Conditions: Acral Lentiginous Melanoma Mucosal Melanoma
Keywords: Melanoma Mucosal Acral Lentiginous Ipilimumab Nivolumab Anti-tumor Potential Predictors Opdivo Yervoy
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 14
Healthy volunteers: no

Brief summary:
Participants with advanced or metastatic mucosal melanoma (cohort A) and acral lentiginous melanoma (cohort B) eligible for treatment with nivolumab in combination with ipilimumab followed by nivolumab therapy will submit tissue blocks from tumors of malignant melanoma for histopathology review and immunohistochemistry analysis at Georgetown University-Lombardi Comprehensive Cancer Center. Pretreatment blood will be drawn and stored in the Melanoma Research Foundation Breakthrough Consortium Virtual Repository at each participating institution. At the end of participation, samples will be sent to Georgetown University-Lombardi Comprehensive Cancer Center for processing and storage. An optional pretreatment biopsy of an accessible tumor lesion will be performed in a subset of enrolled patients. Patients will receive nivolumab in combination with ipilimumab according to the standard FDA approved treatment regimen.

Detailed description:
Immunotherapy with HD-IL-2 has produced durable benefit in 10% of patients with metastatic cutaneous melanoma. The antitumor activity of IL-2 has been limited at least in part by immunosuppressive and immune-regulatory forces within the tumor microenvironment. Antibodies against CTLA4 (e.g. ipilimumab), PD1 and its ligand (PD-L1) produced long-term benefit in approximately 20-40% of patients with advanced melanoma. In addition, the combination of ipilimumab with the anti-PD1 antibody, nivolumab, has shown tumor responses in up to 60% of patients with advanced melanoma. These findings have led to FDA approval of ipilimumab and nivolumab as an indication for treatment of patients with advanced melanoma and nivolumab for other cancers. While these data are exciting, only a few patients enrolled to the prior studies had metastatic MCM or ALM. There is no prospective immunotherapy studies conducted in MCM or ALM-specific population. Therefore the activity of the ipilimumab + nivolumab combination in these subsets or patients remains unknown

Reliable predictive biomarkers for the use of immune checkpoint inhibitors are needed to identify pretreatment those patients most likely to respond and early on in treatment assays could help identify mechanisms of tumor response and resistance necessary to improve therapy. Although tumor PD-L1 expression in tumor confers higher treatment response rate, responses to nivolumab or nivolumab + ipilimumab alone were noted in 55% and 41% of patients, respectively, with PD-L1- tumors. Therefore, more reliable predictive biomarkers are needed.

Recently, extensive studies on metastatic colorectal cancer have demonstrated that a new scoring system as well as density of immune cells infiltrates at the center of the tumor and its invasive margin, described as Immunoscore, could accurately separate a group of patients with high Immunoscore with improved DFS, and OS from those with low Immunoscore where the histopathological staging system cannot. A recent study has also demonstrated relationship between degree of pre-treatment CD8+ tumor infiltrating lymphocytes (TILs) infiltration and PD-L1 expression at the invasive margin of the advanced cutaneous melanoma and improved long-term clinical benefits in patients with advanced melanoma who received pembrolizumab monotherapy. Further, there appeared to be an association between tumor response and clonality of the immune infiltrate based on a next-generation sequencing method used to evaluate T-cell receptor rearrangement pre- and in response to checkpoint inhibitor therapy. Also, high mutational burden correlated with overall survival in patients with cutaneous melanoma treated with ipilimumab or lung cancer treated with anti-PD1. However, the biology of MCM and ALM are distinct from cutaneous melanoma at multiple levels. Consequently, the utility of predictive biomarkers developed for cutaneous melanoma remains unknown.

Arms:
arm 1: All patients will receive nivolumab administered IV over 60 minutes at 1 mg/kg combined with ipilimumab administered IV over 90 minutes at 3 mg/kg every 3 weeks for 4 treatment cycles (Induction) then continue with nivolumab administered IV over 60 minutes at 3 mg/kg every 2 weeks until progression, intolerable toxicity, or a maximum of 48 weeks, whichever comes first (Maintenance). Patients exhibiting complete response (CR) should continue nivolumab monotherapy at least 12 weeks beyond documentation of CR, if possible.

