers.

▪ Progressive dise ase (PD): new lesion, or >25 % increase in a ny disease marker.

12.4 Adequac y of trial: All patie nts who fulfill the e ligibility require me nts a nd receive a first dose of 3F8 will have an adequate tria l.

13.0 CRITERIA FOR REMOVAL FROM STUD Y

13.1 All patie nts come off study if PD de ve lops at a ny time after c yc le 1 (see section 12.3).

13.2 All patie nts come off study if there is a life-threa tening grade 4 toxicity c lea rly attributable to 3F8.

13.3 The investigators will make e very reasonable effort to keep each patie nt in the study until all pla nned treatme nts a nd assessme nts ha ve been performed. The investiga tors may discontinue study drug treatme nt for the following reasons:

• Adve rs e e ve nts , including unacceptable toxic ity or e xacerbation of underlying disease, associated with study drug administration a nd necessitating discontinuation of treatme nt. Patie nts who are removed from the study due to adverse e ve nts will be treated a nd followed according to established, acceptable medica l practic e. All pertine nt informa tion conce rning the outcome of suc h treatme nt will be e nte red in the Case Report Form or on the Serious Eve nt Report, as applicable.

• Withdrawal of co ns e nt. The patie nt's desire to withdraw from the study may occ ur at any time. The investiga tor should carefully consider whethe r the patie nt’s withdra wal of conse nt is due to an adverse e ve nt, a nd if so, record the adverse eve nt as the reason for withdrawa l.

• Withdrawal by the phys ician for c linica l reasons not related to study drug treatme nt, for e xa mple, c linica l need to administer a concomita nt medication that is e xc luded by the protocol, in the abse nc e of an adve rse e ve nt.

• Vio lationof the s tudy pro tocol, including fa ilure to return for required treatme nts or

assessme nts.

14.0 BIOSTATISTICS

This tria l concerns the a nti-NB activity of 3F8/GM-CSF. The response e ndpoints are relapse- free surviva l for patie nts treated in C R/VGP R (Groups 1 a nd 2) a nd CR of BM disease for patie nts with prima ry refractory NB (Group 3). Response will be assessed a nd compa red to historica l data, in the xxxxx xt of the large e xpe rie nce with the 03-077 predecessor study (section 3.7).

We will treat NB patients with 3F8/GM-CSF provided the y mee t e ligibility criteria of this protocol. This protocol will re ma in ope n until such a time tha t 3F8 is licensed for broader use (i.e., as an FDA- approved drug). All enrolled patie nts who receive a ny 3F8 will be conside red in the e va luation of safety/toxic ity.

15.0 RESEAR CH PAR TICIPANT REGISTRATION AND RANDOM IZATION PROCEDURES

15.1 Research Participant Re gistratio n

Confirm e ligibility as defined in the section entitled Inc lusion/Exc lusion Xxxxx xxx. Obtain informed conse nt, by following procedures defined in section entitled Informed Conse nt Procedures. During the registration process registering individuals will be required to comple te a protocol specific Eligibility Checklist. The individua l signing the Eligibility Checklist is confirming whether or not the partic ipa nt is e ligible to e nroll in the study. Study staff are responsible for e nsuring tha t all institutiona l require me nts necessary to e nroll a partic ipa nt to the study ha ve been completed. See re la xxx X xxxxxxx Research Polic y a nd Procedure #401 (Protocol Partic ipa nt Registra tion).

16.0 DATA MANAGEM ENT ISSUES

A Research Study Assista nt (RSA) will be assigned to the study. The responsibilities of the RSA include project complia nce, data collection, abstraction a nd e ntry, data reporting, regulatory monitoring, problem resolution a nd prioritiza tion, and coordina te the activities of the protocol study team. The data collec xxx for this study will be entered into a sec ure database. Sourc e doc ume ntation will be a va ilable to support the computerized patie nt record.

16.1 Quality Ass urance

Registra tion reports will be ge xxxx xxx by the RSA to monitor patie nt accruals a nd comple xx xxxx of re gsitra tion data. Routine data qua ilty reports will be ge xxxx xxx to assess missing data a nd inconsiste nc ies. Accrua l rates and exte xt and acc urac y of e va lua tions a nd follow-up will be monitored periodica lly throughout the study period and pote ntia l proble ms will be brought to the atte ntion of the study team for disc ussion a nd action.

