Phase II Study of Anti-GD2 3F8 Antibody and GM-CSF for High-Risk Neuroblastoma
Phase: PHASE2
Conditions: Neuroblastoma
Sponsor: Memorial Sloan Kettering Cancer Center

Memorial Sloan Kettering Cancer Center 
IRB Number: 03-077 A(22) 
 
 
 
 
 
 
 
 
ma 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Please Note: A Consenting Professional must have completed the mandatory  Human 
Subjects Education and Certification Program. 
 
 
 
 
 
 
 
 
 
 
 
 
 
Memorial Sloan-Kettering Cancer Center 
1275 York Ave. 
New York, NY 10021 
 
 
Amended: 04/24/12 
Approval date: 11-Mar-2020 
Phase II study of anti-GD2 3F8 antibody and GM-CSF for high-risk neuroblasto
THERAPEUTIC/DIAGNOSTIC  PROTOCOL 
Principal  Investigator: 
Brian  H. Kushner, M.D. 
Department  of Pediatrics 
212-639-6793 
Co-Principal  Investigator(s): Shakeel Modak, M.D., Department of Pediatrics 
Investigator(s): 
Ellen Basu, MD, PhD., Department  of Pediatrics 
Nai-Kong V. Cheung, M.D., Ph.D 
Stephen Roberts, M.D., Department  of Pediatrics 
Andrew Vickers, Ph.D., Department of Epidemiology and 
Biostatistics 
Consenting Professional(s): 
Ellen  Basu, MD, PhD. 
Brian H. Kushner, M.D. 
Nai-Kong V. Cheung, M.D., Ph.D. 
Shakeel Modak, M.D. 

Memorial Sloan Kettering Cancer Center 
IRB Number: 03-077 A(22) 
Approval date: 11-Mar-2020 
 
 
 
Table of Contents 
1.0 
PROTOCOL SUMMARY AND/OR SCHEMA ................................................................ 2 
2.0 
OBJECTIVES AND SCIENTIFIC AIMS .......................................................................... 3 
3.0 
BACKGROUND AND RATIONALE ............................................................................... 3 
4.1 
OVERVIEW OF STUDY DESIGN/INTERVENTION ................................................... 11 
4.2 
DESIGN ........................................................................................................................... 11 
4.3 
INTERVENTION ................................................................................................. 11 
TABLE 4. PRETREATMENT EVALUATION AND DURING THE STUDY ......................... 13 
WHAT? ......................................................................................................................................... 13 
COMPLETE HISTORY AND PHYSICAL EXAMINATION ........................................ 13 
5.0 
THERAPEUTIC/DIAGNOSTIC AGENTS ..................................................................... 14 
6.0 
CRITERIA FOR SUBJECT ELIGIBILITY ..................................................................... 15 
6.2 
SUBJECT EXCLUSION CRITERIA ................................................................... 16 
7.0 
RECRUITMENT PLAN ................................................................................................... 16 
8.0 
PRETREATMENT EVALUATION (TABLE 4) ....................................................... 17 
9.0 
TREATMENT/INTERVENTION PLAN ................................................................................ 17 
10.0 
EVALUATION DURING TREATMENT/INTERVENTION (TABLE 4) ..................... 18 
11.0 
TOXICITIES/SIDE EFFECTS ......................................................................................... 19 
12.0 
CRITERIA FOR THERAPEUTIC RESPONSE/OUTCOME ASSESSMENT ................ 19 
13.0 
CRITERIA FOR REMOVAL FROM STUDY ................................................................ 20 
14.0 
BIOSTATISTICS ................................................................................................................... 21 
15.1 
SUBJECT REGISTRATION AND RANDOMIZATION PROCEDURES ...................... 22 
15.2 RESEARCH PARTICIPANT REGISTRATION ................................................... 22 
16.1 
DATA  MANAGEMENT ISSUES .................................................................................. 22 
16.2 
QUALITY ASSURANCE ......................................................................................... 23 
16.3 
DATA AND SAFETY MONITORING ............................................................... 23 
17.0 
PROTECTION OF HUMAN SUBJECTS ............................................................................ 23 
17.2 SERIOUS ADVERSE EVENT (SAE) REPORTING .......................................... 24 
18.0 
INFORMED CONSENT PROCEDURES ........................................................................ 25 
19.0 
REFERENCE(S) .................................................................................................................... 26 
 
 
 
 
 
 
 
 
 
 
 
 
1.0 
PROTOCOL SUMMARY AND/OR SCHEMA 
 
This  phase  II  trial  of  the  anti-GD2  murine  IgG3  monoclonal  antibody  3F8  combined  with 
granulocyte-macrophage  colony  stimulating  factor  (GM-CSF)  will  assess  response  of  minimal 
 
Amended: 04/24/12 
Page 2 of 34 

Memorial Sloan Kettering Cancer Center 
IRB Number: 03-077 A(22) 
Approval date: 11-Mar-2020 
Amended: 04/24/12 
Page 3 of 34 
 
 
 
 
residual disease (MRD) in patients with high-risk neuroblastoma (NB) and help  establish  the 
optimal way to use GM-CSF. The study will focus on two groups of patients: Group 1 patients 
have primary refractory NB in bone marrow  (i.e.,  incomplete  response  to  standard treatment), 
and Group 2 patients have no evidence of NB by standard studies but are at high risk for relapse. 
We will apply real-time quantitative RT-PCR methodology targeting GD2 synthase mRNA for 
monitoring MRD; we will test the hypothesis that early BM response has prognostic import. The 
method of using GM-CSF to achieve optimal antitumor biologic effect is not known but will be 
studied by comparing anti-NB effects in patients with the subcutaneous (sc) usage of GM-CSF in 
this protocol versus results achieved with the intravenous (iv)  usage  of GM-CSF  in the  prior 
study. We hypothesize that the sc route will augment overall immune function with consequent 
improved anti-NB activity. Changing to the simple sc usage, as compared to the prior 2-hr iv 
method, will vastly simplify treatment, both for patients and for  medical staff,  and  will reduce 
costs; the ultimate  result will be promoting wider availability of this treatment. 
 
2.1 
OBJECTIVES AND SCIENTIFIC AIMS 
 
• 
Assess the efficacy of  subcutaneous  GM-CSF  in  enhancing  3F8-mediated 
ablation of BM disease. 
 
• 
Apply real-time quantitative RT-PCR to test the hypothesis that minimal residual 
disease content of BM after the first treatments with 3F8/GM-CSF has significant 
prognostic  impact on relapse-free survival. 
 
• 
Simplify treatment with consequent reduction in cost. 
 
3.1 
BACKGROUND AND RATIONALE 
 
3.2 
Dise ase background: Neuroblastoma (NB) is the  most  common  extracranial 
solid tumor of childhood; 50-60% of patients present with an unresectable primary tumor and 
metastases in bone  marrow  (BM).1  Intensive  induction  chemotherapy  and  aggressive  surgery 
have improved remission rates in young  patients2-4; results have been less impressive  in 
adolescents and adults in whom NB is especially chemoresistant.5,6 Realization of an effective 
strategy for eradicating minimal residual disease (MRD) has remained a formidable challenge. Post-
surgical use of local radiotherapy helps control MRD in the primary site.7 Myeloablative therapy 
(with stem-cell support) has been the most common approach for eradicating MRD in distant 
sites.   The long-term relapse-free survival rate in the most recent national study was only 
~20%, although the vitamin A derivative 13-cis-retinoic acid (isotretinoin) helped to prolong 
relapse-free survival.8 These results, plus the potentially severe toxicities of chemotherapy and 
radiotherapy,  are compelling reasons for pursuing novel therapeutic  approaches. 
 
3.3 
Rationale for  immunothe rapy:  Various  strategies  have  undergone  clinical 
testing to induce or augment  immune-mediated  attack  against  cancer.  However,  few  clinical 
trials  have  used  monoclonal  antibody-targeted  immunotherapy against  solid  tumors  in children9- 
18 or adults,19-22 especially with antibody-dependent cellular cytotoxicity (ADCC) as the principal 
underlying  immune  cytotoxic  mechanism.     Also,  an  antineoplastic  role  for  granulocytes  has 

Memorial Sloan Kettering Cancer Center 
IRB Number: 03-077 A(22) 
Approval date: 11-Mar-2020 
Amended: 04/24/12 
Page 4 of 34 
 
 
 
 
received scant attention.23 
A treatment program combining 3F8 and GM-CSF thus represents an 
attempt to help fill a gap in the emerging field  of immunotherapy. 
 
