Conditions: Melanoma
Keywords: Melanoma Malignant Melanoma
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 10
Healthy volunteers: no

Brief summary:
The purpose of this study is to assess the safety and efficacy of combined treatment with Ipilimumab and all-trans retinoic acid (ATRA) in melanoma patients.

Detailed description:
The successful treatment of melanoma with immune checkpoint inhibitors, such as anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and Programmed cell death protein 1 (PD-1) antibodies, has altered our thinking and approach to immunotherapy for solid tumors. Despite these advances, only a portion of patients experience a durable response suggesting that there is room for improvement via enhanced immunomodulatory approaches. Anti-CTLA-4 (Ipilimumab) significantly improves overall survival and achieves long-lasting complete responses in some melanoma patients, the number of patients that achieve durable clinical benefit is limited and could be improved by a combined immunomodulatory approach. The objectives of this study are to assess the safety and efficacy of combined treatment with Ipilimumab and all-trans retinoic acid (ATRA) in melanoma patients. We hypothesize that combined treatment with Ipilimumab and ATRA will improve patient responses, increase tumor antigen-specific T cell responses, and decrease immunosuppressive myeloid-derived suppressor cells (MDSCs) in melanoma patients compared to patients treated with Ipilimumab alone.

Arms:
arm 1: Arm A (No VESANOIDTherapy) will receive the standard of care treatment with ipilimumab only, receiving the standard 4 doses of either 3 or 10 mg/kg ipilimumab every 3 weeks. arm 2: Arm B (VESANOID Therapy) will receive the standard 4 doses of either 3 or 10 mg/kg ipilimumab every three weeks plus the supplemental treatment of 150 mg/m2 of VESANOID orally for 3 days surrounding each dose of ipilimumab (day -1, day 0, day +1) for a total of 12 days of VESANOID treatment.

Interventions: intervention 1: VESANOID intervention 2: Ipilimumab
Intervention descriptions: intervention 1: All-trans retinoic acid (ATRA) is a vitamin A derivative that binds the retinoic acid receptor on MDSCs and differentiates immature monocytes into more mature dendritic cells (12). VESANOID is a standard treatment for patients with acute promyelocytic leukemia (APL). intervention 2: Ipilimumab is current standard of care treatment for melanoma.

Protocol document:
Prot_SAP_000.pdf: 
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w 
COMIRB Protocol 
 
PROTOCOL #: 14-0948 
 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490  TELEPHONE: 303-724-1055 Fax: 303-724-0990 
 
 
 
 
Project Title: 
Ipilimumab and All-Trans Retinoic Acid 
Combination Treatment of Advanced Melanoma  
Principal Investigator:  
 
Martin D. McCarter, M.D. 
 
 
Version Date: 
 
 
7 September 2016 
 
I. Hypotheses and Specific Aims 
The successful treatment of melanoma with immune checkpoint inhibitors, such as anti-
CTLA-4 and PD-1 antibodies, has altered our thinking and approach to immunotherapy for 
solid tumors. Despite these advances, only a portion of patients experience a durable 
response suggesting that there is room for improvement via enhanced immunomodulatory 
approaches. Anti-CTLA-4 (ipilimumab) significantly improves overall survival and achieves 
long-lasting complete responses in some melanoma patients, the number of patients that 
achieve durable clinical benefit is limited and could be improved by a combined 
immunomodulatory approach. The objectives of our study are to assess the safety and 
efficacy of combined treatment with ipilimumab and all-trans retinoic acid (ATRA), brand 
name VESANOID, in melanoma patients. We hypothesize that combined treatment with 
ipilimumab and VESANOID will improve patient responses, increase tumor antigen-
specific T cell responses, and decrease immunosuppressive myeloid-derived suppressor 
cells (MDSCs) in melanoma patients compared to patients treated with ipilimumab alone. 
 
Specific Aims: 
 
1. To establish the safety and tolerability of ipilimumab and VESANOID 
combination therapy in advanced melanoma patients 
 
2. To demonstrate the reduction in MDSC frequency and suppressive function in 
peripheral blood of Advanced melanoma patients undergoing ipilimumab and 
VESANOID combination therapy 
 
3. To determine if the addition of VESANOID to standard ipilimumab treatment 
increases the frequency of tumor-specific T cell responses  


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II. Background and Significance 
 
Melanoma uniquely elicits profound immune responses and is one of the few tumors for 
which immunotherapies have been successful (1). However, melanoma counters current 
immunotherapeutic approaches by inducing immune escape mechanisms, a critical 
barrier that may contribute to low response rates and treatment failure (1). Melanoma 
continues to be a leading cause of death among adult cancer patients (2, 3). Furthermore, 
incidents of melanoma continue to increase across all age groups (4).  
 
The antibody therapy specific for CTLA-4, an inhibitory molecule expressed on activated T 
cells and constitutively expressed on Tregs, has recently been shown to significantly 
improve overall survival and induce durable, long-lasting responses of greater than 5 
years in approximately 20% of treated patients (5, 6). While the results of this monumental 
study highlight a crucial advantage of immunotherapies in achieving long-term immunity 
that is resistant to disease relapse, increased response rates to ipilimumab could greatly 
impact survival and clinical outcomes for patients with advanced-stage melanoma. One 
proven mechanism of action for this drug is enhancing the frequency and function of 
tumor-specific T cells (7). However, immunosuppression may limit the development of 
effective immune responses in some patients treated with ipilimumab. Improving clinical 
responses to ipilimumab by enhancing other immune-mediated mechanisms in a 
combinatorial approach is essential for the future success of immunotherapeutic 
strategies. 
 
MDSCs are a mixed population of immunosuppressive myeloid cells that are key 
contributors to tumor-induced immunosuppression. Increased numbers of MDSCs are 
associated with larger tumor burdens, increased tumor progression, and shorter overall 
survival in several cancers, including melanoma (8-11). These data suggest that targeting 
MDSCs in a combinatorial approach may improve tumor-specific immune responses, 
increase clinical responses to immunotherapy, and ultimately increase survival in 
melanoma patients. In this proposal, we aim to decrease the frequency and/or function of 
MDSCs in melanoma patients treated with all-trans retinoic acid (ATRA). 
 