Ra ndom-sa mple data qua lity and protocol complia nce a udits will be conduc xxx by the study teamon anongoing basis.

16.2 Data and Safe ty Monito ring

The Data a nd Safety Monitoring (DSM) P la ns at Memorial Xxxxx-Xxxxxxxxx Ca ncer Center were approved by the Nationa l Ca ncer Institute in September 2001. The plans address the ne w polic ie s set forth by the NCI in the doc ume nt entitled “Polic y of the Nationa l Ca ncer Institute for Data a nd Safety Monitoring of Clinica l Trials” which can be found at:

xxxx://xx ncertria ls. nc x.xxx.xxx/xxxxxxx hers/dsm/inde x.html. The DSM Pla ns at MSKCC were established a nd are monitored by the O ffice of Clinica l Researc h. The MSKCC Data a nd Safety Monitoring P la ns can be found on the MSKCC Intra ne t a t : xxxx://xxxxxx0 /c linresearch/Docume nts/MSKCC%20Data%20a nd%20Safety%20Monitoring% 20Pla ns.pdf

There are xx xxxx l differe nt mec ha nisms by which c linica l trials are monitored for data, safety and qua lity. The re are institutiona l processes in pla ce for qua lity assura nce (e.g., protocol monitoring, complia nce a nd data verification audits, therapeutic response, and staff educa tion on c linica l research QA) a nd departme nta l procedures for qua lity control, plus there are two institutiona l committees tha t are responsible for monitoring the activities of our c linical tria ls progra ms. The committees: Data and Safety Monitoring Committee (DSMC) for P hase I a nd II c xxxxxx x xxxx ls, a nd the Data and Safety Monitoring Board (DSMB) for P hase III clinica l tria ls, report to the Ce nter’s Research Counc il and Institutiona l Review Board.

During the protocol de ve lopme nt a nd review process, each protocol w ill be assessed for its le ve l of risk a nd degree of monitoring required. Every type of protocol (e.g., NIH sponsored, in- house sponsored, industria l sponsored, NCI cooperative group, etc.) will be addressed a nd the monitoring procedures will be established at the time of protocol activation.

17.0 PROTECTION OFHUMAN SUBJECTS

The investiga tor a grees to conduc t this study in accorda nce with the Interna xxxxx x Xxxxxxx nce on Harmonization (ICH) princ iples of Good C linica l P ractice a nd with the Declaration of He lsinki (1989). The investigator will conduct all aspects of this study in accorda nce with all na tiona l, state, a nd loca l laws of the applic able re xxxx xxxx a ge ncies.

Most patie nts will be childre n, adole sce nts, and young adults beca use of the nature of t hese tumors. Patie nts of both se xes a nd all ethnic backgrounds are e ligible for this study. Alternative treatme nts are ava ilable a nd will be disc ussed with patie nt or le ga l gua rdia n. Patie nts are responsible for the costs of physic xxx visits a nd usua l labora tory tests, hospita xxxx xxxxx, a nd outpa tie nt care. The y are not responsible for the cost of 3F8. If the re is an injury as a result of this research study, eme rge nc y care, xxxxxxx xxxx xxxx, a nd outpatie nt care will be made a va ilable by Memorial Hospit al and billed to the patie nt as part of the medica l e xpe nses. No mone y will be provided by Memoria l Hospita l as compe nsation for researc h-re lated injury.

17.1 Privacy

MSKCC’s Privac y O ffice may a llow the use a nd disc xxxxxx of protected health informa tion pursua nt to a completed a nd signed Research Authorization form. The use a nd disc xxxxxx of protected hea lth informa tion will be limited to the individuals described in the Research Authorization form. A Research Authorization form must be comple xxx by the Princ ipal Inve stigator a nd approved by the IRB a nd Privac y Board (IRB/PB).

