Conditions: Melanoma
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 8
Healthy volunteers: no

Brief summary:
Randomized trial to determine if neo-adjuvant subcutaneous GM-CSF restores the host regional lymph node immunity

Detailed description:
The sentinel lymph nodes in patients with melanoma are immunosuppressed and the investigators have shown this occurs early in the disease process. This regional nodal immunosuppression precedes nodal metastasis and may be required for nodal spread. Administration of GM-CSF has been used to alter the immune response to metastatic melanoma. The investigators propose to assess whether administration of a short course of GM-CSF preoperatively to patients about to undergo wide local excisions and sentinel lymph node dissection can alter the immune environment of the sentinel lymph node and restore an immune surveillance profile in the sentinel lymph node.

Arms:
arm 1: Patients will be treated with 14 days of GM-CSF self-administered subcutaneously daily for 14 days in a dose of 125 µg/m2 beginning on the day of enrollment. Patients will be instructed in the self-administration of GM-CSF and after they have demonstrated competency with the procedure, they will self-administer the treatment at home. Patients will undergo surgery within 1 day to 5 days after cessation of the GM-CSF therapy. arm 2: no neo-adjuvant therapy prior to surgical intervention

Interventions: intervention 1: GM-CSF intervention 2: Standard of Care
Intervention descriptions: intervention 1: 14 days in a dose of 125 µg/m\^2 intervention 2: No neo-adjuvant therapy prior to surgical intervention

Protocol document:
Prot_SAP_000.pdf: 
 
 
Neoadjuvant GM-CSF and 
Modulation of the Immune Cell 
Profile of the SLN in Primary 
Cutaneous Stage I-III Melanoma 
 
NCT# 02451488 
 
October 5, 2016 


1 
 
 
Neoadjuvant GM-CSF and Modulation of the Immune Cell Profile  
of the SLN in Primary Cutaneous Stage I-III Melanoma 
 
 
1Aodhnait S Fahy B.M.B.Ch., Ph.D, 1Travis E. Grotz, M.D., 2Svetomir N. Markovic M.D. 
Ph.D., Vera Suman, Ph.D, Alexey Leontovich, Ph.D, 2Rachel Maus,  1James W. Jakub, M.D 
 
1Department of Surgery, 2Division of Hematology in the Department of Medicine and 
3Department of Oncology, Mayo Clinic, Rochester. 
 
VERSION #3:     October 5, 2016 
 
CONTENTS 
Protocol Synopsis 
Introduction 
  
Background 
Aims 
 
Hypothesis 
GM-CSF Intervention 
 
GM-CSF Immunological Effects 
GM-CSF Clinical Safety 
GM-CSF Details of Drug-Related AEs 
 
GM-CSF:  Drug Related SAEs 
GM-CSF: Drug-related deaths 
GM-CSF - Clinical Efficacy 
GM-CSF - Overall Risk/Benefit Assessment 
Study Objectives 
 
Primary objectives 
 
Secondary objectives 


2 
 
Study Design 
 
Inclusion Criteria 
 
Exclusion Criteria 
 
Study Scheme 
Details of Procedures 
 
Recruitment 
 
Preoperative treatment 
 
Surgery 
 
Tissue analysis 
 
Followup 
 
Timeline 
GM-CSF Administration and Safety 
 
Storage of GM-CSF 
 
Handling of GM-CSF 
 
Administration of GM-CSF 
 
Disposal of GM-CSF 
Trial Safety 
 
Adverse event reporting  
 
 
Serious adverse events  
Nonserious adverse events 
Outline of frequency and nature of likely side effects for GM-CSF 
   
Outline of frequency and nature of likely side effects of additional SLN harvest 
Safety Assessments  
Stopping rules for patients 
Stopping rules for the trial 
Prohibited therapies 
Statistical considerations 
 
Randomization 
 
Primary endpoint 


3 
 
 
Sample size and power 
 
Analysis Plan 
Projected Results 
References 
Appendix 
GM-CSF Pharmacology 
GM-CSF Preclinical Toxicology 
GM-CSF Pharmacokinetics in Normal Subjects 
 
 
PROTOCOL SYNOPSIS 
Protocol Title 
Neoadjuvant GM-CSF treatment and modulation of the immune 
cell profile of the SLN in  melanoma (as above) 
# Site and Names: 
One Site: Mayo Clinic Rochester 
Study Schema: 
Twenty patients with a primary melanoma 1-4mm Breslow depth 
will be randomized to either undergo standard care (10 patients), 
or be treated with 14 days of GM-CSF administered 
subcutaneously daily in a dose of 125 µg/m2 preoperatively (10 
patients), prior to sentinel lymph node biopsy. The regional lymph 
nodes will undergo detailed analysis defining their immune 
features  
Trial Objective: 
To characterize and compare the regional nodal immune features 
of the GM-CSF treated patients, including the Th1/Th2 
environment, with the regional immune environment of the 
control cohort  
 
Study Design: 
Open-label, single-center, randomized controlled trial 
Accrual Goal: 
20 patients 
Accrual Rate: 
1-2 patients per month 


4 
 
Followup: 
30 days postoperatively 
Inclusion Criteria: 
• Histologically confirmed, 1-4mm Breslow depth 
melanoma 
• Clinical stage I or II disease 
• Undergoing sentinel lymph node biopsy 
Exclusion Criteria 
• Clinical stage III or IV disease 
Immunosuppressive therapy in the past 6 weeks (e.g. 
high-dose steroids, methotrexate, anti-TNF, IL-1Ra 
antagonist) 
• Pregnant or nursing women 
• Patients under 18 
Analysis 
Immunologic features of the sentinel lymph node will be 
evaluated by FACS and RT-PCR. 
Patient safety of GM-CSF will be evaluated using the NCI CTCAE 
criteria, version 4.0. Patients will be encouraged to keep a diary of 
events during GM-CSF administration and will be encouraged to 
report any symptoms or adverse events to the study team.  
 