Investigational product: All-trans retinoic acid (ATRA) is a vitamin A derivative that binds 
the retinoic acid receptor on MDSCs and differentiates immature monocytes into more 
mature dendritic cells (12). VESANOID is a standard treatment for patients with acute 
promyelocytic leukemia (APL). 
 
VESANOID (tretinoin) is an FDA-approved commercially available retinoid that induces 
maturation of APL cells in culture (Appendix B). It is not approved in combination with 
Ipilimumab or for the treatment of patients with melanoma.  It is available in a 10 mg soft 
gelatin capsule for oral administration. The recommended dose (for APL) is 45 mg/m2/day 
administered as two evenly divided doses until complete remission is documented. 
Therapy should be discontinued 30 days after achievement of complete remission or after 
90 days of treatment, whichever occurs first. 
 
VESANOID has been investigated in 114 previously treated APL patients and in 67 
previously untreated (“de novo”) patients in one open-label, uncontrolled single 
investigator clinical study (Memorial Sloan-Kettering Cancer Center [MSKCC]) and in two 
cohorts of compassionate cases treated by multiple investigators under the auspices of 
the National Cancer Institute (NCI). 


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Virtually all patients with APL experience some drug-related toxicity, especially headache, 
fever, weakness, and fatigue. These adverse effects are seldom permanent or irreversible 
nor do they usually require interruption of therapy. Some of the adverse events are 
common in patients with APL, including hemorrhage, infections, gastrointestinal 
hemorrhage, disseminated intravascular coagulation, pneumonia, septicemia, and 
cerebral hemorrhage.  There are no reported side effects in patients with solid tumors 
treated with a short course of 150 mg/m2 VESANOID combined with high dose IL-2 or a 
dendritic cell vaccine (13, 14).   
 
Pre-clinical findings: Pre-clinical data suggest that VESANOID abrogates the 
suppressive effects of MDSCs isolated from both human cancer patients and mouse 
cancer models in vitro (15-17). Furthermore, mice treated with VESANOID have 
decreased frequencies of MDSCs and increased frequencies of dendritic cells that 
improve T cell responses against tumors (18-20).  
 
Treatment justification: Although VESANOID is a standard treatment for patients with 
acute promyelocytic leukemia, it has also been used to treat patients with solid tumors. 
Renal cell carcinoma patients treated with VESANOID followed by high dose IL-2 had 
reduced frequencies of circulating MDSCs with reduced immunosuppressive function 
during the VESANOID phase of the treatment regimen (14). However, the subsequent 
high-dose IL-2 treatment abrogated these effects and resulted in a lack of clinical benefit 
overall (14). In another recent trial, VESANOID was used in combination with a dendritic 
cell vaccine targeting p53 mutations in small cell lung cancer patients (13). The survival 
outcomes have not yet been reported; however, the study demonstrated that the 
frequency and function of MDSCs was reduced following VESANOID treatment and that 
p53-specific T cell responses were increased in more patients receiving the combinatorial 
therapy compared to patients receiving the dendritic cell vaccine alone (13).  
 
Description of population to be studied: Advanced melanoma patients undergoing 
standard ipilimumab treatment. 
 
Study rationale: Reducing immunosuppression by MDSCs during ipilimumab treatment 
may maximize T cell activation and expansion, increasing the overall effectiveness and 
success of ipilimumab treatment. This pilot clinical trial will demonstrate whether 
combinatorial immunotherapeutic approaches that augment tumor-specific T cells and 
simultaneously block immunosuppressive mechanisms are beneficial in melanoma 
patients. 


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Preliminary Studies 
 
Our preliminary research has identified a population of immunosuppressive MDSCs 
(Lineage negative, HLA-DR negative, CD11b positive, and CD33 positive) that are 
increased in Stage IV melanoma patients relative to healthy donors. To establish MDSCs 
as a potentially important contributor to immunosuppression in melanoma patients, we 
compared the frequency and function of immunosuppressive MDSCs in the peripheral 
blood of healthy donors, Stage I, and Stage IV melanoma patients (11). We found 
significant increases in the frequency of MDSCs in the peripheral blood of Stage IV 
melanoma patients compared to healthy donors. We showed that the frequency of these 
cells correlates with an increase in regulatory T cells and an increased level of IL-6 and IL-
8 cytokines in the peripheral blood. Furthermore, patients with clinical disease progression 
had an increased frequency of these cells.  We also found that patients with a high 
frequency of MDSCs had decreased overall survival and an increased risk of death 
(hazard ratio 4.83, p = 0.016). This increased risk of death was independent of other 
clinical factors including age, gender, and treatment regimens. Finally, we demonstrated 
that MDSCs isolated from Stage IV melanoma patients were significantly more 
immunosuppressive than those isolated from healthy donors. Overall, these data suggest 
that MDSCs may be a significant contributor to immunosuppression in melanoma patients 
and that targeting these cells in a combinatorial approach may improve immune 
responses against melanoma.  
 
VESANOID induces the differentiation of immature monocytes into more mature cells of 
the dendritic cell lineage (16, 17, 21). In agreement with these results, our preliminary 
data suggest that VESANOID induces maturation of in vitro-derived MDSCs. We cultured 
monocytes from a healthy donor with GM-CSF/IL-4 or GM-CSF/IL-6 to generate mature 
monocytes of the dendritic cell lineage (a control population) or MDSCs, respectively (22, 
23).  In contrast to the control population, MDSCs express low levels of HLA-DR and 
maintain high expression of CD14, consistent with the phenotype of an immature 
monocyte. Furthermore, MDSCs express increased levels of molecules that inhibit T cell 
responses such as Arginase I, NOX1, iNOS, and PD-L1. Conversely, MDSCs cultured 
with VESANOID are phenotypically more similar to the control dendritic cell population, 
expressing decreased levels of inhibitory molecules and CD14 and increased levels of 
HLA-DR.  Most importantly, MDSCs derived in vitro with GM-CSF and IL-6 suppress T cell 
proliferation. However, treatment of these MDSCs with VESANOID eliminates their 
suppressive effects and restores T cell proliferation. Finally, MDSCs express higher levels 
of retinoic acid receptors RAR and RXR, suggesting that VESANOID treatment may 
selectively target these inhibitory cells. These preliminary data and our previous 
publications demonstrate that our research team has established the protocols necessary 
to accomplish the experiments outlined in this protocol. 
 