Interventions: intervention 1: Nivolumab intervention 2: Ipilimumab
Intervention descriptions: intervention 1: nivolumab administered IV over 60 minutes at 1 mg/kg every 3 weeks for 4 treatment cycles (Induction) then continue with nivolumab administered IV over 60 minutes at 3 mg/kg every 2 weeks intervention 2: ipilimumab administered IV over 90 minutes at 3 mg/kg every 3 weeks for 4 treatment cycles (Induction)

Protocol document:
Prot_SAP_000.pdf: 
Protocol Number: CA209763 
Version dated:  January 31, 2019 
TITLE: 
A Study To Estimate the Anti-Tumor Activity And Identify Potential Predictors of Response in 
Patients with Advanced Mucosal or Acral Lentiginous Melanoma Receiving Standard 
Nivolumab in Combination with Ipilimumab followed by Nivolumab Monotherapy 
 
NCT number:  
NCT02978443 
 
Document Date:  
31Jan2019 
 
 
 


Version dated: January 31, 2019 
2
Clinical Protocol CA209763 
A Study To Estimate the Anti-Tumor Activity And Identify Potential Predictors of Response in 
Patients with Advanced Mucosal or Acral Lentiginous Melanoma Receiving Standard 
Nivolumab in Combination with Ipilimumab followed by Nivolumab Monotherapy 
 
 
 
The Melanoma Research Foundation Breakthrough Consortium (MRFBC) 
 
Lombardi Comprehensive Cancer Center 
 
Suthee Rapisuwon, MD (PI) (WHC) 
Michael B. Atkins, MD (Co-PI) MGUH) 
 
Andrew Pecora MD (HUMC) 
 
 
 
 
 
 
  
 
University of Colorado 
 
 
 
Karl D. Lewis, MD 
Vanderbilt University  
 
 
 
Douglas B. Johnson, MD  
Columbia University  
 
 
 
Richard D. Carvajal, MD 
              
 
Yale Cancer Center 
 
 
 
 
Mario Sznol, MD 
Moffit Cancer Center  
 
 
 
Zeynep Eroglu, MD 
Memorial Sloan Kettering Cancer Center 
 
Alexander Shoushtari, MD 
Northwestern University 
 
 
 
Jeffrey A. Sosman, MD 
Dana-Farber Harvard Cancer Center   
 
Ryan J. Sullivan, MD (MGH) 
 
 
 
 
 
 
 
F. Stephen  Hodi, MD (DFCI 
 
 
 
 
 
 
 
David  F. McDermott MD (BIDMC) 
 
 
 
 
 
 
Laboratory Investigators 
 
 
 
Suthee Rapisuwon, MD (LCCC) 
 
 
 
 
 
 
 
Arash Radfar, MD PhD (LCCC) 
 
 
 
 
 
 
 
Brent Harris, MD PhD (LCCC) 
 
 
 
 
 
 
 
William Robinson, MD PhD (CU) 
 
 
 
 
 
 
 
Alexander Lazar, MD PdD (MDACC) 
 
 
 
 
 
 
 
Jennifer Wargo, MD, MMSc (MDACC) 
 
 
 
Statistician 
 
 
 
Ming Tan, PhD 
Supporters 
 
 
 
Alison Martin, MD (MRFBC) 
Robyn Burns, Ph.D (MRFBC) 
Bristol-Myer Squibb  
 
 
 
 
 
 
 
 
 
 


Protocol Number: CA209763 
Version dated: January 31, 2019 
 
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Protocol Number: CA209763 
Version dated: January 31, 2019 
 
4
INDEX 
Page 
 
Schema 
 
 
 
 
 
 
 
 
3 
 
1.0 
Introduction 
 
 
 
 
 
 
 
 
4 
 
2.0 
Objectives 
 
 
 
 
 
 
 
 
14 
 
3.0 
Selection of Patients  
 
 
 
 
 
 
15 
 
4.0 
Registration Procedures 
 
 
 
 
 
 
16 
 
5.0 
Study Design  
 
 
 
 
 
 
 
16 
 
6.0  
Patient Monitoring 
 
 
 
 
 
 
 
17 
 
7.0 
Evaluation and Management of Toxicity 
 
 
 
 
19 
 
8.0 
Criteria for Removal From Study 
 
 
 
 
 
19 
 
 
 