Statistics 
The study will be a double-arm open label randomized study 
including 20 evaluable subjects. A sample size of 10 in each group 
will have 80% power to detect an effect size of 1.325 on mRNA 
expression and cell type prevalence in the sentinel node using a 
two group t-test with a 0.050 two-sided significance level. The 
effect size is the expected difference in the means divided by the 
pooled standard deviation.  It is estimated that accrual will be 
completed in 18 months. 
 
 
INTRODUCTION 


5 
 
 
Background 
The prognosis for patients with melanoma and lymph node involvement is highly 
variable, with the 5-year survival ranging from 40-78% (Nading et al., 2010). A 
proportion of this variability is thought to be attributable to the host immune response 
to the disease process, with high T cell infiltration correlating with improved survival 
(Cochran et al., 2006). The sentinel lymph node (SLN) is in direct communication with 
the primary tumor via the afferent lymphatics (Cochran et al., 2006).  This site of direct 
downstream drainage of tumor associated antigens should allow for a robust host 
immune response; however, we have demonstrated evidence of Th2 polarization and 
early signs of immunodysfunction within the SLN of patients with melanoma (Grotz et 
al., 2012; Mansfield et al., 2011).  This proposal builds off of our ongoing research and is 
the next logical step in not only defining but reversing this immune perturbation.  This 
study aims to reverse this early immune dysfunction and preserve the immune 
regulation in melanoma, potentially leading to better clinical outcomes.  
 
We have previously demonstrated that SLNs in melanoma are fundamentally 
immunologically distinct from normal lymph nodes; the SLNs consistently have less 
expression of Th1 transcription factor T-bet and almost complete absence of cytotoxic T 
cells  and mature antigen presenting cells (CD86+) (Grotz et al., 2012; Mansfield et al., 
2011). Th2 cytokines depress macrophage and T cell activation and function, and as such 
are independent predictors of poor prognosis in melanoma (Cochran et al., 2006).  We 
have shown this regional nodal immune dysfunction occurs in cases with and without 
evidence of lymph node metastases (Mansfield et al., 2011). In particular, 
immunohistochemical analysis indicated that cytotoxic T cell accumulation within the 
SLN declines early in melanoma and that the degree of CD8 depletion corresponds to 
the risk of disease recurrence (unpublished data). Furthermore, there is a Th2 skewed 
cytokine expression within the SLNs of patients with melanoma compared to control 
lymph nodes. These changes precede SLN metastasis and we theorize are necessary for 
regional spread of disease. Other studies have confirmed that exposure of peripheral 


6 
 
blood monocytes to melanoma produces a Th2-type cytokine profile. Cytokine analysis 
of SLN from melanoma demonstrates an initial increase in GM-CSF observed in SLNs 
could lead to the attraction of a high number of dendritic cells (DC to SLNs). However, 
the presence of immunosuppressive molecules, such as IL-10 and COX-2, could block 
their maturation and prohibit their ability to become efficient antigen presenters. 
Restoring the immune profile of the SLN to a normal state preoperatively may allow a 
robust host response and offer improved regional control, disease free survival and 
potentially overall survival to patients with melanoma. In this study we plan to build off 
of our prior work and improve the immunologic state of the SLN in patients undergoing 
definitive surgery for primary melanoma by administering neoadjuvant therapy with 
granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF has been shown 
to be safe for human administration and has been previously postulated to exert an 
anti-tumor effect by activating macrophages, T cells and dendritic cells (Pan et al., 
2004). Analysis within animal models has demonstrated GM-CSF can prompt reversal of 
the early melanoma SLN immunomodulation in mouse B16 melanoma models (Pan et 
al., 2004). Furthermore, Vuylsteke et al. found that preoperative intratumoral injections 
of GM-CSF resulted in larger T-cell areas within the SLNs resulting in upregulation of 
expression of dendritic cell markers and improved outcomes (Vuylsteke et al., 2004), 
while Vaquerano showed regression of in-transit melanoma metastases with 
intralesional GM-CSF (Vaquerano et al., 1999). Most recently, our group has 
demonstrated improved overall survival in patients with resected advanced stage III 
disease who received adjuvant GM-CSF compared with a control population who 
underwent similar surgical management but were observed (Grotz et al., 2013).  
Therapies that reverse peripheral tolerization, eradicate suppressive regulatory cells, re-
establish a Th1 cytokine milieu and/or prevent lymphangiogenesis are likely to make a 
profound impact on the outcomes of patients with melanoma. Immune modulation of 
the SLNs and regional lymphatics offers a promising treatment option for patients. The 


7 
 
use of these agents before surgical resection and SLN biopsy presents a unique 
translational setting to study the immune enhancing effects of these therapies in vivo. 
Hypothesis 
We hypothesize that the regional immune cell profile of the SLN will be modified 
preoperatively by neoadjuvant use of GM-CSF, and that this preoperative modulation 
will restore the host regional immune surveillance towards a healthy Th1 dominant 
state.  
 
Aims 
- 
To characterize the immune cell environment of the SLN in patients with 
melanoma following the preoperative administration of GM-CSF  
- 
To determine if the SLN Th1/Th2 ratio is restored in patients with clinically node 
negative melanoma receiving a short course of neoadjuvant GM-CSF compared 
with the immune environment of the SLN in a control untreated population.  
 