 


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III. Study Design and Research Methods 
 
This is a Phase 2 investigator-initiated randomized open label clinical investigation aimed 
at evaluating the safety of combined VESANOID and ipilimumab treatment versus 
standard treatment with ipilimumab alone, and the effect of this combined treatment on 
the frequency and function of peripheral MDSCs in advanced melanoma patients being 
treated at the University of Colorado Cancer Center. This trial will be conducted in 
compliance with this protocol, Good Clinical Practice, and local and federal regulations. 
 
Anticipated total number of enrolled research subjects: 60 
Anticipated number to complete study: 48 
 
  
Outcome Measures 
• 
Safety and tolerability of ipilimumab and VESANOID combination therapy in 
advanced melanoma patients will be established using the Bayesian approach. 
 
• 
MDSC frequency in peripheral blood of advanced melanoma patients undergoing 
ipilimumab and VESANOID combination therapy will be measured by flow 
cytometry. 
 
• 
MDSC suppressive function in peripheral blood of advanced melanoma patients 
will be measured through the activation and proliferation of T cells in the presence 
of isolated MDSCs. 
 
• 
Changes in the frequency of tumor-specific T cell responses attributable to the 
addition of VESANOID to standard ipilimumab therapy will be determined by the 
frequency of IFN-gamma producing cells after stimulation with melanoma 
antigens. 
 
• 
Subjects with unresectable Stage III and Stage IV disease will be followed for 
RECIST 1.1 
 
 
 
 
 
 
 
 
 
 
 


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Description of Population to be Enrolled 
We will identify patients undergoing treatment for melanoma in the cutaneous oncology 
clinic at the University of Colorado Cancer Center who are eligible for treatment with 
ipilimumab. Up to 60 patients will be enrolled with 48 patient anticipated to complete the 
study.  
 
Patient Screening 
All patients being evaluated for melanoma in the cutaneous oncology clinic for advanced 
disease are eligible for the trial based on the following criteria: 
Inclusion Criteria 
• 
Patients over the age of 18 year. 
• 
Patients diagnosed with advanced melanoma. 
• 
Patients that are considered candidates for ipilimumab therapy. 
• 
Patients able to understand and willing to sign a written informed consent 
documents. 
• 
Patients willing to have regular blood draws, one before treatment and four during 
or after treatment. 
 
Exclusion Criteria 
• 
Patients under the age of 18. 
• 
Patients with Stage I or II, melanoma who are not candidates for Ipilimumab. 
• 
Patients that have received systemic treatments within four weeks prior to the 
beginning of treatment. 
• 
Women that are pregnant or nursing. 
• 
Patients taking immunosuppressive medications. 
• 
Patients with active autoimmune disease. 
• 
Patients with known sensitivity to retinoic acid derivatives.  
• 
Patients with aspartate aminotransferase (AST), alanine aminotransferase (ALT), 
or bilirubin > 2.5 × ULN. 
 
 
Informed Consent Process 
 
Following patient identification, patients may be approached by the Principal Investigator, 
Study Coordinator, or Sub-Investigator to discuss study participation. Treatment options 
and research-specific procedures will be carefully reviewed with the patient prior to 
enrollment in the study.  
 
 
Allocation of Treatment 
 
Treatment allocation will be determined by the breaking of a randomization envelope. 
Subjects will be randomized into two groups with 30 patients in each group. Arm A (No 
VESANOID Therapy) will receive the standard of care treatment with ipilimumab only, 
receiving the standard doses of 3 or 10mg/kg ipilimumab approximately every 3 weeks or 
longer according to standard of care protocols. Arm B (VESANOID Therapy) will receive 


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the standard 4 doses of 3 or 10mg/kg ipilimumab approximately every three weeks or 
longer according to standard of care protocols plus the supplemental treatment of 150 
mg/m2 VESANOID orally for 3 days surrounding each of the first four infusions of 
ipilimumab (day -1, day 0, day +1) for a total of 12 days of VESANOID treatment (Figure 
1).  The total daily dose of VESANOID may be taken anytime on the day before the 
ipilimumab infusion (day -1) according to the patient’s preferences, prior to the ipilimumab 
infusion on the day of the infusion (day 0), and anytime on the day after the ipilimumab 
infusion (day 1) according to the patient’s preferences. 
 
 
 
 
Drug Distribution 
 
Ipilimumab is standard of care for this patient population.  VESANOID will be supplied by 
the Investigator through the University of Colorado Cancer Center pharmacy. 
VERSANOID is supplied as 10 mg capsules, two-tone (lengthwise), orange-yellow and 
reddish-brown and imprinted VERSANOID 10 ROCHE. It is packaged in high-density 
polyethylene opaque bottles of 100 capsule with child-resistance closure. 
 
 
Patient Compliance 
 
Patients in Arm B will self-administer VERSANOID capsules in the outpatient setting. At 
subsequent follow-up visits, patients will be asked about any noticeable side effects or 
intolerance to the VERSANOID.  If patients were not able to take all the prescribed 
capsules, they will be instructed to bring any remaining pills to the clinic during each visit.  
Pharmacy personnel will count and record the number of used and unused drug at each 
visit on the Drug Compliance Assessment for Oral Investigational Product Document. 
 
Figure 1. Diagram of the treatment and blood draw schedule for this study.  Patients in Arm A and B will receive an infusion 
of ipilimumab approximately every three weeks for 12 weeks (a total of 4 infusions).  Patients in Arm B will also receive 3 days of 
VESANOID treatment surrounding each infusion of ipilimumab, beginning one day prior to the infusion (a total of 12 days of 
VESANOID).  Three tubes of peripheral blood (approximately 20 ml) will be drawn at enrollment (pre-treatment), prior to each 
infusion of ipilimumab, and three weeks after the final ipilimumab infusion, for a total of 5 time points.  Three additional tubes of 
peripheral blood (for a total of approximately 40 ml) will be drawn at enrollment, at the 3rd infusion, and within 30 days of the final 
infusion for additional functional experiments. *(Note: prior to dosing is ≤72 hours) 


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Study Procedures 
Study assessments and sample collection time points are summarized in Table 1. 
Following the standard protocols for ipilimumab treatment, peripheral blood will be drawn 
at baseline and prior to each dose of ipilimumab to monitor electrolytes, liver function, 
thyroid function, and complete blood counts (CBCs). These assays will be performed in 
the clinical laboratories at the University of Colorado Hospital and monitored by the 
treating medical oncologists and study team, led by our collaborator and Director of the 
Melanoma Research Clinics at the University of Colorado Denver, Dr. Rene Gonzalez.  
 