 
 
  
 
 
 
9.0 
Laboratory Studies   
 
 
 
 
 
 
19 
 
10.0 
Statistical Considerations 
 
 
 
 
 
 
27 
 
11.0  Records to be Kept   
 
 
 
 
 
 
30 
 
12.0 
Patient Consent and Peer Judgment  
 
 
 
 
30 
 
13.0 
Publication Plan 
  
 
 
 
 
 
 
30 
 
14.0 
References 
 
  
 
 
 
 
 
 
31 
 
Appendices 
A. 
Tissue Specimen Shipment Form 
B. 
Blood and Stool Specimen Shipment Form 
C.  
(Optional) On-Treatment Tissue Specimen Shipment Form 
D. 
RECIST v.1.1 criteria 
E. 
Bristol-Myer Squibb Worldwide Safety Adverse Event Reporting 
F.         Suggested Schedule of Events 
G.  
Correlative Sample Submission Schedule 
H. 
Laboratory Manual (Separate document) 
 
 


Protocol Number: CA209763 
Version dated: January 31, 2019 
 
5
 
 
 
 
 
SCHEMA 
 
 
Patients with advanced or metastatic mucosal melanoma (cohort A) and acral lentiginous 
melanoma (cohort B) eligible for treatment with nivolumab in combination with ipilimumab 
followed by nivolumab therapy will submit tissue blocks from tumors of malignant melanoma 
for histopathology review and immunohistochemistry analysis at GU-LCCC.  Pretreatment blood 
will be drawn and stored in the MRFBC Virtual Repository at each participating institution. At 
the end of participation, samples will be sent to GU-LCCC for processing and storage.  An 
optional pretreatment biopsy of an accessible tumor lesion will be performed in a subset of 
enrolled patients.  Patients will receive nivolumab in combination with ipilimumab according to 
the standard FDA approved treatment regimen. Commercial drugs will be used in this protocol 
and will not be supplied by the study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Protocol Number: CA209763 
Version dated: January 31, 2019 
 
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1.0 
INTRODUCTION 
 
1.1 
Background 
 
In 2014, it has been estimated that over 76,000 new patients with melanoma were diagnosed in 
the United States and over 9,700 succumbed to their disease. Over the past several decades, a 
significant increase in the incidence of melanoma has been observed.  The annual increase in 
the incidence rate varies between regions throughout the world, but in general, the increase in 
the Caucasian population has been approximately 3% to 7% per year.  The number of deaths 
due to melanoma has also increased in populations with light skin complexions throughout the 
world in the past four decades (1). 
1.2 
Mucosal Melanoma (MCM) 
Primary mucosal melanoma represents approximately 1.3% of all melanoma diagnoses in the 
U.S. (2). While the incidence of cutaneous melanoma has increased over the past several years, 
the incidence of MCM remains relatively unchanged. Additionally, the pathogenesis of MCM is 
poorly understood in part due to its rarity, the endodermal origin of the mucosa and relative 
paucity of mucosal melanocytes compared to cutaneous tissues (3). Although BRAF mutation is 
a common somatic alteration in cutaneous melanomas, being documented in approximately 
50% of tumors, it is relatively infrequent (3%) in MCM. On the other hand, 10-22% of patients 
with MCMs have tumors containing activating KIT mutations or amplifications (4-7). While 
vigilant dermatologic evaluation can lead to early detection and facilitate early treatment of 
patients with cutaneous melanoma, MCM often presents at a late stage, with a higher propensity 
for deep invasion and distant metastases. MCM is a heterogeneous group of diseases that is 
comprised of primary tumors arising in the head and neck (55.4%), anorectal (23.8%), 
vulvovaginal (18%), and urinary track (2.8%). Each subtype demonstrates distinct natural 
history 
 
1.3 
Acral Lentiginous Melanoma (ALM) 
ALM is an uncommon variant of melanoma with an estimated incidence between 1-7% of all 
melanomas (8). The tumor most commonly arises from palmoplantar area and less commonly in 
subungual and mucous membrane areas. While the incidence is relatively low in Caucasians, 
ALM is more common in Hispanic, Asian and African populations, representing up to 50-70% 
of all melanomas in these groups (8-10). Somatic KIT aberration was found in 36% of ALM, 
with the majority being enhancement or amplification of KIT expression (6). Although somatic 
BRAF mutation rate has been reported to be up to 15-20%(11, 12), these results are likely 
confounded by non-acral lentiginous cutaneous melanoma at acral sites. In fact, a small Spanish 
study found that 87% of ALMs contain somatic alterations including AURKA, CCND1, CDK4, 
NRAS as well as somatic copy number variations, but no somatic BRAF mutations. A larger 
pool of samples is required to determine the nature of the molecular changes in ALM and their 


Protocol Number: CA209763 
Version dated: January 31, 2019 
 
7
clinical relevance (13). 
 