 
GM-CSF INTERVENTION 
 
GM-CSF Immunologic Effects 
GM-CSF (sargramostim, Leukine, Genzyme Corporation, Cambridge, MA) is a 
hematopoietic growth factor which stimulates proliferation and differentiation of 
hematopoietic progenitor cells and is approved for this purpose (Leukine, Bayer 2007). 
In addition, GM-CSF has immunologic activities that play a vital role in various diverse 
functions of the immune system. These include its ability to activate macrophages, 
which distinguish tumor cells from normal cells and kill only the tumor cells (Fidler et al., 
1984), stimulation of peripheral blood monocytes in vitro to become cytotoxic for 
human melanoma cells (Grabstein et al., 1986; Thomasson et al., 1989), production of 
monocyte activation and tumoricidal activity following in vivo administration (Chachoua 
et al., 1994; Demir et al., 2003) and stimulation of production of an angiogenesis 
inhibitor by macrophages (Dong et al., 1997). GM-CSF also serves as the principal 


8 
 
mediator of proliferation, maturation and migration of dendritic cells (Young et al., 
1995; Szabolcs et al., 1995; Szabolcs et al. 1996).  Dendritic cells are antigen presenting 
cells that play a major role in the induction of primary and secondary T-cell immune 
responses.  
The recommended dose of GM-CSF for the FDA-approved use of neutrophil recovery 
after chemotherapy in acute myelogenous leukemia is 250μg/m2/day administered 
intravenously or subcutaneously (Leukine, Bayer 2008). However, the dose required to 
achieve the goal of activation of macrophages and monocytes is lower (Demir et al., 
2003; Grabstein et al., 1986). GM-CSF has been used in several clinical trials of adjuvant 
therapy of melanoma and in almost all of these trials the dose has been 125 μg/m2/day 
for 14 consecutive days followed by 14 days off therapy for a period of a year (Daud et 
al., 2008; Elias et al., 2005; Lutzky et al., 2009; Spitler et al., 2000). In one study, the GM-
CSF was given in the same dosing schedule for 3 years (Spitler et al, 2009). A cooperative 
group trial (ECOG 4697), in which 800 patients were randomized, has just been 
completed and used the 125ug/m2/day dose of GM-CSF for a period of 1 year (Lawson 
et al., 2006). 
 
We propose to use the dose of GM-CSF that has been used in the clinical trials of 
adjuvant therapy of melanoma, 125 μg/m2/day for 14 consecutive days before patients 
undergo their definitive surgery (wide local excision and sentinel lymph node biopsy).  
 
GM-CSF Pharmacology/pharmacokinetics/Toxicology - see appendix 
 
GM-CSF Clinical Safety 
GM-CSF has been under clinical investigation since 1987 in many countries and has been 
marketed in the United States under the trade name Leukine® since 1991. Thus, there 
has been extensive experience with the use of this product and it has generally been 
regarded as safe. The side effects described in the Package Insert are for doses of GM-
CSF higher than those proposed for this trial. Moreover, the Package Insert describes 


9 
 
side effects for administration of GM-CSF by the intravenous route. In the current trial, a 
lower dose of GM-CSF will be used and it will be administered subcutaneously, not 
intravenously. Accordingly, the side effects described in the Package Insert aren’t those 
expected in this trial.  
 
Ravaud et. al. administered a slightly higher dose of GM-CSF (5 μg/kg) to patients with 
metastatic melanoma in a similar 21-day cycle of 14 days on followed by 7 days rest 
(Ravaud et al., 2001).  This was a randomized Phase II trial in which administration of 
GM-CSF monotherapy was compared to administration of combination therapy of DTIC 
with GM-CSF. Toxicity was manageable and there were no Grade 4 adverse events. 
Additionally, this study was conducted in France with the E.Coli-derived version of GM-
CSF (molgramostim), which has been reported to be more toxic than the yeast-derived 
version (sargramostim) to be used in our study. In studies in patients with hormone 
refractory prostate cancer, Small et. al. used a dose of GM-CSF of 250 μg/m2 for 14 days 
and reported that the data suggest the GM-CSF may have antitumor activity in advanced 
prostate cancer. This is twice the dose that will be used in our study and there were no 
untoward side effects (Small et al., 1999). In a follow-on study, Fong et. al. treated 24 
patients with metastatic, castration-resistant prostate cancer in a phase I trial where 
sequential cohorts were treated on a 28-day cycle with increasing doses of ipilimumab 
and subcutaneous injections of GM-CSF at a dose of 250 μg/m2 for the first 14 days of 
the 28-day cycle (Fong et al., 2009). Toxicity was manageable, even in combination with 
ipilimumab and a GM-CSF substantially higher than our trial.  
 
In a clinical trial including 98 patients with melanoma at high-risk of recurrence, 
adjuvant GM-CSF therapy was well tolerated administered for 14 days on followed by a 
14 day break, for 3 consecutive years (Spitler et al., 2009). Eighty patients (82%) had at 
least one Grade 1 (74%) or Grade 2 (8%) treatment-related adverse event. The most 
common side effects were mild injection site reactions (68%), erythema at the injection 


10 
 
site (57%), and flu-like symptoms (54%). Six patients experienced Grade 3 or 4 adverse 
events but these were all thought to be unrelated to the study drug. 
 
Leukocytosis is expected in patients receiving GM-CSF, even in short courses such as our 
trial, but, to our knowledge, has not been reported to be associated with adverse clinical 
side effects, other than sternal pain associated with bone marrow stimulation. We are 
not aware of thrombocytosis as a result of GM-CSF therapy and have not seen that in 
our experience. Institutionally we have administered GM-CSF at this dose off protocol 
for patients with resected stage III and IV melanoma and published our results in over 
150 patients (Grotz et al., 2013).  Our standard is for patients to receive an education on 
self-administration by an oncology RN and proceed home with the drug and self-
administer.  Off protocol these patients are typically on the drug for months to years 
and we are not aware of any acute toxicity within the first 2 weeks, aside from minor 
irritation at the injection site. 
 
Taken together, these reports indicate that the dose of 125 μg/m2 of GM-CSF for 14 
days is very safe and tolerable. 
 
GM-CSF - Overall Risk/Benefit Assessment 
GM-CSF has been marketed since 1991 and has been used in thousands of patients. It 
has an excellent safety record. It is not thought to provide clinical benefit as 
monotherapy in patients with metastatic melanoma that cannot be surgically excised. 
However, it may provide benefit as monotherapy in the adjuvant setting for patients 
with melanoma after all gross disease is resected (Spitler et al., 2000) and also may 
provide benefit in patients with metastatic melanoma when used in combination with 
other agents (O’Day et al., 2002; Weber et al., 2005). 
 