 
 
Table 1: Data and Sample Collection Schedule 
 
Lab draws will be performed at regularly scheduled clinic visits as outlined in Figure 1. 
Peripheral blood mononuclear cells (PBMCs) will be isolated and flow cytometry will be 
used to quantify MDSCs and T cells.  
Tests and 
procedures 
Baseline 
Active Treatment 
Discontinuati
on of 
Treatment 2 
≤4 wks prior 
to start 
Prior to each 
infusion1 
Midway (prior to 
second infusion) 
≤ 30 days 
after final 
infusion 
Medical History, 
exam, weight, 
physical signs, & 
adverse events 
assessment 
X 
X 
 
X 
X 
Hematology group 
(WBC, ANC, Hgb, 
PLT)7 
X 
X 
 
X 
 
Chemistry group and 
thyroid function 3,7 
X 
X 
 
X 
 
Research 
correlates 
 
 
 
 
 
Phenotypic 
characterization 
(3 yellow tops) 
X 
X5 
 
 
 
Functional 
characterization (+3 
yellow tops) 
X 
 
X 
X 
 
Tumor Measurement 
(CT, MRI, etc.) 4 
X6 
 
 
X8 
X 
1Infusions of ipilimumab will occur approximately every 3 weeks or longer as per hospital standard of care. 
2See “Protections against risk” below for conditions necessitating discontinuation of treatment.  If treatment is 
discontinued, it is not necessary to repeat the specified evaluations. 
3Creatinine, total bilirubin, AST, ALT, alkaline phosphatase, Ca, K, Na, albumin, total protein, TSH (with additional thyroid 
testing as clinically indicated) 
4Baseline assessment prior to enrollment.  Measurements will be taken using RECIST criteria. 
5Except for the first infusion where the baseline blood draw will be used.  
6Baseline tumor measurement window ≤90 days 
7Labs drawn within 72 hours prior to infusion 
8timeframe for scans to be consistent with standard of care practice 


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- 
Baseline immune response determination. Participants in each arm of the study 
will be asked to provide 40 mls of blood prior to initiating treatment to evaluate 
baseline immune responses and immune cell frequencies.  
 
- 
Immune response monitoring. Repeated blood samples of 20 mls will be drawn 
prior to the administration of the 2nd and 4th doses of ipilimumab for continued 
measurement of immune cell frequencies.  
 
- 
Post-treatment immune response determination. Participants will also be 
asked to provide 40 mls of blood prior to the administration of the 2nd dose and 3 
weeks after the 4th dose of ipilimumab for evaluation of post-treatment immune 
responses.  
 
- 
Cytokine analysis. Up to 20 ml of plasma will be stored for five years following 
study completion for subsequent cytokine analysis. 
 
Following the standard of care for ipilimumab treatment, will use standard imaging 
techniques (RECIST criteria version 1.1) every 3 months to monitor the appearance of 
new metastatic lesions and the size of existing lesions.  
 
 
Specimen Analysis 
 
Functional assays will be performed to assess the ability of isolated MDSCs to suppress T 
cell responses.  
 
- 
Determination of tumor-specific T cell response frequency. Peripheral blood 
mononuclear cells (PBMCs) will be isolated over a ficoll gradient and five million 
cells will be banked to analyze the frequency of MDSCs and T cells using flow 
cytometry as previously published. Approximately five million PBMCs will be used 
to determine the frequency of tumor-specific T cell responses by stimulating the 
cells with a mixture of peptides derived from known melanoma tumor antigens and 
measuring IFN-gamma production by flow cytometry.  
 
- 
T cell suppression assay. The remaining PBMC will be used to perform T cell 
suppression assays using magnetically separated MDSC populations, comparing 
the suppressive function of MDSCs throughout treatment. Any remaining MDSCs 
will be flash frozen and banked for gene expression studies analyzing the 
expression of known suppressive molecules by real-time quantitative PCR.  
 
- 
Cytokine analysis. Multiplex cytokine array will be used for subsequent cytokine 
analysis. Concentration of cytokines related to the function of MDSCs (GM-CSF, 
VEGF, MIP-1 alpha, MIP-1 beta, IL-10, IL-6, and IL-8) and T cells (IFN-gamma, 
TNF-alpha, IL-1-beta, IL-2, IL-4, IL-5, IL-12p70, IL-17) will be analyzed. 
 
 
 
 
 
 


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Study Completion and Follow-up 
 
Following the standard of care for ipilimumab treatment, standard imaging techniques will 
be used according to RECIST criteria version 1.1 approximately every 3 months to 
monitor the appearance of new metastatic lesions and the size of existing lesions.  
 
Patients who complete or discontinue the study will have a final clinic visit and will be 
followed by office visit or phone contact every 2 months for two years. 
IV. Anticipated Risks 
 
The characterization of potential side effects and toxicities associated with combined 
ipilimumab and VESANOID treatment is an important objective of this protocol. As 
VESANOID has not been administered in combination with ipilimumab, there are no 
clinical or safety data concerning this drug combination. No adverse events related to 
VESANOID treatment in patients with solid tumors have been reported. However, patients 
with acute promyelocytic leukemia treated with VESANOID may develop Retinoic Acid 
Syndrome, otherwise known as Differentiation Syndrome, characterized by fever, 
dyspnea, weight gain, hypotension, and pulmonary infiltrates. 
Expected adverse events known to occur in a minority of patients treated with ipilimumab 
include immune-mediated enterocolitis, hepatitis, dermatitis, neuropathies, and 
endocrinopathies. A recent study showed that of the patients treated with ipilimumab 
monotherapy, 14.5% reported grade 3 or higher immune-related adverse events (irAEs) 
(24). The same study showed that of the patients treated with ipilimumab combined with 
the gp100 vaccine, 10.3% reported grade 3 or higher irAEs (24). Most of these events 
occurred within the 12 week treatment schedule, with only 1 patient (0.15%) reporting a 
grade 3 irAE >70 days after the final dose of ipilimumab (24). Most severe adverse events 
were successfully managed using published protocol-defined guidelines (administration of 
corticosteroids, delays in treatment schedule, or discontinuation of treatment); 1.1% of 
deaths were attributable to irAEs.  
 