3.4 
Rationale for 3F8: 3F8 is a murine IgG3 MoAb that is well suited for targeted 
immunotherapy.  The reasons are several.  First, the intensive chemotherapy required to produce 
the minimal disease state optimal for immunotherapy in NB patients results in prolonged severe 
lymphopenia. This setting is unfavorable for active immunotherapy  but  allows  passive 
immunotherapy since the patient will be unable to reject allogeneic, xenogeneic, or genetically- 
engineered antibodies. Second, 3F8 recognizes the ganglioside GD2.24 This target  antigen is 
expressed at high density on NB (but has restricted distribution in normal human tissues), is not 
modulated from the cell surface when bound by antibodies, and is genetically stable (unlike tumor-
associated antigens such as immunoglobulin idiotypes on lymphoma cells).25-27 Third, scintigraphy 
using 131I-3F8 confirms that 3F8 localizes selectively  to  GD2(+) tumor deposits in patients.28 The 
excellent targeting potential of 3F8 was  evidenced  by its  high tumor  to  non- tumor ratio, the 
high percent injected dose per gram uptake, and the limited, if any, nonspecific liver and spleen 
uptake.28,29 Fourth,  3F8  mediates destruction in vitro  of GD2(+) human solid tumor cells by 
human complement30 and by human lymphocytes, cultured monocytes, and neutrophils.31-33 Finally, 
the capacity of 3F8 to activate  complement  on NB cells (which lack decay accelerating factor34) 
raises the possibility, not only of complement-mediated lysis in patients, but also  of the  release  
of complement  fragments that may elicit an inflammatory influx of granulocytes capable of lysing 
3F8-labeled tumor cells. Furthermore, the deposition of complement fragments C3b and iC3b on 
NB cells may enhance ADCC because the receptor for iC3b – variously called Mac-1, CR3, 
CD11b/CD18, or άMβ2-integrin – is a key element in anti- GD2 MoAb-mediated  tumor cell kill 
by neutrophils,35,36 which are the most abundant circulating class of leukocyte. 
 
3.5 
Rationale for GM -CSF: GM-CSF has the  potential  for  amplifying  3F8 
antitumor activity in patients via effects on granulocytes and tissue-based macrophages. Reasons 
for combining GM-CSF with 3F8 include the following. First, granulocyte production is only 
transiently suppressed with chemotherapy and GM-CSF  increases  numbers  of  circulating 
neutrophils and eosinophils, does not affect complement levels, and is well tolerated compared to 
other   cytokines   such   as   interleukin-2.37 
Second, granulocytes from patients receiving 
chemotherapy and from normal volunteers are effective in mounting ADCC against NB cells via 
non-oxidative mechanisms, and GM-CSF enhances this cytotoxicity.36,38-42 Third, eosinophilic 
infiltration of some cancers has favorable prognostic significance, and eosinophils exhibit potent 
antitumor activity in animal models.43,44 Fourth, activated monocytes-macrophages efficiently 
phagocytose NB cells, and exposure in vitro or in vivo to GM-CSF primes monocytes- 
macrophages for greater antineoplastic cytotoxicity.33,39,45-50 Fifth, GM-CSF enhances the 
proliferation, maturation, and function of  antigen-presenting  cells,  including  antigen  processing 
and presentation by macrophages and dendritic cells37,51,52 - effects that might promote induction, 
or antitumor activity,  of an idiotypic network.53-56 Finally,  GM-CSF  is not a growth factor for 
NB cells in vitro.57 
 
3.6 
GM -CSF-me diate d activation of polymorphonucle ar ne utrophils (PM Ns): 
PMNs are the predominant class of circulating leukocytes;  chemotherapy  only  transiently 
decreases  their  numbers  and  largely  spares  their  cytotoxic  capabilities.     These  features  are 

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IRB Number: 03-077 A(22) 
Approval date: 11-Mar-2020 
Amended: 04/24/12 
Page 5 of 34 
 
 
 
 
advantageous for immune-based attack against cancer.23 We and others have shown that Mac-1 
(CD11b/CD18), FcRII, and FcRIII are required for optimal ADCC in a  system using human 
PMNs as effectors, human NB cells as targets, and clinically active anti-GD2 MoAbs as 
mediators.35,36 Efficient tumor-cell kill occurs despite the large size  of  the  targets  (which 
precludes a role for phagocytosis) and their relative resistance to reactive oxygen species.41 GM- 
CSF enhancement of ADCC in this system correlates with upregulation of Mac-1 and  with 
increased exocytosis of azurophil (primary) granules which contain cathepsin G and  defensins 
capable of lysing NB cells.42 To elucidate the cytotoxic  mechanisms  of  anti-GD2  MoAb- 
mediated ADCC, we used blocking MoAbs, PMNs devoid of Mac-1 (from donors with 
leukocyte adhesion deficiency), and PMNs that do not generate reactive  oxygen species (from 
donors with chronic granulomatous disease),35 while others used blocking MoAbs, electron 
microscopy,  and PMNs from healthy  donors as well as from patients.36 
 
3.7 
Rationale for quantitating BM dise ase by re al-time RT-PCR: Curative 
strategies for stage 4 NB must encompass control of both the primary mass and distant disease. 
Dose-intensive chemotherapy has had a favorable impact on resectability of primary tumors and 
surgical removal of all visible tumor, followed by local radiotherapy, reduces the risk of relapse 
in the  primary  site  to  <10%.4,7  Dose-intensive  chemotherapy  eliminates  histologically  evident 
BM involvement in 90% of high-risk cases.3 These encouraging results have shifted the focus of 
curative  strategies to the problem of MRD – which is readily assessed in BM. 
Adjuvant treatments, such as immunotherapy with monoclonal antibodies, may make 
gradual and not quantum changes in MRD, which by definition is beyond the sensitivity of 
conventional histologic58  or  radiographic  techniques.  The  availability of more sensitive methods 
for identifying and quantitating occult tumor cells in sequential BM specimens can allow 
determination of the efficacy and optimal duration of adjuvant treatment. Thus, the absence of 
detectable tumor cells in BM can serve as a surrogate end point  for  the  adjuvant  treatment 
strategy, while quantitative data on BM disease will help  detect  relapse  earlier  and  indicate  a 
need for a change in treatment. 
We59-65 and others66-70 have identified MRD in BM by immunological and molecular 
methods. The limit of detection for immunocytology is 1/105 to 1/106 cells; RT-PCR has slightly 
superior sensitivity with a range of 1/105 to 1/107 cells. A drawback to immunocytology is the 
need for freshly collected samples and the  technique’s  labor  intensive  aspect  (counting  ce llls 
under a microscope). In contrast, molecular monitoring of tumor cells by RT-PCR uses cryo- 
preserved mononuclear cells and experiments can be repeated multiple times and  for  multiple 
markers as they are being developed. Pitfalls in the use of molecular-based  assays  of MRD 
include false positivity due  to the presence of the “tumor-specific” markers in normal ce llls, and 
the process of illegitimate  transcription (i.e., the transcription of any gene in any cell type). 
Development of a real-time quantitative RT-PCR assay broadens the potential  for 
monitoring MRD. The advantages of this technique include a wide  linear  dynamic  range  and 
superior sensitivity and accuracy, which allows  good  intra-  and  inter-assay reproducibility,  plus 
high throughput capacity, speed, and elimination of lengthy post-PCR handling steps, which 
prevents potential carryover contamination. Real-time quantitation of GD2 synthase  mRNA 
provides information  on expression of the key enzyme  in the biosynthesis  of GD2.63,64 
 
3.8 
Re sults using re al-time quantitative RT-PCR of GD2 synthase mRNA: Most 
recently, we have used this method to assess molecular response in BM from patients with high- 

Memorial Sloan Kettering Cancer Center 
IRB Number: 03-077 A(22) 
Approval date: 11-Mar-2020 
Amended: 04/24/12 
Page 6 of 34 
 
 
 
 
risk NB treated with 3F8 plus GM-CSF by 2-hr iv infusion; we now intend to apply this method 
for comparing anti-NB activity of the subcutaneous versus iv route of GM-CSF usage. In our 
retrospective study, the patients fell into four groups before treatment with 3F8 and iv GM-CSF: 
33 patients were in complete/very good partial remission, 32 had primary refractory disease 
(incomplete response to induction), 11 had secondary refractory disease (incomplete response to 
treatment for relapse), and 16 had progressive disease. Molecular detection of tumor cells was 
evident in pretreatment BMs of all four groups (32%, 59%, 64%, and 75%, respectively) with 
mean GD2 synthase transcript levels  of 7.5, 22.3, 54.7, and 436.3, respectively (Table 1). 