STUDY OBJECTIVES  
Primary objectives: 


11 
 
• To characterize and compare the regional nodal immune features of the GM-CSF 
treated patients, including the Th1/Th2 environment, with the regional immune 
environment of the control cohort  
 
STUDY DESIGN 
Twenty adult (>18 years old) patients undergoing lymphatic mapping and sentinel lymph 
node biopsy for cutaneous melanoma, Breslow depth 1.00 – 4.00 mm of the trunk or 
extremity, with clinically negative regional lymph nodes will be enrolled. Ten patients 
will be randomized to an intervention arm. These 10 patients will be treated with 14 
days of GM-CSF administered subcutaneously in a dose of 125 µg/m2.The timing of 
starting the drug will be coordinated with an ending date within 1-5 days of their 
surgical date.  
 
Eligible patients will not have a primary or secondary immunodeficiency. Patients who 
are taking immunosuppressive drugs including high-dose steroids, TNF inhibitors, 
methotrexate or IL-1Ra antagonists within the previous six weeks will be excluded. 
Patients who have had prior GM-CSF therapy or chemotherapy within the previous 3 
months will be excluded. Patients with evidence of metastatic disease will be excluded.  
 
All inclusion criteria must be met for a subject to be eligible for participation in this 
study. Patients will be thoroughly informed about all aspects of the study including 
administration of the drug and all regulatory requirements before informed consent is 
sought.  
 
Inclusion Criteria 
To be eligible for the study, patients must satisfy the following criteria: 
 
1) Histologically confirmed primary cutaneous malignant melanoma 
2) 1-4mm Breslow depth  


12 
 
3) Scheduled for sentinel lymph node biopsy as part of their standard surgical 
management  
4) Man or woman, age >/= 18 years 
5) Women of childbearing potential (WOCBP) must be using an adequate method 
of contraception to avoid pregnancy throughout the study and for up to 2 weeks 
after the study in such a manner that the risk of pregnancy is minimized. WOCBP 
include any female who has experienced menarche and who has not undergone 
successful surgical sterilization (hysterectomy or bilateral oophorectomy) or is 
not postmenopausal. Sexually active WOCBP must use an effective method of 
birth control during the course of the study, in a manner such that risk of failure 
is minimized. All WOCBP must have a negative pregnancy test prior to first 
receiving GM-CSF. 
6) Men must agree to use and utilize an adequate method of contraception 
throughout treatment and for at least 2 weeks after study drug is stopped 
7) Patients on the following immunosuppressive medications can be considered for 
accrual (but use of these medications will be documented by the study 
coordinator if used within 6 weeks of surgery) 
a. Aspirin (daily) 
b. Ibuprofen 
c. NSAIDs 
d. Low dose steroids (≤5mg/day) 
e.  
8) All patients must be willing and able to give written informed consent.  
 
Exclusion Criteria 
Subjects meeting any of the following criteria are ineligible for study entry 
1) Clinical stage III or IV disease 
2) Autoimmune disease: Patients with a history of inflammatory bowel disease are 
excluded from this study as are patients with a history of immunologic disease 
(e.g. rheumatoid arthritis, scleroderma, systemic lupus erythematosus, 
autoimmune vasculitis, motor neuropathy considered of autoimmune origin) 
3) Any underlying medical conditions which, in the opinion of the investigator, will 
make the administration of GM-CSF hazardous or obscure the interpretation of 


13 
 
adverse events; such as, psychological, familial, sociological or geographical 
conditions potentially hampering compliance with the study protocol and 
followup schedule 
4) Any vaccination therapy within 4 weeks prior to GM-CSF administration 
5) Concomitant therapy with any of the following within the past 3 months: GM-
CSF, interferon, other non-study immunotherapy regimes; cytotoxic 
chemotherapy 
• 
Immunosuppressive mediations within the past 6 weeks including: 
o High dose steroids (>5mg/day) 
o TNF-inhibitors 
o Methotrexate 
o IL-1Ra antagonist 
6) Active or chronic infection with HIV, hepatitis B or hepatitis C 
7) WOCBP who are unwilling or unable to use an acceptable method to avoid 
pregnancy for the entire study period and 2 weeks after cessation of the study 
drug.  
8) Prisoners or subjects who are compulsorily detained 
 
Study Scheme 
 
 
 
 


14 
 
Patients will be randomized to control or intervention group. Patients will be treated 
with 14 days of GM-CSF self-administered subcutaneously daily for 14 days in a dose of 
125 µg/m2. Patients will be instructed in the self-administration of GM-CSF and after 
they have demonstrated competency with the procedure, they will self-administer the 
treatment at home. Patients will undergo surgery within 1 day to 5 days after cessation 
of the GM-CSF therapy.  
 
Surgical intervention will follow our institutional standard of care, including wide local 
excision of the primary melanoma according to National Comprehensive Cancer Center 
(NCCN) guidelines and SLN biopsy (7).   
 
DETAILS OF PROCEDURES 
This study is divided into five phases (Recruitment, Preoperative treatment, Surgery, 
Laboratory analysis, Followup) with associated procedures that will be performed at 
specific time points as described within the following sections.  
 
Recruitment 
As soon as a patient is considered for this study and prior to any study procedures, the 
patient will have the nature of the study explained to him or her, and will be asked to 
give written informed consent and Health Insurance Portability and Accountability 
(HIPAA) authorization. Informed consent must be obtained prior to any procedures that 
do not constitute part of the patient’s normal care.  
 
Patients will be introduced to the project at their meeting with their surgeon. (S)he will 
outline the project to them and answer any questions. The study coordinator will meet 
with the patient to discuss the trial further and formal consent obtained if the patient 
wishes to be part of the study. Following informed consent and after the patient decides 
to participate in the study, the patient will be randomized into either the intervention or 
control arm.  