Following standard protocols for ipilimumab treatment, patients will be closely monitored 
for expected signs and symptoms of diarrhea, pain, mucus or bloody stool, fever, rash, 
muscle weakness, sensory alterations, headache, fatigue, mental status changes, 
hypotension, and visual disturbances.  
 
Potential VESANOID Side Effects 
Virtually all patients experience some drug-related toxicity, especially headache, fever, 
weakness, and fatigue. These adverse effects are seldom permanent or irreversible nor 
do they usually require interruption of therapy. Some of the adverse events are common 
in patients with APL, including hemorrhage, infections, gastrointestinal hemorrhage, 
disseminated intravascular coagulation, pneumonia, septicemia, and cerebral 
hemorrhage. The following describes the adverse events, regardless of drug relationship, 
that were observed in patients treated with VESANOID (tretinoin). 
 
Typical Retinoid Toxicity 
The most frequently reported adverse events were similar to those described in patients 
taking high doses of vitamin A and included headache (86%), fever (83%), skin/mucous 
membrane dryness (77%), bone pain (77%), nausea/vomiting (57%), rash (54%), 


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mucositis (26%), pruritus (20%), increased sweating (20%), visual disturbances (17%), 
ocular disorders (17%), alopecia (14%), skin changes (14%), changed visual acuity (6%), 
bone inflammation (3%), visual field defects (3%). 
 
Body as a Whole 
General disorders related to VESANOID (tretinoin) administration and/or associated with 
APL included malaise (66%), shivering (63%), hemorrhage (60%), infections (58%), 
peripheral edema (52%), pain (37%), chest discomfort (32%), edema (29%), disseminated 
intravascular coagulation (26%), weight increase (23%), injection site reactions (17%), 
anorexia (17%), weight decrease (17%), myalgia (14%), flank pain (9%), cellulitis (8%), 
face edema (6%), fluid imbalance (6%), pallor (6%), lymph disorders (6%), acidosis (3%), 
hypothermia (3%), ascites (3%). 
 
Respiratory System Disorders 
Respiratory system disorders were commonly reported in APL patients administered 
VESANOID (tretinoin) . The majority of these events are symptoms of the RA-APL 
syndrome (see boxed WARNINGS). Respiratory system adverse events included upper 
respiratory tract disorders (63%), dyspnea (60%), respiratory insufficiency (26%), pleural 
effusion (20%), pneumonia (14%), rales (14%), expiratory wheezing (14%), lower 
respiratory tract disorders (9%), pulmonary infiltration (6%), bronchial asthma (3%), 
pulmonary edema (3%), larynx edema (3%), unspecified pulmonary disease (3%). 
 
Ear Disorders 
Ear disorders were consistently reported, with earache or feeling of fullness in the ears 
reported by 23% of the patients. Hearing loss and other unspecified auricular disorders 
were observed in 6% of patients, with infrequent (<1%) reports of irreversible hearing loss. 
 
Gastrointestinal Disorders 
GI disorders included GI hemorrhage (34%), abdominal pain (31%), other gastrointestinal 
disorders (26%), diarrhea (23%), constipation (17%), dyspepsia (14%), abdominal 
distention (11%), hepatosplenomegaly (9%), hepatitis (3%), ulcer (3%), and unspecified 
liver disorder (3%). Incidents of suspected treatment-related hepatic toxicity will be closely 
monitored until resolution.  If LFT abnormalities do not resolve with cessation of treatment 
and/or steroid therapy, consideration will be given to hepatitis B and C testing. 
 
Cardiovascular and Heart Rate and Rhythm Disorders 
Arrhythmias (23%), flushing (23%), hypotension (14%), hypertension (11%), phlebitis 
(11%), cardiac failure (6%) and for 3% of patients: cardiac arrest, myocardial infarction, 
enlarged heart, heart murmur, ischemia, stroke, myocarditis, pericarditis, pulmonary 
hypertension, secondary cardiomyopathy. 
Central and Peripheral Nervous System Disorders and Psychiatric 
Dizziness (20%), paresthesias (17%), anxiety (17%), insomnia (14%), depression (14%), 
confusion (11%), cerebral hemorrhage (9%), intracranial hypertension (9%), agitation 
(9%), hallucination (6%) and for 3% of patients: abnormal gait, agnosia, aphasia, asterixis, 
cerebellar edema, cerebellar disorders, convulsions, coma, CNS depression, dysarthria, 
encephalopathy, facial paralysis, hemiplegia, hyporeflexia, hypotaxia, no light reflex, 
neurologic reaction, spinal cord disorder, tremor, leg weakness, unconsciousness, 
dementia, forgetfulness, somnolence, slow speech. 
Urinary System Disorders 


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Renal insufficiency (11%), dysuria (9%), acute renal failure (3%), micturition frequency 
(3%), renal tubular necrosis (3%), enlarged prostate (3%). 
Miscellaneous Adverse Events 
Isolated cases of erythema nodosum, basophilia and hyperhistaminemia, Sweet's 
syndrome, organomegaly, hypercalcemia, pancreatitis and myositis have been reported. 
Cardiovascular 
Cases of thrombosis (both venous and arterial) involving various sites (eg, 
cerebrovascular accident, myocardial infarction, renal infarct) have been reported rarely. 
Hematologic 
Rare cases of thrombocytosis have been reported. 
Skin 
Genital ulceration 
Miscellaneous Adverse Events 
Rare cases of vasculitis, predominantly involving the skin, have been reported. 
  
The toxicity profile of VESANOID and Ipilimumab may overlap causing difficulty in 
determining the causative agent. Our investigators have many years of prior experience 
treating patients with Ipilimumab and their clinical judgement will be used to assess the 
toxicity and any potential relationship.  
 