1.4 
Treatment of Metastatic Melanoma 
Approximately half of patients with cutaneous melanoma have tumors with activating mutations 
in BRAF and another 20% have mutations in NRAS making these tumors sensitive to selective 
BRAF and/or MEK inhibitors. However, such small molecule inhibitors have limited to no use 
in patients with MCM or ALM where such mutations are rare. In contrast, activating KIT 
mutations are present in the tumors of 10-22% of patients with MCM and a much smaller 
percentage of patients with ALM.  Further, while therapy with imatinib, a tyrosine kinase 
inhibitor, can inhibit a subset of activating KIT mutations, this treatment only produces an 
overall response rate of approximately 40%, a median duration of response of 4.7 months (2.6-
27.1 months), and a median progression-free survival of 10-15 weeks (5, 14, 15) in such 
patients.  
 
The use of biochemotherapy combinations has been extensively evaluated, as well. The results 
of the largest available Phase III trial comparing cisplatin, vinblastine, and dacarbazine (CVD) 
with concurrent rhIL-2 and interferon- versus CVD alone, showed that biochemotherapy had 
slightly higher objective response rates (ORR) and significantly longer median progression-free 
survival (PFS) than CVD alone, but failed to show an improvement in median overall survival 
(OS). Given the lack of activity together with its associated toxicity and complexity, 
biochemotherapy combinations are generally not recommended for patients with metastatic 
melanoma (16). 
 
Reported ORR using chemotherapy combination in the second-line setting has been 
approximately 10%. For example, in a Phase III randomized study, the ORR reported for a 
paclitaxel and carboplatin combination was 11%, without any impact on OS. Addition of 
bevacizumab to cisplatin and paclitaxel in a randomized Phase II study also did not meet the 
primary end point in PFS and OS(17, 18). 
 
Recently, treatment options for patients with advanced melanoma have expanded greatly with 
the FDA approval in 2011-2013 of the CTLA4 blocking antibody, ipilimumab, and the highly 
selective inhibitors of BRAFV600E, vemurafenib and dabrafenib. In addition, the MEK 
inhibitor, trametinib, also received FDA approval in 2013 for the treatment of patients with 
BRAFV600E mutant melanoma.  In addition the combination of dabrafenib and trametinib 
received FDA approval in 2014 based on a randomized Phase II study in which the combination 
showed superior efficacy relative to dabrafenib alone (19). Finally, the combination of 
vemurafenib + cobimetinib (a MEK inhibitor) received FDA approval in late 2015 based on an 
phase III trial showing the combination was superior to vemurafenib alone.  
 
 
1.5 
PD-1 inhibitor monotherapy in Metastatic Melanoma 
Nivolumab (BMS-936558; anti-PD-1 monoclonal antibody) is a fully human monoclonal 
immunoglobulin (Ig) G4 antibody that binds to the PD-1 cell surface membrane receptor, a 