15 
 
 
The following checks will be completed by the surgeon and by the study coordinator.  
• Eligibility confirmation 
• Histologic diagnosis of malignant melanoma (based on original biopsy, no new 
biopsies required) 
• Medical history 
• Physical examination  
• Concomitant medication review 
 
A surgical date will be set and based on this, patients enrolled in the GM-CSF arm will be 
given written directive on the start and stop date of the GM-CSF.  
 
We will prospectively record the demographic, clinical and pathological data of all 
patients including age, sex, primary location, lymph node basin, if the SLN and non SLN 
analyzed were positive or negative for metastatic disease, Breslow depth, Clark’s level, 
mitotic rate, ulceration, presence of regression, and tumor infiltrating lymphocytes, if 
any residual disease is present at the primary site at the time of WLE and time period 
between original biopsy confirming melanoma and the sentinel lymph node procedure. 
The 10 second radioactive count of the SLNs and if the SLN is blue or not will be 
captured. If the SLN is positive the size of the metastatic focus will be recorded. All SLNs 
will be sent to pathology as is our standard. Only tissue from SLN#1 and if applicable 
SLN#2 will be submitted for research purposes. All data will be stored in a secure file on 
the Mayo intranet.  
 
Preoperative treatment 
Patients who have been randomized to undergo GM-CSF treatment preoperatively will 
be educated in administration of subcutaneous GM-CSF.  
 


16 
 
The intervention group will be issued a 14 day supply of GM-CSF to self-administer daily. 
They will be provided phone numbers and details of who to contact with any concerns 
for adverse events, as well as a diary in which to record time of administration, whether 
entire vial was injected, and any adverse events. Patients will be provided a list of 
adverse events and to call immediately for any grade 3 or high adverse event.  Grade I-II 
AEs will be logged into their diary.  The diary will be collected on the day of surgery. 
 
Patients in the control arm, who have been randomized to not undergo preoperative 
GM-CSF, will not receive GM-CSF in the preoperative phase. 
 
Surgery 
Patients will undergo formal consent for their operative procedure which will be 
separate from the consent for trial enrollment. Patients will undergo sentinel lymph 
node biopsy and the sentinel lymph nodes will be sent to pathology. The lymph node 
(LN) will undergo gross sectioning along the long axis at 1-2mm intervals until the LN is 
exhausted. The sections are then placed on a cryotome where they freeze from the 
bottom up. Prior to freezing, the top of one of these 2mm sections will be divided in half. 
One half will be reviewed by pathologists, the other half will be submitted for research. 
The remaining sections of the lymph node will be examined by standard pathological 
analysis. Sentinel lymph node number one will be the preferred SLN from which tissue 
will be collected, unless it is < 7mm in greatest dimension, in which cases sequential 
SLNs will be evaluated for gross size and ½ of a top will be taken from the next SLN with 
a gross size >7mm as described above.  If all SLNs are <7mm tissue will not be 
collected for research. In addition, if there is any fresh or frozen tissue in excess of 
diagnosis and not critical to the pathologic interpretation this should be submitted for 
research purposes as well. If there is no remaining fresh or frozen tissue then paraffin 
fixed tissue will be collected after pathological diagnosis for this study. Only tissue from 
SLN #1 and if applicable SLN #2 will be submitted for research purposes. The tissue 
specimen will be submitted fresh for research purposes as described below and as is our 
practice with TRAG approval for our currently active study (IRB 10-000806).  
 


17 
 
Laboratory analysis 
Lymph node tissue analysis 
 
The SLN(s) will be dissected free of surrounding fat and then undergo gross sectioning 
along the long axis at 1-2mm intervals.  The sections are then placed on a cryotome 
where they freeze from the bottom up. Prior to freezing, one of these 2mm sections will 
be divided in half  and submitted for research. The remainder of the lymph node will be 
processed, examined and stored per the institutional standard. The fresh tissue 
obtained for research purposes will be collected in a sterile fashion and resuspended in 
RPMI buffer that will be provided by the co-investigator.  The lymph node samples will 
be processed into a single cell suspension using a Milltenyi gentleMACS dissociator 
(Milltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s 
directions. Immune cell profiling and activation statuses will be performed utilizing flow 
cytometry and potentially other phenotyping methods. One approach to assess Th1/Th2 
biasing in T cells of the LN cells, will include lymphocyte suspensions which will be 
stimulated with PMA (5ng/ml) and Ionomycin (0.1 µg/ml) ex vivo and incubated for 4 h 
at 37oC with 5% CO2.  After stimulation, the cells will be positively selected for CD4 T 
cells using Miltenyi magnetic beads as prescribed by the manufacturer (Miltenyi Bergish 
Gladbach, Germany).  The purity of the T cell population will be validated using flow 
cytometry. Total RNA will be isolated from the isolated cell populations using the Qiagen 
RNA extraction kit (Qiagen, Germantown, MD). The quality of the RNA will be evaluated 
by obtaining electropherograms on Agilent 2100 Bioanalyzer and RNA integrity number 
using 2100 Expert software (Agilent Technologies, Santa Clara, CA). cDNA will be reverse 
transcribed from a total of 0.3 µg of RNA using RT2 First strand Kit (Qiagen) per 
manufacturer’s instructions. To make the PCR cocktail the RT2 SYBR Green ROX qPCR 
Mastermix (Qiagen) will be used.  The Th1/Th2/Th3 RT-qPCR arrays (SABioscience, 
Valencia CA) will be used to quantify RNA expression of Th1 and Th2 mRNAs as well as 
housekeeping controls.   
 