Dose Modifications for VESANOID 
 
If patients experience grade 3 or 4 toxicity that is believed to be related to the VESANOID, 
then the VESANOID will be held until the toxicity resolves to grade 0 or 1. If the toxicity 
appears to be related to a combination of Ipilimumab and VESANOID, the VESANOID will 
be held. After the toxicity has resolved, the VESANOID will be restarted at the next 
scheduled time at a reduced does of 100mg/m2. If the grade 3 or 4 toxicity returns, the 
VEASOIND will be held indefinitely. 
 
The severity of the adverse events will be rated on a scale of Grade 1-4 and reported 
according to the Data and Safety Monitoring Plan using CTCAE version 4.0. Medical 
oncologists at the University of Colorado Hospital Cutaneous Oncology Clinic specialize in 
melanoma and have extensive experience treating patients with immune-based therapies 
that cause immune-related adverse events. The primary investigator will be notified of all 
adverse events that occur during this trial and will work with Dr. Gonzalez and the treating 
oncology team to determine the likelihood of VESANOID’s contribution to the adverse 
event.  
 
Risks to the patient include potential disclosure of protected health information, pain and 
bruising due to standard risks with venipuncture, and adverse events associated with 
ipilimumab and/or ipilimumab combined with VESANOID. The risks associated with 
participation in this trial will be discussed in full detail during the consent process and will 
be clearly presented to the patient in advance. All potential subjects have will have the 
option of receiving standard of care or supportive care/hospice care. 
 
 
Protections Against Risk 
 


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Patients will be screened prior to study enrollment and monitored for adverse events 
during the study. Participants will be instructed to notify the clinical provider immediately 
for any adverse events.  
- 
Subject eligibility determination: Study applicants will undergo complete 
laboratory tests and physical exams to determine their eligibility and safety of their 
participation in this study. Study applicants will be excluded if they do not meet the 
eligibility criteria. Subjects will also be excluded if other clinical problems that may 
contraindicate their participation in this study exist.  
 
- 
Adverse event assessment: During the treatment phase of the study, 
participants will be asked about adverse events at each clinic visits and vital signs 
will be obtained before administering the study medication. Patients will not 
receive the medication if they have any signs of symptoms that may contraindicate 
its administration. All adverse events occurring during the course of the study will 
be collected, documented, and reported to the principal investigator. Each clinic 
visit, a study investigator will review the adverse events since the previous visit. 
The study investigators will follow all adverse events to the point of a satisfactory 
resolution. A study participant may have their medication discontinued or may be 
withdrawn from the study if the medically responsible investigator determines it is 
the best decision in order to protect the safety of a participant. All adverse events 
will be assessed to determine if they meet criteria for a severe adverse event. 
Adverse event collection will conclude at the final study visit, within 30 days of their 
final ipilimumab infusion. 
 
- 
Serious adverse event assessment: Severe adverse events, as defined by the 
FDA, will be systemically evaluated at each clinic visit. Any severe adverse event, 
whether or not related to the study medication, will be reported to the principal 
investigator, the COMIRB, and the UCCC DSMC. Initial severe adverse event 
reports will be followed by submission of a completed severe adverse event 
reports to the above parties. In the event that a patient withdraws from the study or 
the investigator discontinues a patient due to severe adverse events, the patient 
will have the appropriate follow-up medical monitoring. Monitoring will continue 
until the problem has resolved or stabilized with no further change expected, is 
clearly unrelated to the study medication, or results in death. A summary of the 
severe adverse events that occurred during the previous 6 months will be included 
in the data safety monitoring report to the DSMC. 
 
 
VESANOID Related Toxicity 
 
Significant VESANOID related toxicity is defined as any >= grade 3 non-hematologic 
toxicity related to VESANOID, or other grade VESANOID related adverse events that lead 
to treatment discontinuation. An exception is VESANOID related headache not optimally 
managed with supportive care therapies (acetaminophen or non-steroidal anti-
inflammatory medication). 
 
 
 
Toxicity and Study Discontinuation Criteria 


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The trial stopping criteria outlined in Tables 2 and 3 below will be followed to avoid posing 
undue risk to additional patients. 24 patients will potentially be recruited in each arm of our 
study as necessary to fulfill the efficacy endpoint. Toxicity in Arm B will be monitored using 
the Bayesian approach (25, 26) as extended by Thall and Sung (27). Toxicity is defined as 
AEs of grade 3 or higher, any >= grade 3 non-hematologic toxicity related to VESANOID 
or combined Ipilimumab and VESANOID toxicity of grade 3 or higher.. Historical data of 
similar patients show an overall toxicity rate of 10-15% for ipilimumab treatment alone 
(Arm A). It is expected for the current trial that Arm A and B will have toxicity rates at a 
maximum of 15% and 25%, respectively.  
 
The prior probabilities of toxicity for Arm A and B are modeled by minimally informative 
prior distributions (Beta (0.3, 1.7)). Denoting the historical probabilities of the rate of 
toxicity in Arm A by p(TOX, H), and the probabilities of the rate of toxicity in Arm B by 
p(Tox, Arm B), the following formula will be applied to calculate the trial stopping decision 
criteria: for Arm B, patient recruitment will stop if Prob{p(TOX, H) + dTOX < p(TOX, Arm B)| 
data} > 0.8, where dTOX = 0.1.  
Patients in Arm B will be monitored according to the stopping boundaries for toxicity 
shown in Tables 2 and 3. If recruitment to Arm B is stopped due to toxicity, recruitment to 
Arm A will also be stopped. Patients with significant irAEs will be treated using protocol-
defined guidelines. 
 