17.2 Se rious Adve rs e Eve nt (SAE) Re porting

Any SAE must be reported to the IRB/PB as soon as possible but no later tha n 5 cale ndar days. The IRB/PB require s a Clinica l Research Database (CRDB) SAE report be submitted e lectronica lly to the SAE O ffice at xxx@xxxxx.xxx. The report should conta in the following information:

Fie lds populated from CRDB:

• Subje ct’s na me (ge ne rate the report with only initials if it will be se nt outside of MSKCC)

• Medica l record number

• Disea se/histology (if applicable)

• Protocol number a nd title Data needing to be e ntered:

• The date the adve rse e ve nt occ urred

• The adverse e ve nt

• Re la tionship of the adverse e ve nt to the treatme nt (drug, device, or interve ntion)

• If the AE was expected

• The se verity of the AE

• The interve ntion

• Deta iled te xt that include s the following

o A e xpla na tion of how the AE was ha ndled

o A description of the subjec t’s condition

o Indication if the subjec t remains on the study

o If an a me ndme nt will ne ed to be made to the protocol a nd/or conse nt form. The PI’s signa ture a nd the date it was signed are required on the completed report.

For IND/IDE protocols:

The C RDB AE report should be completed as above and the FDA assigned IN D/IDE number written at the top of the report. If appropriate, the report will be forwarded to the FDA by the SAE staff through the IND Office.

17.2.1

SAEs are defined as grade 4 toxic ities othe r than fatigue, weight loss or ga in, anore xia, na use a, a nxiety, constipation, urinary rete ntion from opioid a na lge sics, somnole nce, ha lluc inations, disorie ntation, confusion, agitation, a nxie ty from a ntihista mine a nd opioid

pre medications, hypomagnese mia, fe ver, rash, dry skin from isotre tinoin, urtica ria from 3F8 or GM-CSF, mye losuppression from the combination of 3F8 and GM-CSF, pain a nd tra nsie nt hypoxia from opioids, and breath-holding with or without tra nsie nt oxyge n require me nt. P re- existing conditions, e.g., hearing loss, abnorma l liver func tion tests from total pare nte ral nutrition, a nd alopec ia, are not counted as SAEs. Hospita lizations tha t arise from complications of c he motherapy are conside red part of sta nda rd care and therefore will not be reported.

18.0 INFORMED CONSENT PROCEDUR ES

Before protocol-spec ified procedure s are carried out, conse nting professiona ls will e xpla in full deta ils of the protocol a nd study procedures as well as the risks involved to partic ipa nts prior to the ir inc lusion in the study. Partic ipa nts will also be informe d that they are free to withdraw from the study at any time. All partic ipa nts must sign an IRB/PB-approved conse nt form indica ting the ir conse nt to partic ipa te. This consent form meets the require me nts of the Code of Federal Re gulations a nd the Institutiona l Review Board/Privac y Board of this Center. The conse nt form will inc lude the following:

1. The nature a nd objectives, potentia l risks a nd bene fits of the intended study.

2. The le ngth of study a nd the like ly follow-up required.

3. Alternatives to the proposed study. (This will include a va ilable sta ndard a nd investigationa l therapies. In addition, patie nts will be offered an option of supportive care for therape utic studies.)

4. The na me of the investiga tor(s) responsible for the protocol.

5. The right of the partic ipa nt to accept or refuse study interve ntions/interac tions a nd to withdraw from partic ipation at a ny time.

Before any protocol-spec ific procedures can be carried out, the conse nting professiona l will fully e xpla in the aspects of patie nt privacy concerning r esearch specific information. In addition to signing the IRB Informed Consent, all patie nts must a gree to the Research Authorization compone nt of the informed conse nt form.

Each partic ipa nt a nd conse nting professiona l will sign the conse nt form. The partic ipa nt must receive a copy of the signed informed conse nt form.