18 
 
Furthermore, we will utilize flow cytometry to further investigate the immune cell 
phenotype using the following extracellular, anti-human monoclonal antibodies to 
important immune cell subsets including: anti-CD3 PE-Cy 7, anti-CD8 PE-Cy 5, anti-CD4 
FITC, anti-CD62L PE-Cy 5, anti-CD69 FITC, anti-CD152 (CTLA-4) PE, anti-CD11c PE-Cy 5, 
anti-CD86 PE-Cy 7, anti-HLA-DR PE, anti-CD3 FITC, anti-CD14 FITC, anti-CD16 FITC, anti-
CD19 FITC, anti-CD123 PE-Cy 7, anti-CD141 PE, anti-CD68 PE-Cy 5, anti-CD20 PE, anti-
CD56 PE, anti-CD279 (PD-1) APC, anti-CD11b PE, anti-CD64 FITC (BD Pharmingen, San 
Jose, CA). The human monoclonal antibodies, anti-CD4 PE-Cy 5 and anti-CD25 PE, will be 
purchased from Biolegend (Sand Diego, CA) and used in conjunction with intracellular 
staining with anti-FoxP3 FITC for the quantification of regulatory T cells.  Cell death will 
be determined by propidium iodine antibody (eBioscience, San Diego, CA). Four-color 
flow cytometry will be performed on a Guava 8HT flow cytometer (EMD Millipore, 
Billerica, MA) capturing 25,000 events for all samples. Results will be analyzed using 
Guava Soft Incyte (EMD Millipore, Billerica, MA). 
 
Followup 
All patients will be offered a postoperative visit as is standard and all patients will 
receive a 30 day follow-up phone call from the surgical RN as is our standard.   
 
 
 
 
 
 
Timeline 
 


19 
 
 
 
GM-CSF ADMINISTRATION 
Storage of GM-CSF 
GM-CSF will be stored in a secure area in the pharmacy.  Pharmacy will dispense the 
GM-CSF and necessary supplies for self-administration (syringes, needles, alcohol wipes, 
and sterile water for injection) only after the patient has been consented and 
randomized to the treatment arm. Vials of GM-CSF will be stored refrigerated at 2-8C. 
Lyophilized drug is stable for 36 months at 2-8C.  
 
Handling of GM-CSF 
Patients will be educated on the proper handling and preparing of GM-CSF.   It should be 
prepared using standard precautions for the safe handling of agents applying aseptic 
technique.  
 
Recombinant human GM-CSF should be reconstituted in water for injection USP 
containing 0.9% benzyl alcohol and stored at 2-8C prior to administration. Aseptically 
inject 1.0ml water for injection USP into the vial to dissolve the lyophilized power. The 
diluent should be directed against the side of the vial and the contents gently swirled to 
avoid foaming during dissolution. The reconstituted solution is stable for 14 days at 2-8C. 
Recombinant GM-CSF may be administered immediately or held for a period of no more 
than 14 days in the original vial. Do not freeze after reconstitution. If the GM-CSF 


20 
 
concentrate or solution comes into contact with skin or mucosa, immediately and 
thoroughly wash with soap or water.  
 
Administration of GM-CSF 
Patients will be educated on handling and administration of GM-CSF. The patients will 
be encouraged to call with any questions during the 14 day course of self-administration.  
The patients in the intervention arm will maintain a log, documenting their daily 
injection, time of injection and if the total dose was injected.  If they miss a dose they 
are not to double up the next day.  They will bring the administration log with them the 
day of surgery. 
 
Disposal of GM-CSF 
After final drug reconciliation, unused GM-CSF should be disposed at the site following 
procedures for disposal of anticancer drugs. The investigator will ensure that the 
arrangements have been made for disposal and that procedure for proper disposal are 
present according to the applicable regulations, guidelines and institutional procedures.  
 
TRIAL SAFETY 
Adverse Event Reporting 
We project minimal toxicity from GM-CSF administration, but we will grade for and 
measure as per the National Cancer Institute Common Terminology Criteria of Adverse 
Events, version 4.0 (http://ctep.cancer.gov). An adverse event is defined as any new 
untoward medical occurrence or worsening of a pre-existing medical condition in a 
patient or clinical investigation subject administered an investigational product. Patients 
will be provided phone numbers and details of who to contact with any concerns for 
adverse events, as well as a diary in which to record time of administration, whether 
entire vial was injected, and any adverse events. Patients will be provided a list of 
adverse events and to call immediately for any grade 3 or high adverse event.  Grade I-II 
AEs will be logged into their diary.  The diary will be collected on the day of surgery.  


21 
 
Once the 14 day course is stopped no AE’s from the GM-CSF are expected. All patients 
will be offered a postoperative visit as is standard and all patients will receive a 30 day 
follow-up phone call from the surgical RN as is our standard.   
 
All adverse events will be graded according to the NCI CTCAE version 4.0. The following 
categories and definitions of casual relationship to investigational product as 
determined by a physician should be used for adverse events: 
RELATIONSHIP 
ATTRIBUTION 
DESCRIPTION 
Unrelated to investigational 
agent/intervention 
Unrelated 
The AE is clearly NOT related to the intervention 
Unlikely 
The AE is doubtfully related to the intervention 
Related to investigational 
agent/intervention 
Possible 
The AE may be related to the intervention 
Probable 
The AE is likely related to the intervention 
Definite 
The AE is clearly related to the intervention 
 
  
 
Adverse events can be spontaneously reported or elicited during open-ended 
questioning. The following information will be captured for all AEs: onset, duration, 
intensity, seriousness, relationship to investigational product, action taken and 
treatment required. The investigator will supply the DSMB with any additional 
information.  
 
The following hospitalizations are not considered SAEs in this study 
• Elective surgery planned before signing the consent 
• Admissions per the protocol for the planned medical or surgical procedure 
• Routine health assessment requirement and admission for baseline/trending of 
health status 
 
All AEs will be reported to the department of surgery DSMB at Mayo Clinic, Rochester, 
MN. Any morbidity caused by GM-CSF administration within this cohort will be captured 
and reported from the day of administration through 30 days postoperatively. Specific 


22 
 
risks are detailed below that have been experienced in patients taking long term GM-
CSF use.  It is not anticipated patients will experience side effects beyond local injection 
site irritation and ‘first treatment effect’ as described below, during this 14 day 
administration. 
 