Table 2: Calculated Trial Stopping Criteria for Ipilimumab 3mg/kg 
 
 
 
Table 3: Calculated Trial Stopping Criteria for Ipilimumab 10mg/kg 
 
Safety stopping boundary based on 95% exact lower confidence interval >=true grade 3 or 4 AEs 
rate 
Number of 
enrollment 
Stop for the number of AEs 
assuming the true AEs rate is 54%  
(single drug arm A) 
Stop for the number of AEs 
assuming the true AEs rate is 
60% 
(combo drug arm B) 
7 
7 
No stop 
8 
8 
8 
9 
9 
9 
10 
>=9 
10 
11 
>=10 
11 


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12 
>=11 
>=11 
13 
>=11 
>=12 
14 
>=12 
>=13 
15 
>=13 
>=14 
16 
>=13 
>=14 
17 
>=14 
>=15 
18 
>=15 
>=16 
19 
>=15 
>=16 
20 
>=16 
>=17 
21 
>=17 
>=17 
22 
>=18 
>=19 
23 
>=18 
>=19 
24 
>=19 
>=20 
 
 
 
V. Data Handling and Record Maintenance 
 
Specimen labeling: All collected specimens will be labeled with the study number, 
patient number, and date. Specimens will be collected by the research staff, processed 
according to protocol-specific procedures, and stored in locked freezers until assays are 
performed. To maintain confidentiality patient names and hospital numbers will not be 
used. Only the clinical provider, the study coordinator, and data manager will have access 
to the subject identities.  
 
Clinical data collection: Clinical data will be collected during each visit by the clinical 
provider and stored in a locked office or cabinet with access limited to the study 
coordinator and/or data manager. Age, gender, pathologic features of the melanoma, co-
morbid conditions, and concomitant medications will be recorded. 
Paper forms (Appendix 1) will be used for the initial recording of clinical data. Data will be 
recorded legibly in black ballpoint. Corrections will be made legibly, initialed, and dated. 
Correction fluid and covering labels will not be used. Data quality will be monitored by 
random inspection of the completed forms by a research assistant and any problems will 
be discussed with the PI.  
 
Database: Study data will be collected and managed using REDCap (Research 
Electronic Data Capture). REDCap is a secure web application designed to support data 
capture for research studies, providing user-friendly web-based case report forms, real-
time data entry validation (e.g. for data types and range checks), audit trails and a de-
identified data export mechanism to common statistical packages (28). The database is 
hosted at the University of Colorado–Denver Development and Informatics Service Center 
(DISC), which will be used as a central location for data processing and management.  
REDCap data collection projects rely on a thorough study-specific data dictionary defined 
in an iterative self-documenting process by all members of the research team with 
planning assistance from the DISC. The research team can create and design surveys in 
a web browser and engage potential respondents using a variety of notification methods.  
 


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The database will be designed and maintained by the study coordinator and the data 
manager.  The study coordinator will enter the following data at the conclusion of each 
clinic visit: 
- 
Subject information form (name, hospital identification number, study identification 
number, date of birth, gender, ethnicity, and Stage of disease) 
- 
Subject visit form (date of visit, treatment received, and pill counts) 
- 
Adverse event form (signs and symptoms, onset, severity, toxicity grade, action 
taken, outcome, likeliness of being study related) 
 
The data manager will enter the following data at the completion of the study: 
- 
Disease history (primary diagnosis, disease progression) 
- 
Treatment history (previous treatments) 
- 
Sample Information (blood volume, cell counts, storage location, assays 
performed) 
 
 
Data and Safety Monitoring 
The Principal Investigator in collaboration with Dr. Rene Gonzalez, a medical oncologist 
and director of the Melanoma Research Clinics at the University of Colorado Denver, will 
be responsible for monitoring the trial per the trial monitoring plan, monitoring the safety 
and efficacy of the trial, executing the DSM plan and for complying with all reporting 
requirements to local and federal authorities. This oversight will be accomplished through 
additional oversight from the Data and Safety Monitoring Committee (DSMC) at the 
University of Colorado Cancer Center (CU Cancer Center).  The DSMC is responsible for 
ensuring data quality and patient safety for all clinical studies at the CU Cancer Center. 
 
The team of treating medical oncologists and the PI will meet weekly to discuss the 
progress of the clinical trial and any adverse events. The principal investigator will provide 
a Data Safety Monitoring report to the DSMC every 6 months that will include a protocol 
summary; current enrollment numbers; summary of toxicity data to include specific SAEs, 
UAPs and AEs; any dose modifications; all protocol deviations; and protocol amendments. 
The DSM report to the DSMC will also include, if applicable, the results of any efficacy 
data analysis conducted. Results and recommendations from the review of this six month 
report by the DSMC will then need to be submitted by the site to the IRB of record at the 
time of continuing review. A summary of the DSMC activities follows: 
 
• 
Conduct of internal audits 
• 
Ongoing review of all reportable adverse events and all serious/unanticipated 
adverse events  
• 
Has the authority to close and/or suspend trials for safety or trial conduct issues  
• 
May submit recommendations for corrective actions to the CU Cancer Center’s 
Executive Committee 
 
 
Per the CU Cancer Center Institutional DSM Plan, SAEs, UAPs, and reportable AEs are 
reported to the DSMC and the IRB.  All SAEs, UAPs, and reportable AEs are to be 
reported to the DSMC within 5 business days of receiving notification of the occurrence.  
 


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The principal investigator will be notified of any recommendation in order that he may alert all 
investigators involved in the trial with regard to the action plan.  
Each subject’s treatment outcomes will be monitored at weekly team meetings by the 
Investigators and Clinical Research Coordinators (CRCs). Toxicity will be addressed in 
real time and communicated to the team, then submitted to DSMC as indicated. Data 
regarding number of subjects, significant toxicities, dose modifications, and responses will 
be discussed and documented in the minutes.  
The PI will provide a DSM report to the UCCC DSMC on a six month basis after the first 
patient is enrolled. DSM reports will contain data from all participating sites. The DSM 
report will include summaries of minutes taken at monthly meetings, the participants’ 
demographic characteristics, expected versus actual recruitment rates, treatment 
retention rates, any quality assurance or regulatory issues (including a summary of any 
protocol deviations), summary of AEs and SAEs, summary of dose modifications, and any 
actions or changes with respect to the protocol. The DSM report to the DSMC will also 
include, if applicable, the results of any efficacy data analysis conducted. Results from 
these reviews will be provided to all participating investigators to submit to their IRBs at 
the time of continuing review. 
 