19.0 REFERENCES

1a. Search Xxxxx n Drug Designations a nd Approva ls. US Department of Health a nd Huma n Services, US Food a nd Drug Administration.

xxxx://xxx.xxxxxxxxxx.xxx.xxx/xxxxxxx/xxxxxxxxxx/xxxx /OOPD_Results_2.cfm?Inde x_Number=26880

8. Accessed Nove mber 19, 2013.

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2. Xxxxxxx A, Xxxxx xxxx CR, Xxxxx XX, et al: High-dose rapid a nd standard induc tion che mothe rapy for patients a ged over 1 year with sta ge 4 neuroblastoma : A ra ndomised tria l. Lancet Oncol 9:247-256, 2008

3. Xxxxxxx XX, Xxxxxx K, LaQ ua glia MP, Xxxxx S, Xxxxxxx ne K, Xxx xxx N-KV: Reduc tion from se ve n to five c yc les of inte nsive induc tion che motherapy in childre n with high-risk ne uroblastoma. J Clin O ncol 22:4888-4892, 2004

4. Xxxx XX, Xxxxx XX, Xxxxxxx XX, et al: P ilot induc tion regime n incorpora ting pharmacokinetica lly guided topoteca n for treatme nt of ne wly dia gnosed high risk ne uroblastoma : A Childre n’s Oncology Group study. J Clin O ncol 29:4351-4357, 2011

5. Xxxxxx XX, Xxxxx n A, See ge r RC, et al: Ne uroblastoma in adults a nd adolescents. An indole nt course with poor surviva l. Cancer 79:2028-35, 1997

6. Xxxxxxx XX, Kra mer K, LaQ ua glia MP, Xxxxx S, Xxxxxx N-KV: Neuroblastoma in adolesce nts a nd adults: The Memoria l Sloa n-Kette ring e xperie nce. Med Pediatr Oncol 41:508-515, 2003

7. Xxxxxxx XX, Xxxxx n S, XxXxx glia MP, Kra me r K, Xxxxxx D, Xxxxx r G, Xxx xxx N -KV: Hyperfractionated low-dose (21 Gy) radiotherapy for high-risk ne urobla stoma following intensive che mothe rapy a nd surge ry. J Clin Oncol 19:2821-2828, 2001

8. Mattha y KK, Xxxxxxxx nca JG, See ger RC, et al: Treatme nt of high-risk ne uroblastoma with inte nsive c he motherapy, radiotherapy, a utologous bone marrow tra nspla ntation, a nd 13 -cis-retinoic ac id. N Engl J Med 341:1165-73, 1999

9. Xx A, Xxxxx n AL, O zka ynak MF, et al: Anti-GD2 a ntibody with GM-CSF, inte rle ukin-2, and isotretinoin for ne uroblastoma. N Engl J Med 363:1324-1334, 2010

10. Di Carlo E, Xxxxx G, Xxxxxxx X X, et al: The intriguing role of polymorphonuclear ne utrophils in a ntitumor reactions. Blood 97:339-345, 2001

11. Xxxxxxx XX, Xxx xxx IY, Kra mer K, Xxxxx S, Xxxxxx N-KV: High-dose c yc lophospha mide inhibition of humora l immune response to murine xxxxx xxxx l antibody 3F8 in ne uroblastoma patie nts: Broad implica tions for immunotherapy. Pedia tr Blood Ca ncer 48:430-434, 2007

12. Xxx xxx NKV, Xxxxxxxx UM, Xxxxx XX, et al: Monoclona l a ntibodies to a glycolipid antige n on human ne uroblastoma ce lls. Ca ncer Res 45:2642-2649, 1985

13. Xxxxxx G, C he xxxx DA, Xxxxx NM, et al: Detection of ga nglioside GD2 in tumor tissues a nd sera of ne uroblastoma. Ca nce r Res 44:5914-5920, 1984

14. Xxxxxxx H, Xxxx va H, Rapola J, Xxxxxxx n UM: Cell-surface ga nglioside GD2 in the immunohistoc hme ical detection a nd differe ntia l diagnosis of ne uroblastoma. Am X X xxx Pathology 96:248-252, 1991

15. Xxxxxx K, Xxxx xx WL, Xxxxxxx XX, Xxxxxx XX, Ha me ed M, Xxx xxx N-KV: Xxxxx xxxx nglioside GD2 loss following xxxxx xxxx l a ntibody therapy is rare in ne uroblastoma. C lin Cancer Res 4:2135-2139, 1998