Adverse effects from surgery 
This study involves removing 1 half of one (1-2mm) tissue section from the center of the 
lymph node prior to pathological analysis.  Harvesting alternating 1-2 mm sections of 
sentinel lymph nodes for H&E, IHC and research studies is something that has been 
done in numerous clinical studies and as is part of some institutional standard practices.  
It is accepted that only a small percentage of a lymph node is ever pathologically 
examined.  Much more gets wasted down the cryotome or not seen histologically 
because of sampling issues or stored in blocks then would be submitted in this study for 
research purposes. The research tissue would not be evaluated by the pathologist, 
therefore creating a small risk (1-2%) of understaging patients. A micrometastatic foci, 
while unlikely, could be missed in these cases if the metastasis is less than 1mm in size. 
However, the SLN tissue being reviewed is removed from the patient, therefore there is 
not an increased risk of leaving disease behind as the micrometastatic foci would still be 
removed as part of the surgical procedure. We do not see this risk as any different than 
if the patients were not on study and these shaves were not placed on a slide for 
pathologic review.  
 
Data Safety Monitoring Board 
The Mayo Clinic Department of Surgery data safety monitoring board (DSMB) will meet 
and review the study every six months to assure the safety of the procedure. Specifically, 
the DSMB will evaluate the progress of the trial, including an interim assessment of 
participant recruitment and accrual. We will also report to the DSMB the feasibility of 
the trial, in terms of how many consented patients received the drug from pharmacy, 
were educated on the self-administration technique, how many doses they 
administered per their self-assessment log and how many withdrew. The demographics 


23 
 
of the participants are expected to match the demographics of our melanoma 
population. 
 
Safety assessments 
Safety will be evaluated for all treated patients using the NCI CTCAE version 4.0. Safety 
assessments will be based on medical review of AE reports. Safety will be reviewed by 
the Department of Surgery DSMB with reference to stopping rules for the study as 
below.  
 
Stopping rules for patients 
For GM-CSF, toxicity is assessed according to the NCI CTCAE, Version 4. The major non-
hematologic toxicities expected for GM-CSF are injection site reactions and a flu-like 
syndrome. GM-CSF must be discontinued if the patient has an allergic reaction which, in 
the opinion of the investigator would pose an unacceptable hazard to the patient. GM-
CSF will also be discontinued for any grade 3 or greater AE or any unacceptable toxicity 
attributed to GM-CSF at the request of the patient.  
 
Subjects must be discontinued from any further study therapy for the following reasons: 
• Any clinically significant adverse event (grade 3 or grade 4) 
• Any clinical adverse event which in the opinion of the investigator indicates that 
continued treatment is not in the best interest of the patient 
• Intercurrent illness which would, in the judgement of the investigator require 
discontinuation of the drug 
• Noncompliance with respect to taking the drug  
Patients who have a drug related Grade 3 or greater adverse event at the time of 
discontinuation from study treatment will continue to be followed until resolution of 
the event or until the event is considered irreversible. 
 


24 
 
Subjects must be discontinued from the study therapy AND withdrawn from the study 
for the following reasons: 
• Withdrawal of informed consent for any reason 
• Patient request to withdraw from the study 
• Pregnancy 
• Termination of the study 
• Imprisonment or the compulsory detention of the patient.  
 
Stopping rules for the study 
The trial will be stopped if 3 consecutive patients or at any time after 6 patients have 
been treated, if 30% of patients, have been required to be removed from the study due 
to drug-related toxicities, accrual will be halted and the study reviewed by the DSMB.  
 
Prohibited therapies 
Patients may not use any of the following during the study: 
• Immunosuppressive agents, except if needed to treat AEs or provided during 
anesthesia administration 
• Chronic systemic corticosteroids 
• Any non-oncology vaccine therapies used for the prevention of infectious 
diseases 
• Other investigational agents 
 
STATISTICAL CONSIDERATIONS 
Randomization: Patients will be randomized in a 1:1 fashion using a varying permuted 
block randomization (block sizes of 2 and 4).  
Primary endpoint: The primary endpoint is the Th1/Th2 ratio. Th1/Th2 ratio will be 
compared between the two groups. The trial will be considered a success if the Th1/Th2 
ratio is statistically significantly higher in the GM-CSF treated group.  


25 
 
Sample Size and power: There will be 10 patients in each group. It is expected that this 
treatment will have a considerable effect on the Th1/Th2 ratio. If it does not, it will not 
be of further interest. A sample size of 10 in each group will have 80% power to detect 
an effect size of 1.325 using a two group t-test with a 0.050 two-sided significance level. 
The effect size is the expected difference in the means divided by the pooled standard 
deviation. 
Analysis plan: The primary endpoint will be compared between the two groups using a 
two-sample t-test. If the distribution of the Th1/Th2 ratios is considerably skewed, the 
analysis will be done on the log transformed values. If the log transformed distribution is 
also skewed, then a rank-sum test will be used to compare the Th1/Th2 values between 
the two groups. Given the small sample size, all other analyses will be exploratory. 
Continuous measurements will be summarized with means and standard deviations and 
compared between groups using a two-sample t-test (on log transformed data if 
necessary). Categorical variables will be summarized as frequencies and relative 
frequencies and will be compared between groups with a Fisher’s exact test. We will 
also summarize disease-free survival with Kaplan-Meier curves. The adverse event data 
will be reported as frequencies and relative frequencies. The number/proportion of 
patients with Grade 3+ AEs will be compared between the two groups using Fisher’s 
exact test. All tests will be two-sided and a p-value of 0.05 will be considered statistically 
significant.  
 
PROJECTED RESULTS 
We will compare demographic, clinical and pathological variables between the two 
cohorts. Given the unselected nature of this study we anticipate that the two groups will 
be comparable.  
 