Protocol Number: CA209763 
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8
negative regulatory molecule expressed by activated T and B lymphocytes. Inhibition of the 
interaction between PD-1 and its ligands promote immune responses and antigen-specific T cell 
responses to both foreign and self antigens. PD-1 receptor blockade by nivolumab is a new 
approach for immunotherapy of tumors. Results from a Phase 1/2 study (CA209-003) indicate 
that nivolumab is active in multiple tumor types including advanced melanoma, non-small cell 
lung carcinoma (NSCLC) and renal cell carcinoma (RCC) (20-22).  
Pembrolizumab (MK-3475) is a selective humanized monoclonal antibody (mAb) of the 
IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-
L1 and PD-L2. The variable region sequences of a mouse antihuman PD-1 antibody were 
attached into a human IgG immunoglobulin with a stabilizing S228P Fc alteration, where the 
IgG4 subtype does not engage Fc receptors or activate complement, therefore avoiding cytotoxic 
effects of the antibody when it binds to the T cells that are intended to activate.  Results from a 
Phase 1 study (Keynote-001) demonstrated that pembrolizumab is active in patients with 
advanced melanoma (23)  
In CA209003 (MDX1106-03), the clinical activity of nivolumab was demonstrated in a variety 
of tumor types and across a range of doses (0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg and 10 
mg/kg). As of the clinical cut-off date of 05-Mar-2013, a total of 306 subjects with melanoma, 
RCC, and NSCLC have been treated with nivolumab. All subjects initiated treatment at least one 
year prior to analysis. A response of either CR or PR, as determined by investigator assessed 
tumor evaluations based on modified RECIST 1.0, has been reported at all dose levels. Among 
107 patients with advanced melanoma who received nivolumab, the objective response rate was 
33/107 (31% [95% CI 22, 41]). Responses occurred at each dose level, with 6/17 (35%), 5/18 
(28%), 11/35 (31%), 7/17 (41%), and 4/20 (20%) melanoma subjects responding at 0.1, 0.3, 1, 3, 
and 10 mg/kg, respectively (24).The 1-, 2-, and 3-year survival rates were 63%, 48%, and 41%, 
respectively, based on Kaplan Meier estimates. The median overall survival (OS) was 17.3 
months (95% CI: 12.5, 36.7). Overall survival at 3 mg/kg dose was 20.3 months (95% CI: 7.2, 
NE [not estimable]. The PFS rate in subjects with melanoma at 48 weeks was 38% [95% CI: 28, 
47]) and at 96 weeks 29% [95% CI: 20, 39]. Median PFS was 3.7 months in all melanoma 
subjects (95% CI: 1.9, 9.3) and 9.7 months (95% CI:1.8, 16.4) for subjects treated at the 3 mg/kg 
dose level.  In CA209037, the clinical activity of nivolumab was compared with investigator’s 
choice of chemotherapy (ICC), dacarbazine or paclitaxel with carboplatin, in a 2:1 randomized, 
phase 3 trial in a total of 405 participants with unresectable or metastatic melanoma who 
progressed after ipilimumab, and BRAF inhibitor if they had BRAF (V600) mutation. With a 
minimum follow-up of 24 weeks, confirmed objective response rate was 31.7% (38/120) in 
nivolumab group versus 10% (5/47) in ICC group. At the time of the primary analysis, 87% 
(33/38) of nivolumab response were continuing on treatment without progression. Median 
progression-free survival was 4.7 months (95%CI 2.3-6.5) for the nivolumab group and 
4.2months (2.1-6.3) for the ICC group (HR 0.82; 99.99% CI 0.32-2.05) (25) 
Similarly, In KEYNOTE-002 (26), a randomized cross-over phase 2 trial in subjects with 
advanced melanoma who had confirmed disease progression after ipilimumab and if BRAF 
(V600) mutant positive, previous treatment with a BRAF or MEK inhibitor or both, 180 patients 