16. Yeh SDJ, Xxxxxx XX, Xxxx h L, Xxxxxxx XX, Xxxx R, Xxxxxx S, Xxx xxx N -KV: Radioimmunodetec tion of ne uroblastoma with iodine -131-3F8: Correla tion with biopsy, iodine-131- metaiodobe nzylgua nidine a nd sta nda rd dia gnostic modalities. J N uc l Med 32:769-776, 1991

17. Xxxxxx XX, Xxxxx C, Xxxxxxx G, et al: Monoclona l antibodies: Basic princ iple s – radioisotope conjuga tes. In: De Vita VT, He llma n S, Xxxx nberg SA, (eds.): Biologic Therapy of Cancer – Princ iples a nd Practice. Philade lphia, X.X. Xxxxxxxxxx Co., 2000, pp 396-412

18. Saarine n UM, Xxxxxx XX, Ge rson SL, Pe lle y R, Xxx xxx N-KV: Eradica tion of ne uroblastoma cells in vitro by xxxxx xxxx l antibody a nd huma n comple me nt: Method for purging autologous bone marrow. Ca ncer Res 45:5969-5975, 1985

19. Xxxx XX, Xxx xxx N-KV: Interle ukin-2 enha nceme nt of xxxxx xxxx l antibody- mediated cellular cytotoxic ity a ga inst huma n melanoma. Ca ncer Res 47:6600-6605, 1987

20. Xxxxxxx XX, C he xxx N-KV: GM-CSF enha nces 3F8 xxxxx xxxx l antibody-depe nde nt ce llular cytotoxic ity a ga inst huma n melanoma a nd ne uroblastoma. Blood 73:1936-1941, 1989

21. Xxxx XX, Xxx xxx NK : Pha goc ytosis of tumor ce lls by huma n monocytes cultured in recombina nt macropha ge colony-stimula ting factor. J Exp Med 172:231-237, 1990

22. Xxx xxx N-KV, Xxxxx r EI, Xxxxx-Me nsa h WH, et al: Decay-acce le rating factor protects huma n tumor ce lls from comple me nt- mediated cytotoxicity in vitro. J Clin Invest 8:1122-1128, 1988

23. Xxxxxxx XX, Xxx xxx N-KV: Absolute requireme nt of CD11/CD18 adhesion molec ules, FcRII, a nd the phosphotidylinositol - linked FcRIII for xxxxx xxxx l antibody- mediated ne utrophil anti-huma n tumor cytotoxicity. Blood 79:1484-1490, 1992

24. Xxxx xxxxx LS, Xxxxxx s SD, S upe r M, et al: Antidisia loga nglioside/gra nuloc yte ma cropha ge colony-stimulating factor fusion protein fac ilitate s ne utrophil a ntibody-depende nt ce llular cytotoxic ity a nd depe nds on Fcγ RII (CD32) and Mac-1 (CD11b/CD18) for enha nced effector cell adhesion a nd azurophil gra nule e xoc ytosis. Blood 99:4166-4173, 2001

25. Are lla no M, Xxxxxx S: C xxxxxx l uses of GM-CSF, a critical appraisa l. Biologics: Targets & Ther 2: 13-27, 2008

26. Xxxxxx E, Xxxxxxx XX, Xx xxxxxxxxxxx R, et al: Effect of a chimeric a nti- ga nglioside GD2 antibody on cell- mediated lysis of huma n ne urobla stoma ce lls. Ca ncer Res 51:144-149, 1991

27. Xxxx xxxx A, Xxxxx R, Xxxxxx L, et al: P xxxx Xx tria l of gra nuloc yte- macropha ge colony- stimula ting factor combined with murine xxxxx xxxx l antibody R24 in patie nt with metastatic me la noma. J Immunol 16:132-141, 1994

28. Xxxxxx A, Ka mps A, Xxxxxxx SD, et al: The Ch14.18 GM-CSF fusion protein is effective at med ia ting antibody-depe nde nt ce llular c ytotoxic ity a nd comple me nt-depe nde nt c ytotoxic ity in vitro. Clin Canc er Res 5:4259-4263, 1999

29. Xxxxxxx XX, C he xxx N-KV: The c linica lly effective xxxxx xxxx l antibody 3F8 media tes nonoxidative lysis of huma n ne uroe ctodermal tumor ce lls. Ca ncer Res 51:4865-4870, 1991