We anticipate that the gene expression phenotype (as analyzed by RT-PCR) and the 
immune milieu (as analyzed by flow cytometry) of the SLN in patients who received 
preoperative GM-CSF will have maintained a more Th1 biased immune profile. We 


26 
 
anticipate that the improved Th1:Th2 profile will not only apply to the SLN but to 
neighboring lymph nodes as well.  
 
Data Management Plan  
 
All data will be stored confidentially on the Mayo Clinic server and access will be 
provided on a password protected basis. Data will be processed as stated in protocol 
section titled Data Analysis, Calculations, and Statistical Power.  
 
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Appendix 
 
GM-CSF Pharmacology  
GM-CSF is a human granulocyte-macrophage colony stimulating factor (rhu GM-CSF) 
produced by recombinant DNA technology in a yeast (S. cerevisiae) expression system. It 
is a glycoprotein of 127 amino acids characterized by three primary molecular species 
having molecular masses of 19,500, 16,800 and 15,500 daltons. The amino acid 
sequence of GM-CSF differs from the natural human GM-CSF by a substitution of leucine 
at position 23, and the carbohydrate moiety may be different from the native protein. 
GM-CSF belongs to a group of growth factors termed colony stimulating factors which 
support survival, clonal expansion, and differentiation of hematopoietic progenitor cells 
(Metcalf et al., 1986). GM-CSF induces partially committed progenitor cells to divide and 
differentiate in the granulocyte-macrophage pathways which include neutrophils, 
monocytes/macrophages and myeloid-derived dendritic cells. GM-CSF is also capable of 
activating mature granulocytes and macrophages. GM-CSF is a multilineage factor and, 
in addition to dose dependent effects on the myelomonocytic lineage, can promote the 
proliferation of megakaryocytic and erythroid progenitors. However, other factors are 
required to induce complete maturation in these two lineages. The various cellular 
responses (i.e., division, maturation, activation) are induced through GM-CSF binding to 
specific receptors expressed on the cell surface of target cells (Park et al., 1986). 


31 
 
 
GM-CSF Pre-Clinical Toxicology  
The biological activity of GM-CSF is species-specific. Consequently, in vitro studies have 
been performed on human cells to characterize the pharmacological activity of GM-CSF. 
In vitro exposure of human bone marrow cells to GM-CSF results in the proliferation of 
hematopoietic progenitors and in the formation of pure granulocyte, pure macrophage 
and mixed granulocyte-macrophage colonies. Chemotactic, anti-fungal and anti-
parasitic activities of granulocytes and monocytes are increased by exposure to GM-CSF 
in vitro (Reed et al., 1987). GM-CSF increases the cytotoxicity of monocytes toward 
certain neoplastic cell lines (Grabstein et al., 1986) and activates polymorphonuclear 
neutrophils to inhibit the growth of tumor cells. Pharmacology/toxicology studies of 
GM-CSF were performed in cynomolgus monkeys. An acute toxicity study revealed an 
absence of treatment-related toxicity following a single IV bolus injection at a dose of 
300 mcg/kg. Two subacute studies were performed using IV injection (maximum dose 
200 mcg/kg/day × 14 days) and subcutaneous injection (SC) (maximum dose 200 
mcg/kg/day × 28 days). No major visceral organ toxicity was documented. Notable 
histopathology findings included increased cellularity in hematologic organs and heart 
and lung tissues. A dose-dependent increase in leukocyte count, which consisted 
primarily of segmented neutrophils, occurred during the dosing period; increases in 
monocytes, basophils, eosinophils and lymphocytes were also noted. Leukocyte counts 
decreased to pretreatment values over a 12 week recovery period. 
 
GM-CSF - Pharmacokinetics in Normal Subjects 
Pharmacokinetic profiles have been analyzed in controlled studies of 24 normal male 
volunteers (Spitler et al., 2012). Liquid and lyophilized GM-CSF, at a dose of 250 
mcg/m2, has been determined to be bioequivalent based on the statistical evaluation of 
AUC. When GMCSF (either liquid or lyophilized) was administered IV over two hours to 
normal volunteers, the mean beta half-life was approximately 60 minutes. Peak 
concentrations of GM-CSF were observed in blood samples obtained during or 


32 
 
immediately after completion of GM-CSF infusion. For liquid GM-CSF, the mean 
maximum concentration (Cmax) was 5.0 ng/mL, the mean clearance rate was 
approximately 420 mL/min/m2 and the mean AUC (0inf) was 640 ng/mL•min. 
Corresponding results for lyophilized GMCSF in the same subjects were mean Cmax of 
5.4 ng/mL, mean clearance rate of 431 mL/min/m 2, and mean AUC (0inf) of 677 
ng/mL•min. GM-CSF was last detected in blood samples obtained at three or six hours. 
When GM-CSF (either liquid or lyophilized) was administered SC to normal volunteers, 
GM-CSF was detected in the serum at 15 minutes, the first sample point. The mean beta 
half-life was approximately 162 minutes. Peak levels occurred at one to three hours post 
injection, and GM-CSF remained detectable for up to six hours after injection. The mean 
Cmax was 1.5 ng/mL. For liquid GM-CSF, the mean clearance was 549 mL/min/m2 and 
the mean AUC (0inf) was 549 ng/mL•min. For lyophilized GM-CSF, the mean clearance 
was 529 mL/min/m 2 and the mean AUC (0inf) was 501 ng/mL•min. The 
pharmacokinetic profile of the EDTA-containing GM-CSF formulation is not the same as 
that of reconstituted lyophilized and previous liquid formulations. These differences 
include an earlier Tmax, a decrease in clearance and two peaks of absorption as 
compared to a single peak of the other formulations. The magnitude of these 
differences is small and unlikely to result in meaningful pharmacodynamic changes. This 
difference is attributed to the presence of EDTA in GM-CSF.