Memorial Sloan Kettering Cancer Center 
IRB Number: 03-077 A(22) 
Approval date: 11-Mar-2020 
Amended: 04/24/12 
Page 7 of 34 
 
 
 
 
 
Table 1.  NB cell detection in BM before 3F8/GMCSF  treatment in 4 groups of patients 
% positivity by 
GD2  synthase 
 
N 
Histology 
QRT-PCR* 
Transcript Level  (mean±SE) 
First CR/VGPR 
33 
0% 
32% 
7.5 ± 2.9 
Primary  Refractory 
32 
68% 
59% 
22.3 ± 5.6 
Secondary Refractory 
11 
82% 
64% 
54.7 ± 34.2 
Progressive  Disease 
16 
73% 
75% 
436.3 ± 232.4 
*GD2  synthase transcript < 5 was defined as negative. 
 
In these 4 groups, there was molecular evidence of BM response in 43%, 67%, 44%, and 0% of 
patients, respectively (Table 2). 
 
Table 2.  Response rates by molecular  testing and by histology in 4 groups of patients 
GD2  synthase mRNA 
 Histology 
Response Rate 
Negative  Transcript* 
Response Rate 
 
 
 
 
 
 
*Pre- and post-treatment BMs were  (-). 
 
The response was consistent over time, that is, disease status after one or two cycles of 3F8/GM- 
CSF correlated with disease status at a later time point during treatment. Transcript levels in 
pretreatment BMs of responders (28.2±6.8) were lower than those of  non-responders 
(139.0±77.7) (Table 3). 
 
Table 3.  GD2  synthase transcript levels* in BM  before and after 3F8/GMCSF 
 
Pre-treatment  sample 
Post-treatment  sample 
N 
mean ± SE 
mean ± SE 
Responders 
24 
28.2 ± 6.8 
0.5 ± 0.2 
Non-responders 
30 
139.0 ± 77.7 
312.4 ± 95.9 
Negative transcript 
33 
0.9 ± 0.3 
1.0 ± 0.3 
*GD2  synthase transcript < 5 was defined as negative. 
 
Excluding the progressive  disease  group,  progression-free  survival  (PFS)  was  statistically 
different among non-responders, responders, and patients with persistently negative transcript 
(p=0.02) (Figure 1).  Among  patients  with  histologically  negative  BMs,  molecular  responders 
had a significantly lower risk of disease progression (p=0.02) (Figure 2). The results suggest that 
GD2 synthase might serve as a sensitive marker of MRD in BM and that its level might reflect 
clinical disease status as well as correlate with histologic  response to immunotherapy. 
First CR/VGPR 
43% (6/14) 
19 
(not applicable) 
Primary  Refractory 
67% (14/21) 
11 
60% (15/25) 
Secondary Refractory 
44% (4/9) 
2 
60% (6/10) 
Progressive  Disease 
0% (0/10) 
1 
0%  (0/10) 

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10 
20 
3 
Proportion surviving progression-free 
 
 
 
 
3.9 
Clinical e xpe rie nce with 3F8 plus GM -CSF: The optimal method of using GM-
CSF with the aim of enhancing ADCC in patients with solid tumors is not known. In the past, 
we chose to administer GM-CSF by a 2-hr iv infusion (followed by a 1-hr interval before 
starting 3F8  infusion)  because  GM-CSF  disappears  rapidly (<2-3 hr) from the blood with that 
1.0 
1.0 
 
p=0.02 
 
.8 
 
 
.8 
responders 
 
p=0.02 
 
neg transcript 
 
.6 
.6 
neg transcript 
 
.4 
responders 
 
non-responders 
.2 
 
.4 
 
 
non-responders 
 
.2 
Figure 1.  PFS as related to RT-PCR  response 
Figure 2.  PFS by RT-PCR  response in 
0.0 
0 
 
20 
40 
 
60 
80 
 
100 
0.0 
0 
histologically (-)  BMs 
 
Months from beginning of 3F8/GM-CSF 
 
Months from beginning of 3F8/GM-CSF 
schedule.71,72 We wished  to avoid  the prolonged  (>12 hr) bioactive levels in blood associated 
with the sc route; our concern was that high serum levels of GM-CSF might impede granulocyte 
trafficking into tissues.73 However, for circulating tumor cells in BM or blood,  this concern is 
dwarfed by the more potent antitumor effects from prolonged cytokine exposure following 
subcutaneous  injection.37 
Major findings included: 1) treatment for progressive disease:  no  major  responses  in 
16/16 patients; 2) treatment  for  primary refractory  disease: CR (by histology) in BM in 21 
(75%) of 28 patients, including 5/5 adolescents/adults, and with CR evident after 1-3 cycles; and 
3) treatment in first complete/very good partial remission post-transplant (n=41): 72.6% (±8.0%) 
relapse-free survival at 18 months from first dose of 3F8 (Figure 3), with many patients receiving 
10 or more cycles and with use of 13-cis-retinoic  acid after cycle 2.6,18,74 
Figure 3.  Patients treated in CR/VGPR 
 
 
 
 
 
1.0 
 
 
 
.8 
 
 
 
.6 
 
 
 
.4 
 
.2 
 
 
0.0 
0 
 
 
 
 
 
10 
20 
30 
40 
3.10 HAM A 
re sponse /idiotypic 
ne twork: 
In a  study of 34  patients  with MRD 
50 
treated  with 3F8  alone,  patients  with a self-limited 
 
Months from start of 3F8/GM-CSF 
(transient)  HAMA  response  had  significantly better 
PFS than patients who 
had 
persistently high 
40 
50 

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Amended: 04/24/12 
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HAMA titers or no HAMA formation.15 This association of a transient low level HAMA/anti- 
idiotype (Ab2) antibody response with better PFS was a seemingly paradoxic finding since 
HAMA/Ab2 blocks 3F8 binding to tumor cells. The  unexpected  observation  regarding 
HAMA/Ab2 and survival led to the hypothesis of a host idiotypic network induced by 3F8 (Ab1) 
and responsible for long-term tumor control. The idiotypic  network  was  first  postulated  by 
Jerne75 for immune regulation and later demonstrated in human disease models,  including cancer 
patients.76-79 Anti-idiotypic antibodies (Ab2) in the HAMA pool may mimic antigen GD2 and 
stimulate an Ab3 antibody response. We previously reported the association of anti-anti-idiotype 
(Ab3) with prolonged survival among patients who underwent myeloablative  therapy  with 
autologous stem-cell rescue.53  Other  investigators  have  postulated  the  development  of  an 
idiotypic network in patients treated with 17-1A antibodies, where both Ab3 antibodies as well 
as anti-idiotypic  and anti-anti- idiotypic  T cells have been demonstrated.54,55 
In our study of 34 patients, consistent with the emergence of an idiotypic network, Ab3 
(anti-anti-idiotype) and Ab3' (anti-GD2) response at 6-14  months after treatment with 3F8 alone 
correlated with improved PFS, and higher Ab3 titers showed a stronger correlation with 
improved outcome, while non-idiotype antibody responses (anti-mouse or anti-tumor nuclear 
antigen) had no apparent impact on outcome.56 Since GD2 is an auto-antigen,  induced Ab3' 
response represented a breakdown in self-tolerance.  We  hypothesized  that  significant  rises  in 
Ab3 or Ab3' would not be possible unless suppressor pathways were removed and naive T or B 
cells  were  allowed to repopulate.  Following intensive chemotherapy that eliminates a large part 
of the lymphoid system, exposure to tumor-selective Ab1s (3F8) might induce unique Ab2s that 
could bias the recovering repertoire towards the GD2 antigen network. The potential for biasing 
the immune system towards specific antigens has been well documented in murine models80 and 
human disease states.81,82 If true, one would expect such  an idiotypic network to  be  most 
successful following intensive 
immunosuppressive 
therapy, 
and 
not easily induced  
in 
immunocompetent patients. In fact, we found that myeloablative therapy suppressed HAMA and 
Ab2 but was followed by the appearance of Ab3 which in turn correlated with prolonged 
survival.53,56 The prerequisite of strongly  immunosuppressive therapy may explain why in most 
clinical studies of MoAbs, such  an  idiotypic  network  is  not  commonly  observed.  However, 
when found in patients with colorectal or ovarian carcinoma treated with specific MoAbs, the 
idiotypic  network gave patients superior  survival  advantage.54,55,76,77,83-87 
 