30. Xxxxxx E, Xxxxxxxx XX: A mec ha nism for ne utrophil- mediated lysis of human ne uroblastoma cells. Ca nce r Res 53:362-367, 1993

31. Xxxxxx RI, Xxxxxxx XX, Xxxxx P : An eosinophil-depe nde nt mec hanism for the a ntitumor effect of interle ukin 4. Scie nce 257:548-551, 1992

32. Xxxxxxxxx XX: Interle ukin-5, eosinophils, a nd dise ase. Blood 79:3101-3109, 1992

33. Ra gnha mmar P, Xxxxxx i G, Xxxxxx X-X, et al: Cytotoxic func tions of blood mononuc xxxx cells in patie nts with colorecta l carcinoma treated with mAb 17-1A and granuloc yte /mac ropha ge- colony-stimulating factor. Ca nce r Immunol Immunother 35:158-164, 1992

34. Xxxxxx, XX, Aga h R, Xxxxxxx r A: Gra nuloc yte - mac ropha ge colony-stimulating factor- induced antibody-depe nde nt cellula r cytotoxicity in bone ma rrow macropha ge s: Application in bone marrow tra nspla ntation. Blood 81:3474-3479, 1993

35. Xxxx xxxx A, Xxxxx R, Xxxxxxxx R, et al: Monoc yte activation following syste mic administration of gra nuloc yte- mac ropha ge colony-stimulating factor. J Immunol 15:217-224, 1994

36. Masucc i G, Xxxxxxx P, Ra gnha mmar P, Me llstedt H: Gra nuloc yte - monoc yte-colony- stimula ting factor a ugme nts the c ytotoxic capac ity of lymphoc yte s a nd monoc ytes in a ntibody- depende nt ce llula r c ytotoxic ity. Ca nce r Immunol Immunother 29:288-292, 1989

37. Xxxxx ssen MJ, Xxxxx XX, Xx xxxx D, et al: Differe ntia l effect of recombina nt gra nuloc yte macropha ge colony-stimula ting factor on xxxx x xxxxx yte s a nd alveolar macropha ge s. Ca ncer Res 49:4086-4089, 1989

38. Ra gnha mmar P, Xxxxxx X-X, Xxxxxx PP, Mellstedt H : Cytotoxic ity of white blood ce lls activa xxx by gra nuloc yte-colony-stimula ting factor, gra nuloc yte/mac ropha ge-colony-stimula ting factor a ga inst tumor ce lls in the presence of va rious xxxxx xxxx l antibodies. Ca ncer Immunol Immunother 39:254-262, 1994

39. Xxxxx XX, S zabolcs P, Xxxxx MA: Ide ntific ation of de ndritic cell colony-forming units a xxxx xxxxx l huma n C D34+ bone marrow proge nitors tha t are e xpanded by c -kit- liga nd and xxx xx pure de ndritic cell colonie s in the prese nce of gra nuloc yte /mac rophage colony-stimulating factor a nd tumor necrosis factor a lpha [published erratum appe ars in J Exp Med 183:1283, 1996]. J Exp Med 182:1111-1119, 1995

40. Szabolcs P, Xxxxx MA, Xxxxx XX: Expa nsion of immuostimula tor y de ndritic ce lls a mong the mye xxxx xxxxx ny of huma n C D34+ bone marrow prec ursors cultured with c-kit liga nd, gra nuloc yte-ma crophage colony-stimulating factor, a nd TNF-alpha. J Immunol 154:5851-5861, 1995

41. Xxx xxx N-KV, Xxx xxx IY, Ca nete A, Xxx XX, K ushner B, Xxxxxxx MA, He ller G, Xxxxxx XX: Antibody response to murine a nti- GD2 xxxxx xxxx l antibodies: Correlation with patie nt surviva l. Cancer Res 54:2228-2233, 1994

42. Ra gnha mmar P, Fa xxxxx xx J, Xxxxxx X-X, et al: Gra nuloc yte/mac ropha ge-colony-stimula ting factor a ugme nts the induc tion of a ntibodie s, especia lly anti- idiotypic antibodies, to therapeutic xxxxx xxxx l a ntibodies. Ca nce r Immunol Immunothe r 40:367-375, 1995