Protocol Number: CA209763 
Version dated: January 31, 2019 
 
9
was randomly assigned to pembrolizumab 2mg/kg, 181 patients to pembrolizumab 10mg/kg, and 
179 patients to chemotherapy of choice (paclitaxel plus carboplatin, paclitaxel alone, carboplatin 
alone, dacarbazine, or temozolomide). With median follow-up duration of 10 months, the study 
showed improvement of median progression-free survival with hazard ratios of 0.57 (95%CI 
0.45-0.73) for pembrolizumab 2mg/kg and 0.5 (95%CI 0.39-0.64) for 10mg/kg compared with 
chemotherapy, with 6-months PFS of 34% and 38% in pembrolizumab 2mg/kg and 10mg/kg 
respective, compared with 16% in chemotherapy control. Objective response rate was 21% and 
25% in pembrolizumab 2mg/kg and 10mg/kg respectively, in comparison to 4% in 
chemotherapy control. 
1.6 
Combined Checkpoint Inhibitors in Metastatic Melanoma 
Immune checkpoint blockade is a rapidly advancing therapeutic approach in the field of 
immuno-oncology, and treatment with investigational agents targeting this mechanism has 
induced regressions in several types of cancer. CTLA-4and PD-1 receptor are two important 
cellular targets that play complementary roles in regulating adaptive immunity. Whereas PD-1 
contributes to T-cell exhaustion in peripheral tissues, CTLA-4 inhibits at earlier points in T-cell 
activation. In preclinical models, combined blockade of PD-1 and CTLA-4 achieve more 
pronounced antitumor activity than blockade of either pathway alone (27). 
Ipilimumab is a fully humanized IgG1 monoclonal antibody (mAb) binding to the anti-cytotoxic 
T-cell lymphoma-4 antigen (CTLA-4). Ipilimumab is an approved therapy for metastatic 
melanoma (Yervoy® Prescribing Information, 2011) and has demonstrated improved overall 
survival as monotherapy and in combination with dacarbazine (28, 29). Ipilimumab has been 
studied in combination with multiple standard of care (SOC) therapies including chemotherapy 
for squamous and non-squamous NSCLC and radiotherapy for hormone resistant prostate cancer. 
Phase 3 studies are ongoing in NSCLC, small cell lung carcinoma (SCLC), and prostate 
carcinoma.  
Ipilimumab has been shown to produce two-year survival of 24% and prolonged median overall 
survival in two randomized phase III trials (30, 31). A composite analysis of 12 clinical studies 
confirmed the potential long-term survival impact of ipilimumab. In this series, 1,257 patients 
were pretreated and 604 were previously untreated for metastatic disease. The dose of 
ipilimumab was 3 mg/kg for 965 patients and 10 mg/kg for 706 patients. The median overall 
survival for the whole patient population was 11.4 months. Most importantly, the survival curve 
reached a plateau of 22% at 3 years, which extended to 10 years, and it was independent of the 
dose. The most frequently reported adverse events (AEs) related to ipilimumab were immune-
related, which could be severe and/or long lasting (28). 
In the Phase 1 dose escalation study CA209-004, the combination of nivolumab and ipilimumab 
has been studied in subjects with unresectable or metastatic melanoma. In this study, a safe dose 
level for the combination of ipilimumab and nivolumab was established for the treatment of 
advanced melanoma. An objective response rate of 53% (9/17) was observed for patients treated 
with 3 mg/kg ipilimumab plus 1 mg/kg nivolumab administered every 3 weeks for four doses 
(induction) and subsequently continued every 12 weeks for up to eight doses (32).  


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In a Phase 2 double-blind randomized study, 142 patients with previously untreated advanced 
melanoma were randomized in a 2:1 ratio to either receive both ipilimumab (3mg/kg) and 
nivolumab (1mg/kg) every 3 weeks followed by nivolumab (3mg/kg), or ipilimumab (3mg/kg) 
and placebo every 3 weeks followed by placebo every 2 weeks (CA209-069), an objective 
response rate (ORR) of 61% (44/72) was observed in patients treated with the combination 
versus 11% (4/37) in ipilimumab monotherapy, with complete responses reported in 22% (16/72) 
in the combination group, and no patients in the ipilimumab monotherapy group. With a 
minimum follow-up period of 11 months, median PFS and median duration of response were not 
reached in the combination group, compared with mPFS of 4.4 months with ipilimumab 
monotherapy. Grade 3 or 4 AEs was higher in the combination group than with ipilimumab 
monotherapy (54% vs. 24%).(33) 
In a Phase III double-blinded randomized study, 945 patients with previously untreated advanced 
or metastatic melanoma were randomized in a 1:1:1 ratio to receive nivolumab monotherapy, 
nivolumab in combination with ipilimumab, or ipilimumab monotherapy. While the study was 
not formally powered to detect statistical difference between the nivolumab monotherapy and the 
combination group, rather to compare the efficacy between nivolumab monotherapy and the 
combination independently to ipilimumab monotherapy group, ORR was observed in 43.7% 
(38.1%-49.3%) in nivolumab monotherapy, 57.6% in the combination group, and 19% in 
ipilimumab monotherapy. With a median follow-up ranging from 12.2 to 12.5 months, a median 
PFS of 6.9 months (95%CI:4.3-9.5), 11.5 months (95%CI: 8.8-16.7), and 2.9 months 
(95%CI:2.8-3.4) were observed in nivolumab monotherapy, nivolumab plus ipilimumab 
combination, and ipilimumab monotherapy respectively. Grade 3 or 4 treatment-related AEs 
occurred in 16.3% of nivolumab monotherapy, 55% in the combination, and 27.3% in the 
ipilimumab monotherapy group. (34) The result from CA209-069 has led to FDA approval of 
Nivolumab/Ipilimumab combination for BRAF-wild type advanced melanoma. 
1.7 
Immunotherapy in Metastatic MCM and ALM 
Immunotherapy with HD-IL-2 produces durable benefit in 10% of patients with metastatic 
cutaneous melanoma. Little data is available about the value of HD IL-2 in patients with MCM 
and ALM. A multicenter, retrospective study in 2013 evaluating 33 patients with advanced 
MCM who were treated with ipilimumab found a treatment response rate roughly comparable to 
that seen in patients with cutaneous melanoma and a durable response rate of 6.7%(4.2-10.9%) 
(35). Another retrospective study of 71 subjects with advanced MCM who received ipilimumab 
as a part of European Expanded Access Program also demonstrated similar objective response 
rate of 12%, with a median progression-free survival of 4.3 months (95%CI:3.4-5.2)(36). 
 