3.11 
Isotre tinoin (13-cis-re tinoic acid) was shown in a randomized national study to 
decrease the risk of relapse in  patients treated  in  complete  remission.8  This  agent  has 
subsequently become standard of care for NB patients in complete remission. It will be used in 
this protocol after patients are evaluated for response (and for toxicity)  to 3F8/GM-CSF. 
 
3.12 
Inte rpre tations and implications: This phase II trial explores the utility of RT- 
PCR for monitoring  anti-NB  activity of immunotherapy,  with an emphasis  on early recognition 
of treatment efficacy. The studies focus on BM; findings  may  not  reflect  total  body tumor 
burden. Metastatic NB is, however, a systemic disease, and,  as  with leukemia,88  BM  status 
appears to approximate the overall disease state. If the RT-PCR method proves to be sensitive, it 
will provide a useful tool for measuring NB-cell content in BM as a patient  goes  through 
treatment. The ability to identify patients not likely to  benefit  from  further  use  of  a  given 
adjuvant therapy will allow a more timely switch to a possibly better treatment. We hypothesize 
that  quantifying  GD2  synthase  transcript  levels  will help  stratify patients  by tumor  load,  define 

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tumor thresholds that correlate with favorable clinical outcome,  and  provide  prognostic 
classification for patients receiving immunotherapy. Risk-related molecular  guidelines  can  be 
rapidly incorporated into future treatment  programs. 
Our RT-PCR method provides a quantitative measure, such that the percentage  of 
patients who achieve CR in BM, and the degree of response with 3F8/GM-CSF, can be 
calculated and compared to other adjuvant treatments. With NB, where randomized trials are 
difficult to carry out because of the small number of affected patients, such quantitative analyses 
acquire even greater utility for rapidly identifying both the best agent and its optimal use in a 
curative  treatment program. 
The findings regarding RT-PCR  utility  in  the  adjuvant  setting,  granulocyte  activation, 
and idiotypic network will have general implications for antibody-based  therapeutic  strategies 
against solid tumors in humans. Also, the ease  of  administration  of  the  treatments  (a 
subcutaneous injection, which is given at home, and a 30-minute intravenous infusion in the 
outpatient clinic), plus their transient acute side-effects, are compatible with widespread usage of 
this regimen beyond our hospital – a strong positive  factor in furthering product development. 

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Amended: 04/24/12 
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4.1 
OVERVIEW OF STUDY DESIGN/INTERVENTION 
 
4.2 
Design 
 
This is a phase II, open-label, single arm study to assess the activity of 3F8/GM-CSF against 
neuroblastoma and to investigate whether subcutaneous (sc) usage of GM-CSF is as good or 
better than short-term (2-hr) intravenous (iv) usage for optimizing anti-neoplastic  activity. 
Group 1 patients have refractory bone marrow disease, and Group 2 patients have  no 
evidence of disease by standard studies, but are at high risk for relapse. Clinical results will 
be compared to those observed in the predecessor trial in which only iv GM-CSF was used 
with 3F8.   Real-time quantitative  RT-PCR63-65   will be used to assess minimal residual disease 
in bone marrow. All patients will receive a minimum  of  4  cycles  of  treatment  and  will 
continue  treatment  through 24 months. 
 
4.3 
Intervention 
 
Road Map/Schema for Group 1 patients (BM positive): The break between end of a cycle of 
3F8/GM-CSF and start of next cycle is 2-to-4-weeks through 4 cycles after achievement of CR 
in BM; subsequent breaks are ~8 weeks.  Please see roadmap below for a patient achieving CR 
in bone marrow after cycle 1. 
Cycle 1 
3F8 (iv) + GM-CSF (subcutaneous  [sc]) (1 wk) 
3-wk interval*   – BM negative 
Cycle 2       3F8 (iv) + GM-CSF (sc) (1 wk) 
3-wk interval*   –  if BM negative  -> 1st cycle of oral isotretinoin  x14 days 
Cycle 3 
3F8 (iv) + GM-CSF (sc) (1 wk) 
3-wk interval –  oral isotretinoin x14 days 
Cycle 4       3F8 (iv) + GM-CSF (sc) (1 wk) 
3-wk interval –  oral isotretinoin x14 days 
Cycle 5 
3F8 (iv) + GM-CSF (sc) (1 wk) 
8-wk interval  – oral isotretinoin x14 days 
Cycle 6 
3F8 (iv) + GM-CSF (sc) (1 wk) 
8-wk interval  –  oral isotretinoin x14 days on, x14 days off, x14 days on (6th/last cycle) 
Cycle 7 
3F8 (iv) + GM-CSF (sc) (1 wk) 
8-wk interval 
Continue  with 8-wk intervals  through 24 months from 1st dose of 3F8. 
* 
assessment of BM response by RT-PCR and by standard histology 
 
Road Map/Schema for Group 2 patients (no evidence of disease): 
 
Cycle 1 
3F8 (iv) + GM-CSF (sc) (1 wk) 
3-wk interval 
Cycle 2 
3F8 (iv) + GM-CSF (sc) (1 wk) 
3-wk interval*   – oral isotretinoin x14 days 
Cycle 3 
3F8 (iv) + GM-CSF (sc) (1 wk)§ 
3-wk interval  – oral isotretinoin x14 days 
Cycle 4 
3F8 (iv) + GM-CSF (sc) (1 wk) 

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8-wk interval  –  oral isotretinoin x14 days on, x14 days off, x14 days on 
Cycle 5 
3F8 (iv) + GM-CSF (sc) (1 wk) 
8-wk interval  –  oral isotretinoin  x14 days on, x14 days off, x14 days on (6th/last cycle) 
Cycle 6 
3F8 (iv) + GM-CSF (sc) (1 wk) 
8-wk interval 
Cycle 7 
3F8 (iv) + GM-CSF (sc) (1 wk) 
Continue  with 8-wk intervals  through 24 months from 1st dose of 3F8. 
* 
assessment of BM status by RT-PCR and by standard histology 

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TABLE 4. PRETREATMENT EVALUATION  AND DURING THE STUDY 
 
WHAT? 
WHEN? 
Complete history and physical examination. 
• Pretreatment  evaluation 
Complete blood count 
1. Pretreatment  Evaluation 
2. On Monday (day 0) and Thursday (day +3). 
If ANC is >10,000/ul, CBC is repeated on next 
day. 
Liver and kidney function Blood Tests 
1. Pretreatment 
2. On day 0 (Monday) and +4 (Friday) of each 
cycle 
C3 and CH50 
• On days 0 and +4 (Monday and Friday) of 
each cycle. 
HAMA (Red Top Tube) (Dr. Cheung, 
Research Lab) 
• ~Every 1-2 months while  on study. 
Bone marrow studies (including 10 ml 
heparinized  for immunocytology and/or RT- 
PCR, Dr. Cheung,  Research Lab) 
1. Pretreatment 
2. At end of cycles 2 & 4 
3. After cycle 6 of Accutane 
4. Subsequently, 
• 
BM studies are repeated with MIBG or 
PET through 2 years for patients with 
history of BM or cortical bone 
involvement, 
• 
But are repeated ~every 6 months in other 
patients (e.g., patients who were stage 4 
by virtue of metastases in distant lymph 
nodes) while on study. 
CT or MRI 
1. Pretreatment: of primary-   tumor site, plus 
other specific  or suspected sites of tumor. 
2. Followup: of primary  site ~every 3 months 
through 1 year. 
Scintigraphic  studies  (99mTc-MDP-bone 
scan, MIBG scan,92 and/or PET scan.93). 
• Pretreatment 
99mTc-MDP-bone scan 
~Every 3 months while  on study until normal 
MIBG or PET scan 
~Every 3 months through  2 years 
Echocardiogram 
Pretreatment 
Urine catecholamines  levels 
1. Pretreatment 
2. ~Every 3 months through  2 years. 
Pregnancy  screen  (females  of  child-bearing 
age) 
Before starting isotretinoin. 