Serious Adverse Events, Unanticipated Problems, and Other Reportable Events 
 
All SAEs will be submitted to the DSMC mailbox within 5 business days of Primary 
Investigator becoming aware of the event. All SAEs and UAPs will be submitted to 
COMIRB within 5 business days of Primary Investigator becoming aware of the event. A 
composite list of all reportable events will be provides to COMIRB with each annual 
review. 
 
An adverse event or suspected adverse reaction is considered "serious" if, in the view of 
the investigator, it results in any of the following outcomes:  
- 
death 
- 
life-threatening adverse event 
- 
inpatient hospitalization or prolongation of existing hospitalization 
- 
persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
- 
congenital anomaly/birth defect.   
 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical 
judgment, they may jeopardize the patient or subject and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition. 
 
Unanticipated Problems (UAPs) include adverse events which in the opinion of the 
principal investigator comprise 
- 
both unexpected and probably or definitely related to the drug 
- 
any unforeseen development that potentially increases the likelihood of harm to 
participants or others in the future 
- 
information that indicates a change to the risks or potential benefits of the research 


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- 
an actual unforeseen harmful or unfavorable occurrence to participants or others 
that relates to the research protocol (injuries, psychological events, drug errors). 
 
Other Reportable Adverse Events include  
- 
death unrelated to study treatment if occurring within 30 days of the last study 
treatment 
- 
death at least possibly related to study treatment at any timepoint 
- 
secondary malignancy 
- 
pregnancy 
- 
fetal death 
- 
neonatal death 
 
VI. Data Analysis Plan 
 
We will compare the frequency of MDSC subsets, the suppressive function of MDSCs, the 
frequency of T cell subsets, the expression of activation markers, and the CTL response 
within each group using one-sided one-group t-tests with an alpha of 0.05. Two-group t-
tests will be used to compare the changes before and after treatment between groups. P 
values and corresponding effect sizes will be reported. Kaplan-Maier overall survival 
curves and interval censored progression-free survival curves for the two arms will be 
generated. Median survival times and 95% confidence intervals will be reported. 
Correlations between the frequencies of MDSCs and T cell subsets or expression of 
activation markers will be evaluated using Pearson Correlation Coefficients. 
 
The characterization of potential side effects and toxicities associated with combined 
ipilimumab and VESANOID treatment is an important objective of this protocol. Safety 
outcomes will be assessed using the Bayesian approach. Study discontinuation criteria 
will be followed as outlined in Table 2. 
 
 
Power Analysis 
 
We powered our study based on the second of our primary objectives, to show that 
VESANOID treatment reduces the frequency of MDSCs in melanoma patients. Our 
previous study showed that a small sample size was sufficient to detect two-fold increases 
in the frequency of MDSCs in Stage IV melanoma patients (n = 34, p = 0.041) (11). 
Furthermore, a recent clinical trial combining VESANOID and a dendritic cell vaccine 
found approximately two-fold decreases in the frequency of MDSCs with 12 patients 
treated with VESANOID (p = 0.02) (13). With a conservative assumption of a large 
variance for the change in MDSC frequency in each group (assume the measurement for 
pre- and post-treatment has a correlation coefficient = 0.1 and used the larger variance of 
the two to obtain a variance = 6 for the change), and a conservative estimate for the 
magnitude of change in the Arm B (1.8, or 10% less than was observed in the previous 
clinical trial using VESANOID), a sample size of 24 patients in each arm will provide 80% 
power to detect differences in the change of MDSC frequency between Arm A and Arm B 
using one-sided two-group t-tests with an alpha of 0.05. This sample size will also provide 


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over 85% power to detect the change of 1.5 in MDSC frequency before- and after-
treatment in Arm B using one-sided one-group t-tests with an alpha of 0.05.   
 
Potential Scientific Problems 
 
Combined treatment with VESANOID and ipilimumab may result in excessive toxicity. To 
reduce this likelihood, we proposed a short course of VESANOID treatment at a dose with 
low reported toxicity. The number of agents approved to treatment advanced melanoma 
has changed significantly in the past few months making projections on enrollment more 
challenging. However, one recent change is the approval of ipilimumab for the adjuvant 
treatment of resected stage III melanoma. We believe this may allow for the recruitment of 
additional patients who would receive ipilimumab as standard of care. Our capable clinical 
research team will closely monitor the safety and recruitment of patients, feasibly 
recruiting 48 patients within the time frame of this study. Due to the expertise and 
proficiency of our laboratory research team, we do not anticipate technical difficulties that 
would limit our ability to complete this project. However, the effect of VESANOID may be 
weaker than the previous study on which we based our patient sample size. Although we 
believe this study will provide valuable information regardless of the outcome, we plan to 
compare the effects of VESANOID in patients with high versus low pre-treatment 
frequencies of MDSCs as a potential biomarker for targeting this combined treatment to 
subsets of melanoma patient’s in future clinical trials. 
 
 
Knowledge to be Gained 
 
Approximately 20% of patients receiving ipilimumab monotherapy respond to treatment 
and have prolonged median survival of approximately 4 months. Patients in both arms of 
the study may potentially benefit from ipilimumab treatment. Our hypothesis is that adding 
VESANOID to ipilimumab will reduce immunosuppression and may potentially increase 
the efficacy of ipilimumab. Therefore, the possible benefits outweigh the risks associated 
with participation in this study. Furthermore, the exploratory research aims of the study 
may guide future therapy by improving our general knowledge about melanoma induced 
immunosuppression regardless of the clinical outcomes.  
 
This trial is testing the general hypothesis that combining drugs that target immune cell 
activation with drugs that target blocking immunosuppression are more beneficial to the 
treatment of melanoma patients. We will also be collecting data on the response of the 
immune system to this treatment both in frequency and in function. The knowledge to be 
gained is how VESANOID affects the immune system, whether it augments the immune 
cell activation caused by ipilimumab, and whether it improves clinical responses to 
ipilimumab. Although we are testing these hypotheses in melanoma patients, this 
knowledge may be applicable to other cancer patients where ipilimumab is indicated. 
Furthermore, these data may help in the design of future combinatorial treatments as new 
drugs that target immune cell activation become available. 
  


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Appendix I 
 
Sample Data Collection Forms 


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Appendix II 
 
Current FDA Approval/ VESANOID Package Insert