43. Xxxxxxxxx J, Ra gnha mmar P, Xxxxx xxxx M, et al: Humora l anti- idiotypic a nd a nti-a nti- idiotypic immune response in ca nce r patie nts treated with xxxxx xxxx l a ntibody 17-1A. Ca ncer Immunol Immunother 42:81-87, 1996

44. Xxx xxx NKV, Xxx XX, He ller G, et al: Induc tion of Ab3 a nd Ab3' antibody was associated with long-te rm surviva l following a nti-GD2 antibody therapy of sta ge 4 ne uroblastoma. C lin Ca ncer Res 6:2653-2660, 2000

45. Xxxxxx XX, Xxxxxx ll MH, Xxxxxx PP, Xxx Xxxx XX: In vitro assessme nt of the effects of gra nuloc yte-ma crophage colony-stimula ting factor on prima ry human tumors a nd der ived lines. Cancer Res 50:6264-6267, 1990

46. Xxx xxx IY, Xxx K, Xxx xxx N-KV: Activa tion of periphe ral-blood gra nulocytes is strongly corre la xxx with patie nt outcome after immunothe rapy with a nti- GD2 xxxxx xxxx l antibody a nd gra nuloc yte-ma crophage colony-stimula ting factor. J Clin O ncol 30:426-432, 2011

47. Cebon J, Bury RW, Liesc hke Gj, Xxxxxxx G: The effects of dose a nd route of administra tion on the pharmacokinetics of huma n gra nuloc yte- macropha ge colony-stimula ting factor. Eur J Ca ncer 26:1064-1069, 1990

48. Xxxxx N, Xxxxx n WL, Sc he ll M, Xxx ns WE: P harmacokinetics of recombina nt huma n gra nuloc yte-ma crophage colony-stimula ting factor in childre n after intra ve nous a nd subc uta neous administration. J P harmace ut Sci 84:824-828, 1995

49. Xxxxxx XX, Xxxxxx A, Xxxxxxxx XX, K im CS, Xxxx man RE: Neutrophil migra tion is defective during recombina nt huma n gra nuloc yte- macrophage colony-stimula ting factor infusion after autologous bone marrow tra nspla nta tion in huma n. Blood 72:1310, 1988

50. Xxx xxx N-KV, Xxx xxx IY, K ushne r BH, Ostrovna ya I, Krame r K, Xxxxx S: Murine a nti- GD2 xxxxx xxxx l a ntibody 3F8 combined with gra nuloc yte- macropha ge colony stimulating factor and 13-c is-retinoic acid is effective a ga inst che more sista nt marrow MRD among high-risk patie nts with sta ge 4 ne urobla stoma in first re mission. J Clin Oncol 30:3264-3270, 2012

51. Xxx L, Xxx D, We itzma n S, et al: Factors influe nc ing surviva l in childre n with recurre nt ne uroblastoma. J Pediatr Hematol O ncol 26:227-32, 2004

52. Santa na VM, Furma n WL, McGre xxx XX, et al: Disease control intervals in high-risk ne uroblastoma. Ca ncer 112:2796-801, 2008

53. Gara va nta A, Xxxxxx S, Xx Xxxxxxxx X, Xxx D, Xxxxxxxx C, Xxxxx M, Xxxxxx F, Xxxxxxxx E, Bia nc hi M, D’Ange l P, Xxxxxx Xx na zzo G, Xxxxx h R, Fa vre C, Ta mburini A, Ha up t R: O utcome of childre n with ne uroblastoma after progre ssion or relapse. A retrospective study of the Ita lia n Neuroblastoma Registry. Eur J Ca nce r 45:2835-2842, 2009

54. London WB, Castel V, Monc la ir T, Xxxxxx XX, Xxxxxxx ADJ, Xxxx XX, Xxxxxxxx F, Xxxxxxxxxx A, Xxxx nste in RL, Ie hara T, Xxxxxx y KK : Clinica l a nd biological features predictive of surviva l after re lapse of ne uroblastoma : a report from the Interna tiona l Neuroblastoma Risk Group project. J Clin O ncol 29:3286-3292, 2011