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Note: If for any reason tests cannot be completed on day 0, they will be completed on 
day 1 
 
 
5.1 
THERAPEUTIC/DIAGNOSTIC  AGENTS 
 
5.2 
3F8 monoclonal antibody (IND number of BB-IND-8449) 
 
5.2.1 Source and Pharmacology: Monoclonal antibody 3F8 is purified  by 
column chromatography. The final product is tested  to  assure  that  it  is  free  of nucleic 
acid, murine viruses, bacteria, fungi, mycoplasma and pyrogens. Plasma concentrations 
achieved after a 5 mg/m2 and 20 mg/m2 IV dose were 1.1 +/- 0.6 and 3.7 +/- 1.4 ug/mL, 
respectively.  Supplier: 3F8 is manufactured  as an investigational agent. 
 
5.2.2 Formulation and Stability: The purified antibody is frozen at –80oC,  at 2 
mg/mL 0.1 M citrate-phosphate pH 4.2 buffer in glass vials. At –80oC it is stable for at 
least 3 years. For IV administration, 3F8 should be diluted into 10 ml 5% human serum 
albumin and millipore  (0.2 um) filtered  before use.  Route of Administration:  IV infusion. 
 
5.3 
Yeast-derived human recombinant 
granulocyte-macrophage colony stimulating 
factor (GM-CSF; LEUKINE (sargramostim))  is produced by Berlex Laboratories, Seattle, WA. 
 
5.3.1 LEUKINE is a sterile,  white,  preservative-free,  lyophilized  powder 
suitable for IV infusion upon reconstitution of 250 mcg vials;  these  vials  should  be 
reconstituted aseptically with 1.0 ml Sterile Water for  Injection,  USP  (without  preservative). 
The reconstituted LEUKINE solutions are clear, colorless, isotonic with a pH of 7.4 ± 0.3, and 
contain 250 mcg/ml of Sargramostim. The single-use vial should not be re-entered or reused. 
Do not save any unused portion for later administration. Vials containing 500 mcg of 
LEUKINE are already in solution and are multiple  dose vials. 
 
5.3.2 
During reconstitution the Sterile Water for Injection, USP should be 
directed at the side of the vial and the contents gently swirled to avoid foaming during 
dissolution.  Avoid excessive  or rigorous agitation;  do not shake. 
 
5.3.3 Dilution for IV infusion should be performed in 0.9% Sodium Chloride 
Injection. USP. If the final concentration of LEUKINE is below 10 mcg/ml, Albumin (Human), 
add 1 mg Albumin (Human) per l ml 0.9% Sodium Chloride Injection, USP (e.g. use 1 ml 5% 
Albumin [Human]  in 50 ml 0.9% Sodium Chloride Injection,  USP). 
 
5.3.4 An in-line  membrane  filter  is not to be used for IV infusion of LEUKINE. 
 
 
5.3.5 LEUKINE contains no antibacterial preservative and therefore should be 
administered as soon as possible,  and  within 6 hours following reconstitution and/or dilution for 
IV infusion. Store LEUKINE powder, reconstituted solutions, or  diluted  solutions  under 
refrigeration at 2-8°C (36-46°F); do not freeze or shake. LEUKINE vials are intended for single 

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use  only;  discard  any  unused  solution  after  6  hours.  Do  not  use  beyond  the  expiration date 
printed on the vial. 
 
5.3.6 In the absence of compatibility and stability  information,  no  other 
medication should be added to infusion solutions containing LEUKINE. Use only 0.9% Sodium 
Chloride Injection,  USP to prepare IV infusion solutions. 
 
5.3.7 Aseptic technique should he employed in the preparation of all LEUKINE 
Solutions. To assure correct concentration following reconstitution, care should be exercised to 
eliminate any air bubbles from the needle hub of the syringe used to prepare the  diluent. 
Parenteral products should be inspected visually for particulate matter and discoloration prior to 
administration whenever  solution and container  permit. 
 
5.3 
13-cis-Retinoic 
Acid 
(NDC 
#0004-0155-01,0004-0169, 
0004-0156-01- 
ISOTRETINOIN, ACCUTANE) 
 
5.3.1 Source and Pharmacology: The exact mechanism of retinoic acid (RA)- 
induced maturation of tumor cells is not known. In neuroblastoma cell lines it has been shown to 
down regulate MYCN RNA and  protein expression,  and  such down regulation correlates with 
the ability of isotretinoin to induce tumor cell growth arrest. Recent studies using  gene 
transfection directly implicate down regulation of MYCN expression  by  isotretinoin  as  a  key 
event in achieving sustained arrest of neuroblastoma cell proliferation. RA also  appears  to 
enhance normal hematopoietic differentiation by increasing the responsiveness of myeloid and 
erythroid  progenitor  cells  to  the  action  of  myeloid  colony  stimulating  activity  and 
erythropoietin,. respectively. Metabolism: RA is 99.9% bound in plasma (almost entirely  to 
albumin) and has a half-life of 10-20 hours. The major metabolite is 4-oxoisotretinoin, and 
excretion is in the urine and feces. A single oral dose of 100mg/m2 isotretinoin will produce peak 
plasma levels of 1-2mM.  The mean peaktime as 3.2 hours after 80mg orally, with a terminal t½ 
of 10 to 20 hours. Administering 160 mg/m2/day to children after autologous bone marrow 
transplantation has been shown to achieve 13-cis-RA levels of 5 to 7 micromular. 
 
5.3.2 Formulation and Stability: Isotretinoin, which is the 13-cis isomer  of 
retinoic acid,  will be  used.  This  is a yellow--orange crystalline powder with a molecular weight 
of 300.44. Isotretinoin is sensitive to light and oxygen, and so it should not be removed from the 
capsule for longer than one hour prior to administration to the patient and it should be kept in 
subdued light  as much as possible. 
 
5.3.3 Guidelines for Administration: PO with fat-containing food or milk to 
enhance absorption. Administration of the entire capsule is to be encouraged and small children 
can be trained to swallow  them using similar  sized candy. 
5.3.4 Supplier: Isotretinoin is available commercially under the trade name 
ACCUTANE (Roche Laboratories) in 10mg, 20mg, and 40mg soft gelatin capsules. See package 
insert for further  information. 
 
6.1 
CRITERIA FOR SUBJECT ELIGIBILITY 
 
Patients  must  have  neuroblastoma. 
They  can  have  evaluable  (bone  marrow  [+]  by 

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histology, abnormal MIBG or PET scan consistent with bone marrow involvement), but not 
progressive, disease (Group 1). Patients in complete/very good partial remission (CR/VGPR; no 
evaluable  or measurable  disease) yet at high risk for relapse are also eligible  (Group 2). 
 
6.2 
Subject Inclusion Criteria 
▪ Diagnosis of neuroblastoma as defined by international criteria,91 i.e., 
histopathology (confirmed by the MSKCC Department of Pathology)  or 
bone marrow metastases plus high urine catecholamine  levels. 
 
▪ Neuroblastoma, as defined by risk-related treatment guidelines and the 
International Neuroblastoma Staging System,  i.e.,  stage  4  with (any age) 
or without (>18 months) MYCN-amplification,93a or MYCN-amplified 
neuroblastoma  other than stage 1.93b,93c 
 
▪ Group 1 patients have neuroblastoma (as defined above) resistant to 
standard therapy, as evidenced by  incomplete  response  in bone marrow, 
but no MIBG-avid soft tissue tumor and no progressive  disease. 
 