55. Xxxxx X, Xxxxxxxx X, Xxxxxxxxx A, et al: Treatme nt a nd outcomes of patie nts with relapsed, high-risk ne uroblastoma : results of Ge rma n trials. Pediatr Blood Cancer 56:578-83, 2011

56. Kreissma n SG, See ger RC, Xxxxxxx XX, London WB, Sposto R, Xxxxx XX, Xxxx-Xxxx n DA, XxXxxxxxx XX, Yu AL, Xxxxx r L, Buxton A, Park JR, Xxxx XX, Xxxxx XX, Xxxxxxxx XX, Villabla nca JG: P urged versus non-purged periphe ral blood ste m-ce ll transpla ntation for high-risk ne uroblastoma (COG A3973): a ra ndomized phase 3 tria l. La nce t O ncol 2013; 14:999-1008.

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58. Xxx xxx IY, Xx Xxxxxxx MS, Xxxxxx r BH, Xxxxxx K, Xxxxxx N-KV: Qua ntitation of GD2 synthase mRNA by rea l-time re verse tra nscriptase polymerase cha in reaction: Clinica l utility in eva luating adjuva nt the rapy in ne uroblastoma. J Clin O ncol 21:1087-1093, 2003

59. Xxx xxx IY, Xx xx Y, Xxxx xx W, Xxx xxx N-KV: Exploiting ge ne e xpression profiling to ide ntify nove l minima l residual disea se markers of ne uroblastoma. Clin Ca nce r Res 14:7020 -7027, 2008

60. Xxx xxx IY, Xxx K, Xxx xxx N-KV: Activa tion of periphe ra l-blood gra nuloc ytes is strongly corre la xxx with patie nt outcome after immunothe rapy with a nti- GD2 monoc lonal antibody a nd gra nuloc yte-ma crophage colony-stimula ting factor. J Clin O ncol 30:426-432, 2011

61. Xxxxxxx XX, Xxxxxx K, Xxx xxx N-KV: P hase II trial of the anti-GD2 xxxxx xxxx l antibody 3F8 and gra nuloc yte-ma cropha ge colony-stimula ting factor for ne uroblastoma. X X xxx O ncol 19:4189- 4194, 2001

62. Xxxxx ur GM, Pritc ha rd J, Xxxxxxxx F, et al: Revisions of the internationa l criteria for ne uroblastoma diagnosis, staging, a nd response to treatme nt. J Clin Oncol 11:1466-1477, 1993

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65. Ba gate ll R, Xxxx-Xxxxxxx X, London WB, et al: Significa nce of MYCN amplifica tion in Interna tiona l Neuroblastoma Staging Syste m sta ge 1 a nd 2 ne uroblastoma : A report from the Interna tiona l Neurobla stoma Risk Group database. J Clin Oncol 27:365-370, 2008

66. Xxxxxxx XX, Xxx SDJ, Xxxxxx K, Xxxxxx XX, C he xxx N-KV: Impact of MIBG scintigraphy on assessing response of high-risk neuroblastoma to dose- intensive induction c he mothe rapy. J Clin Oncol 21:1082-1086, 2003

67. Xxxxxxx XX, Ye xxx HWD, Xxxxxx XX, Xxxxxx K, Xxx xxx N-KV: Exte nding PET scan utility to high-risk neuroblastoma : 18F-fluorodeoxyglucose positron e mission tomography as sole xxx xxxx moda lity in follow-up of patients. J Clin Oncol 19:3397-3405, 2001

68. Xxxxxxx XX, Kra me r K, Xxxxx S, Xxxxxx N-KV: Five-da y course s of irinoteca n as pa lliative therapy for patie nts with ne urobla stoma. Ca ncer 103:858-862, 2005

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71. Xxxxxxx, XX, Xxxxx XX, Xxxxxxxx J, et al: C yc lophospha mide plus topoteca n in childre n with rec urre nt or refractory solid tumors: A Pedia tric O ncology Group pha se II study. J Clin O ncol 19:3463-3469, 2001

20.0 APPENDICES

Appe ndix A: Research Patie nt Dia xxxx