▪ Group 2 patients have no evidence of disease, i.e., are in CR/VGPR from 
neuroblastoma  (as defined  above). 
 
▪ Signed  informed consent  indicating  awareness  of  the  investigational 
nature of this program. 
 
6.3 
Subject Exclusion Criteria 
▪ Existing severe major organ dysfunction, i.e., renal., cardiac, hepatic, 
neurologic,  pulmonary,  or gastrointestinal  toxicity  > grade 3. 
 
▪ Progressive  disease or MIBG-avid soft tissue tumor. 
 
▪ History of allergy to mouse proteins. 
 
▪ Active life-threatening  infection. 
 
▪ Human anti-mouse  antibody (HAMA) titer >1000 Elisa units/ml. 
 
▪ Inability to comply with protocol requirements. 
7.0 
RECRUITMENT PLAN 
 
In each of the past 2 yrs, 23 patients have been treated on the predecessor of this protocol. 
We anticipate an increase in this accrual rate, which will allow rapid realization of preliminary 
conclusions, with completion of the trial in ~3 yrs. Given the favorable prior  efficacy-toxicity 
profile with 3F8/GM-CSF, we expect that all patients will complete the trial. Patients will be 
offered participation in this study by their attending physician in the Department of Pediatrics, 
Memorial Sloan-Kettering Cancer Center. No patient will be identified by chart review or direct 
advertising. The attending physician will be responsible  for  explaining  the  study,  obtaining 
written informed  consent,  and  registering the  patient  on study.    Patients will mainly be children 

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and adolescents because of the nature of neuroblastoma (90% of patients are <6 years old at 
diagnosis).   Patients of both sexes and all ethnic backgrounds are eligible  for this study. 
 
8.1 
PRETREATMENT EVALUATION  (TABLE 4) 
 
8.2 
Complete history and physical examination. 
 
8.3 
Complete blood count, serum creatinine, blood urea nitrogen, serum aspartate 
aminotransferase,  serum alanine  aminotransferase,  and serum total bilirubin 
 
8.4 
Echocardiogram. 
 
8.5 
Urine catecholamine  levels. 
 
8.6 
Bone marrow aspirates from bilateral anterior and bilateral posterior iliac  crests, 
and biopsies from any two separate sites.  The specimens are studied by: 
 
▪ Standard histochemical  methods for the presence of tumor cells. 
 
▪ Immunocytology  using  a  panel  of  monoclonal  antibodies  that  react  with 
antigens  associated with neuroblastoma  (Dr. Cheung,  Research Lab). 
 
 
8.7 
CT or MRI of primary-   tumor site, plus other specific or suspected sites of tumor. 
 
8.8 
Scintigraphic  studies  (99mTc-MDP-bone scan or MIBG scan92 or PET scan.93). 
 
 
9.1 
TREATMENT/INTERVENTION  PLAN 
 
9.2 
Schedule: The total dosage of 3F8 per cycle is the same as in prior trials (100 
mg/m2), administered at 20 mg/m2/day and infused  over ~1.5 hr or less (0.5  hr is customary), 
with analgesics and antihistamines used as needed for expected side-effects. 3F8 is started ~1 hr 
after completion of GM-CSF administration. GM-CSF is dosed at 250 mcg/m2/day from day –5 
to day +1 (Wednesday to Tuesday is customary) , and is 500 mcg/m2/day thereafter (i.e., on days 
+2 to +4; Wednesday to Friday), as in the predecessor protocol.18,74 Patients come off study if 
progressive disease occurs or if there is life-threatening grade 4 toxicity from 3F8; otherwise, 
patients will receive a minimum of 4 cycles of treatment and will continue treatment through 24 
months.    It is expected that patients will receive ~10 cycles. 
For Group 1 patients (enrolled on study for treatment of primary refractory disease), the 
break between end of a cycle of 3F8/GM-CSF and start of next cycle is 2-to-4-weeks through 4 
cycles after achievement of CR in BM; subsequent breaks are ~8 weeks. In this  group, 
isotretinoin is started after documentation of response to, and after >2 cycles of, 3F8/GM-CSF. 
Road map/schema  is in section 4.2. 
For Group 2 patients (enrolled on study in CR/VGPR, i.e., with no evidence of disease), 
the  break  between end  of a  cycle  and  start  of next  cycle  is  2-to-4  weeks through 4 cycles; 

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subsequent breaks are ~8 weeks. Isotretinoin is started after cycle 2 of 3F8/GM-CSF. Road 
map/schema is in section 4.2. Regarding patients in second or greater CR from relapse in the 
central nervous system, if they develop early HAMA which precludes timely completion of the 
minimum of 4 cycles of 3F8/GM-CSF, they are eligible to go off protocol, to be treated with 
low-dose maintenance regimens of irinotecan,94 temozolomide,95  or  the  two  agents combined;96 
they can resume treatment with 3F8/GM-CSF if HAMA becomes negative. 
Patients who develop early HAMA which precludes timely treatments with 3F8/GM-CSF 
(>4 week delay) are eligible to receive low-dose maintenance regimens such  as  irinotecan 
alone,94  temozolomide  alone,95  irinotecan-temozolomide,96   or   cyclophosphamide-topotecan.97 
They can also receive anti-HAMA agents such  as  rituximab  and  cyclophosphamide.  They 
resume treatment  with 3F8/GM-CSF if HAMA becomes negative. 
9.3 
3F8/GM-CSF treatment  schedule (one cycle). 
 
Days -5 to -1: 
GM-CSF 250 mcg/m2/day,  subcutaneously. 
 
Days 0 and +1:   GM-CSF 250 mcg/m2/day,  subcutaneously. 
3F8 20 mg/m2/day by iv infusion over ~1.5 hr or less (usually  0.5 hr). 
 
Days +2 to +4:   GM-CSF 500 mcg/m2/day,  subcutaneously. 
3F8 20 mg/m2/day by iv infusion over ~1.5 hr or less (usually  0.5 hr). 
 
Note: The daily GM-CSF is not administered if the ANC is >20,000/ul. The ANC count 
from the previous treatment day will be used  to  determine  whether  GM-CSF  will  be 
administered  or  not.    If HAMA  develops,  cycles  are  deferred  until HAMA  titer  decreases to 
<1000 Elisa units/ml. Emla cream (lidocaine 2.5% and prilocaine 2.5%) can be used to prevent 
pain from GM-CSF shots. 
 
9.4 
Isotretinoin is administered at 160 mg/m2/d, divided into  two doses,  x14 days. 
This treatment can be repeated after a minimum rest period of 14 days, for a total of 6 cycles. It 
is not taken on the same days as 3F8. All patients receive a minimum of 2 cycles of 3F8/GM- 
CSF before starting isotretinoin (only to be started after cycle 1 if HAMA develops  and 
precludes timely administration of cycle 2). 
10.1 EVALUATION  DURING TREATMENT/INTERVENTION  (TABLE 4) 
 
Note: If for any reason tests cannot be completed on day 0, they will be completed on 
day 1 
 
10.2 
CBC on days 0 and +3 (Mondays and Thursdays). If ANC is >10,000/ul, CBC is 
repeated on next day. 
 
10.3 
C3 and CH50 on days 0 and +4 (Monday and Friday) of each cycle. 
 
10.4 
HAMA is approximately every 1-2 months while on study (Dr. Cheung, Research 
Lab). 
 
10.5 
Liver and kidney function blood tests on days 0 and +4 of each cycle. 

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10.6 
BM studies (section 8.6) at end of cycles 2 and 4 and after the 6th cycle of 
Accutane.  Subsequently,  BM studies are repeated in conjunction with MIBG or PET scan 
(section 10.8) through 2 years in patients with history of BM or cortical bone involvement,  but 
are repeated ~every 6 months in other patients (e.g., patients who were stage 4 by virtue  of 
metastases in distant lymph nodes). 
 
10.7 
CT or MRI of primary site approximately  every 3 months through 1 year. 
 
10.8 
99mTc-MDP-bone   scan   approximately   every   3   months   while   on   study   until 
normal. 
 
10.9 
MIBG or PET scan approximately  every 3 months through  2 years.92,93 
 
10.10 Urine catecholamine  levels  approximately  every 3 months through 2 years. 
 
10.11 Pregnancy screen (females  of child-bearing age) before starting  cis-retinoic  acid. 
 
11.1 
TOXICITIES/SIDE  EFFECTS 
 
11.2 
Toxicities  are  graded  by the  Common Toxicity Criteria  (Version 2.0) developed 
by the National Cancer Institute  (NCI) of the USA. 
 
11.3 
3F8: Reversible side-effects include pain, paresthesia, hypertension, hypotension, 
tachycardia, urticaria, fever, nausea, emesis, and rarely, diarrhea, serum sickness, 
hyponatremia,   somnolence   and   posterior   reversible   encephalopathy    syndrome 
(PRES). Other potential side-effects that may occur are bronchospasm, anaphylaxis, 
peripheral neuropathy, impaired  accommodation of the eye,  and poor reactivity of pupils 
to light. 
 
11.4 
GM-CSF: Common side-effects include  bone pain, flushing,  local reaction at site 
of  injection,  leukopenia  shortly  after  injection,  and  decrease  in  platelet  count.98 
Rare 
side-effects  (predominantly  in  adults)  include  allergic  reactions,  weight  gain,  pleural  or 
pericardial  effusion,  pericardial  embolism,  thrombosis,  and  difficulty  breathing  after  first 
injection. 
 
11.5 
Isotretinoin: 
Dry 
skin, 
cheilitis, 
dry 
eyes, 
hypercalcemia, 
pseudotumor 
cerebri, hepatotoxicity,  teratogenic  effect on fetus, depression, suicidal ideation. 
 
 
12.1 
CRITERIA FOR THERAPEUTIC  RESPONSE/OUTCOME  ASSESSMENT 
 
12.2 
Response duration is calculated  from first day of treatment with 3F8. 
 
12.3 
Group 1 patients (i.e., patients treated for primary refractory disease) are 
considered a treatment  failure  under this protocol if progressive  disease develops. 

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12.4 
Group  2  patients  (i.e.,  patients  in CR/VGPR) are considered a treatment failure 
under this protocol if progressive  disease develops. 
` 
12.5 
Disease  status  is defined  by the International Neuroblastoma Response Criteria,91 
supplemented  by RT-PCR findings  in bone marrow (section 8.6) 
 
▪ Complete response/remission (CR): no evidence of disease. 
 
▪ Very good partial response/remission (VGPR): >90% decrease in all 
disease parameters, except bone scan unchanged or  improved;  bone 
marrow must be free of disease. 
 
▪ Partial response/remission: >50% decrease in  all  disease  parameters, 
except bone scan unchanged or improved; no more than 1 positive bone 
marrow site. 
 
▪ Mixed response: >50% decrease in >1 but not all disease markers. 
 
▪ Stable disease: <50% decrease in all tumor markers. 
 
▪ Progressive  disease: new lesion, or >25 % increase in any disease marker. 
 
12.6 
Adequacy  of  trial:  All patients  who  fulfill the  eligibility requirements  and  receive 
a first dose of 3F8 will have an adequate trial. 
 
 
 
13.1 
CRITERIA FOR REMOVAL FROM STUDY 
 
13.2 
Group 1 patients come off study if progressive disease develops at any time after 
cycle 1. (see section 12.2). 
 
13.3 
Group 2 patients come off study if progressive disease develops at any time after 
cycle 1 (see section 12.3). 
 
13.4 
Life-threatening grade 4 toxicity clearly attributable  to 3F8. 
 
13.5 
The investigators will make every reasonable effort to keep each patient  in the 
study until all planned  treatments  and  assessments  have  been performed.  The  investigators 
may discontinue  study drug treatment  for the following reasons: 
 
• Adverse events, including unacceptable toxicity or exacerbation of underlying disease, 
associated with study drug administration and necessitating discontinuation of treatment. 
Patients who are removed from the study due to adverse events will be  treated  and 
followed according to established, acceptable medical practice. All pertinent information 
concerning the outcome of such treatment will be entered in the Case Report Form or on 
the Serious Event Report, as applicable. 

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• Withdrawal of consent. The patient's desire to withdraw from the study may occur at 
any time. The investigator should carefullly consider whether the patient’s withdrawal of 
consent is due to an adverse event, and if so, record the adverse event as the reason for 
withdrawal. 
 
• Withdrawal by the physician for clinical reasons not related to study drug treatment, for 
example, clinical need to administer a concomitant medication that is excluded by the 
protocol, in the absence of an adverse event. 
 
• Violation of the study protocol, including failure  to return for required treatments  or 
assessments.  Patients who fail to return for treatments  will be withdrawn from the study 
if more than 4 scheduled doses are missed 
 
13.6 Patients will not be removed from study in order to receive radiotherapy. 
 
 
14.0     BIOSTATISTICS 
 
Clinical results: This phase II trial assesses the anti-NB activity of 3F8 plus sc GM-CSF. 
The response endpoints are CR of BM disease for patients with primary refractory NB (Group 
1), and two-year relapse-free survival for patients treated in CR/VGPR (Group 2). We expect to 
accrue 15 patients per year in each group.  This trial can be completed within 3 years. 
 
Group 1: 3F8 plus iv GM-CSF (IRB protocol 94-018) yielded an overall two-year 
relapse-free Kaplan-Meier survival probability of 13% in 31 patients with primary  refractory 
disease. With the new treatment using 3F8 plus subcutaneous GM-CSF, we will consider a two- 
year relapse-free survival probability of 0.13 as not promising and 0.25 as promising. For a one 
stage design with type I error 0.05 and 0.85 power, 70 patients will be required. If 12 or more 
patients are relapse-free at two years, the treatment will be defined as promising, and an 
additional 15 patients (total: 85) will be enrolled  with the goal of strengthening the findings. 
 
Group 2: 3F8 plus iv GM-CSF (IRB protocol 94-018) yielded a two-year relapse-free 
Kaplan-Meier survival probability of 55% for patients treated in first CR/VGPR. With the new 
treatment, we consider a two-year relapse-free survival probability of 0.55 as not promising and 
1.70 as promising. For a one stage design with type I error 0.05 and 0.85 power, 75 patients will 
be required. If 49 or more patients are relapse free at two years, the treatment will be defined as 
promising. Two separate cohorts will be accrued: 75 patients in first CR/VGPR after transplant 
and 75 patients in first CR/VGPR after conventional chemotherapy + 13-cis-retinoic acid and no 
transplant. Up to 85 patients in each cohort will be accrued with the aim of strengthening the 
findings. 
Patients treated in >2nd remission, long-term relapse-free survival is <5%, including  0/7 
treated with 3F8 plus iv GM-CSF (all 7 relapsed within 15 months). Therefore, we consider a 
two-year relapse-free survival probability of 15% as promising and 1% as  not  promising. 
Patients will be divided into two groups, those with and without  prior antibody therapy: 
* 
For those without prior antibody treatment or other immunotherapy, Simon's two- 
stage optimal design will be used with a power of 90% and an alpha of 5%. For a total of 33 

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subjects, 18 will be accrued during stage 1 and 15 during stage 2. The trial stops early if none of 
the first 18 patients survive for two years without relapse. Otherwise, the trial continues until 33 
patients are accrued. Treatment will be considered promising if two or more patients experience 
two-year relapse-free survival and accrual can continue up to 50 patients with the goal of 
strengthening the findings 
* 
Patients with prior antibody treatment or immunotherapy are less common and so 
only a trial with lower power is feasible. We will accrue 19 patients, declaring treatment  as 
promising if two or more experience two-year relapse-free survival. This design has a power of 
80% and an alpha of 5%, a null hypothesis of 1% and an alternate hypothesis of 15%. Only a 
one-stage design is feasible with these characteristics as an observed zero response rate would 
consistent with a true response rate of 15% for an interim analysis conducted at all possible 
stopping points up  to the 18th patient. If the findings are favorable,  accrual can increase to 35, 
with the aim of strengthening  the conclusions. 
* 
Patients who went off protocol to receive therapy for relapse in  the  central 
nervous system, or to receive additional chemotherapy because of HAMA (section 9.1), are not 
included  in the statistical  analyses for these groups. 
 
Utility of RT-PCR: The association between histologic examination and real time RT- 
PCR regarding BM status can be assessed by the Wilcoxon statistic. The clinical significance 
(relapse-free survival) of histologic results and real time RT-PCR findings in both groups can be 
assessed by log-rank test or Cox regression model or time dependent Cox regression model. 
15.1 
SUBJECT REGISTRATION AND RANDOMIZATION PROCEDURES 
 
15.2 Research Participant  Registration 
 
Confirm eligibility as defined in the section entitled  Criteria for Patient/Subject 
Eligibility. 
 
Obtain informed  consent, by following procedures defined in section entitled  Informed 
Consent Procedures. 
 
During the registration process registering individuals  will be required to complete a 
protocol specific  Eligibility Checklist. 
 
All participants  must be registered through the Protocol Participant  Registration (PPR) 
Office at Memorial Sloan-Kettering Cancer Center. PPR is available  Monday through 
Friday from 8:30am – 5:30pm at 646-735-8000. The PPR fax numbers are (646) 735- 
0008 and (646) 735-0003. Registrations  can be phoned in or faxed. The completed 
signature  page of the written consent/verbal script and a completed Eligibility Checklist 
must be faxed to PPR. 
 
16.1 
DATA  MANAGEMENT ISSUES 
A Research Study Assistant (RSA) will be assigned to the study.  The responsibilities  of 
the RSA include  project compliance,  data collection,  abstraction and entry, data 
reporting,  regulatory monitoring,  problem resolution and prioritization,  and coordinate 
the activities  of the protocol study team. 

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The data collected for this study will be entered into a secure database.  Source 
documentation will be available  to support the computerized  patient record. 
 
16.2 
Quality Assurance 
 
Weekly registration reports will be generated to monitor  patient accruals and 
completeness  of registration data.  Routine  data quality reports will be generated to assess 
missing data and inconsistencies.   Accrual rates and extext and accuracy of evaluations 
and follow-up  will be monitored  periodically throughout  the study period and potential 
problems will be brought to the attention of the study team for discussion and action 
 
Random-sample  data quality and protocol compliance  audits will be conducted by the 
study team, at a minimum  of two times per year, more frequently if indicated. 
 
16.3 
Data and Safety Monitoring 
 
Institutional monitoring plan for phase I and phase II trials:  Memorial  Sloan-Kettering 
Cancer Center (MSKCC) has established standard procedures for data safety monitoring 
of clinical research (see appendix). For Phase II trials, these procedures include 
consideration of accrual rates, toxicity, adherence to  dose-escalation schedules,  adverse 
event notification and data recording. Therapeutic responses are logged on a central 
database and approximately 50% are reviewed by an independent committee. An annual 
report of the trial’s progress is sent to the IRB. Alll   of the procedures for Phase II studies 
outlined in MSKCC’s policy are applicable to the current trial and will be followed by 
the investigators. The analysis of safety will include all patients who receive at least one 
dose of study medication. Adverse events, including all toxic effects of treatment, will be 
tabulated individually, and summarized by body system, according to dosage of study 
medication (single dose as well as cumulative doses of 3F8 and GM-CSF), and according 
to severity or toxicity grade. Laboratory data will be tabulated and summarized by 
descriptive  statistics,  as well as on the basis of MSKCC specified normal ranges. 
 
The policies and procedures for data safety and monitoring of clinical research at 
Memorial Sloan-Kettering Cancer Center are detailed in Appendix (Data Safety and 
Monitoring of Clinical  Research). 
 
17.1 
PROTECTION  OF HUMAN SUBJECTS 
 
The investigator agrees to conduct this study in accordance with the International Conference 
on Harmonization (ICH) principles of Good Clinical Practice and with the  Declaration  of 
Helsinki (1989). The investigator will conduct all aspects of this study in accordance with all 
national,  state, and local laws of the applicable  regulatory agencies. 
 
Most patients will be children, adolescents, and young adults because of the nature of these 
tumors. Patients of both sexes and all ethnic backgrounds are eligible for this  study. 
Alternative treatments are available and will be discussed with patient or  legal  guardian. 
Patients are responsible for the costs of physician visits and usual laboratory tests, 
hospitalizations,  and  outpatient  care.    If there  is  an injury as a result of this research study, 

Memorial Sloan Kettering Cancer Center 
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Page 24 of 34 
 
 
 
 
emergency care, hospitalization, and outpatient care will be made available by Memorial 
Hospital and billed to the patient as part of the medical expenses. No money will be provided 
by Memorial Hospital as compensation for research-related injury. 
 
17.2 
Privacy 
 
MSKCC’s Privacy Office may a lllow the use and disclosure of protected health 
information pursuant to a completed and signed Research Authorization form.  The use 
and disclosure  of protected health information will be limited  to the individuals  described 
in the Research Authorization form.  A Research Authorization form must be completed 
by the Principal Investigator  and approved by the IRB and Privacy Board. 
17.3 Serious Adverse Event (SAE) Reporting 
 
Any SAE must be reported to the IRB as soon as possible but no later than 5 calendar 
days. The IRB requires a Clinical Research Database (CRDB) AE report to be  submitted 
electronically to the SAE Office at sae@mskcc.org.  The report should contain the 
following information: 
Fields populated from the CRDB: 
 
• Subject’s name (generate the report with only initia ls  if it will be sent outside of 
MSKCC) 
• Medical record number 
• Disease/histology (if applicable) 
• Protocol number and title 
Data needing to be entered: 
• The date the adverse event occurred 
• The adverse event 
• Relationship  of the adverse event to the treatment  (drug, device, or intervention) 
• If the AE was expected 
• The severity of the AE 
• The intervention 
• Detailed text that includes  the following information: 
o A explanation of how the AE was handled 
o A description of the subject's condition 
o Indication if the subject remains on the study 
o If an amendment  will need to be made to the protocol and/or consent form 
The PI’s signature  and the date it was signed are required on the completed report.  
For IND/IDE protocols: 
The CRDB AE report should be completed as above and the FDA assigned IND/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. 

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All SAEs must be entered into the CRDB SAE form page. 
 
SAEs are defined as grade 4 toxicities other than fatigue, weight loss or weight gain, 
anorexia, nausea, anxiety, constipation, urinary retention from opioid analgesics, 
somnolence/hallucinations/ disorientation/confusion/ 
agitation/anxiety from 
anti- 
histamine and opioid premedications, hypomagnesemia, fever, rash, dry skin from 
accutane, urticaria from 3F8 or GMCSF,  myelosuppression from the  combination 
of 3F8 and GM-CSF and pain and transient hypoxia from opioids, breathholding, 
with or without transient oxygen requirement. Preexisting conditions  e.g.  hearing 
loss, hyperbilirubinemia or elevated LFTs from TPN, alopecia are not counted as 
SAEs. 
 
 
18.1 
INFORMED CONSENT PROCEDURES 
 
Before protocol-specified  procedures are carried out, consenting professionals  will 
explain full details of the protocol and study procedures as well as the risks involved  to 
participants  prior to their inclusion in the study. Participants  will also be informed  that 
they are free to withdraw  from the study at any time. All participants  must sign an 
IRB/PB-approved consent form indicating their consent to participate.  This consent form 
meets the requirements  of the Code of Federal Regulations  and the Institutional Review 
Board/Privacy Board of this Center. The consent form will include  the following: 
 
 
1. The nature and objectives, potential risks and benefits  of the intended study. 
2. The length of study and the likely follow-up  required. 
3. Alternatives  to the proposed study. (This will include  available  standard and 
investigational therapies. In addition,  patients will be offered an option of 
supportive  care for therapeutic  studies.) 
4. The name of the investigator(s)  responsible  for the protocol. 
5. The right of the participant  to accept or refuse study interventions/interactions  and 
to withdraw  from participation at any time. 
Before any protocol-specific  procedures can be carried out, the consenting professional 
will fully explain the aspects of patient privacy concerning research specific  information. 
In addition to signing the IRB Informed Consent, all patients must agree to the Research 
Authorization component of the informed  consent form. 
 
Each participant  and consenting professional will sign the consent form. The participant 
must receive a copy of the signed informed  consent form. 

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14. 
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