In a recent multi-institutional retrospective analysis of 60 patients with advanced acral and 
mucosal 
melanoma 
treated 
with 
anti-PD1 
inhibitor 
monotherapy 
(nivolumab 
or 
pembrolizumab), including patients from published prospective trials, expanded access program 
and standard treatment (following pembrolizumab approval in 2014), with 77% of patients 
received prior ipilimumab treatment, objective response rate was 32%(15-54%) in ALM, and 
23% (10-40%) in MCM respectively, comparable to the response rate in cutaneous melanoma. 
Half of patients (8/16) with objective response developed relapsed disease with median duration 


Protocol Number: CA209763 
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11 
of response of 12.9 months (95%CI: 2.1-15.9), and median progression-free survival of 3.9 
months (unpublished MSKCC data; In Press) 
 
Despite the potential treatment benefit, majority of patients with metastases from these rare 
variant melanomas either do not respond or only have only a transient response and therefore 
require additional or improved therapeutic options.  
 
In a recent pooled analysis of two phase I (CheckMate 003 and 038) and three phase III studies 
(CheckMate 037, 066, and 067) that enrolled 86 patients with mucosal melanoma (among 889 
treated patients) to receive nivolumab monotherapy, at median follow-up of 9.2months (0.3-
62.5), a median PFS of MCM was 3.0 months (95% CI: 2.2-5.4) compared to 5.1 months (95% 
CI:3.9-6.1) for all treated patients. ORR was 23.3% (95% CI: 14.8-33.6) compared with 35.9% 
(95% CI: 32.7-39.1) for all treated patients, with CR in ~6% in both group. Median duration of 
response was not reached (NR) in mucosal melanoma group, and was 22 months (95% CI: 22-
NR)in all treated patients. Data on the activity of the ipilimumab + nivolumab combination in 
patients with ALM and MCM remains lacking and therefore remains to be determined.  
 
1.8 
Correlative Studies 
1.8.1 Biomarkers in Melanoma 
Reliable predictive biomarkers for the use of immune checkpoint inhibitors are needed to 
identify pretreatment those patients most likely to respond and early on treatment assays could 
help identify mechanisms of tumor response and resistance necessary to improve therapy.  
 
1.8.2 Mutational Burden in Metastatic Melanoma 
Malignant melanoma contains the highest prevalence of somatic mutation per megabase, 
especially high base-pair mutation rate attributable to UV exposure, in comparison to other 
cancers. The highly variable prevalence of somatic mutations among each cancer genome, 
between 0.001 per megabase (Mb) to more than 400 per Mb, is likely contributed by known 
mutagenic exposure, mismatch repair machinery defects, or the duration of cellular 
damage/replication error of passenger mutations between the fertilized egg to the time of 
samples analysis in cancers arise from self-renewing tissues (37, 38).  
 
High mutational burden correlated with overall survival in patients with cutaneous melanoma 
treated with ipilimumab (39) or lung cancer treated with anti-PD1 (40).  


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Figure 1 Mutational Landscape of Tumors According to Clinical Benefit from Ipilimumab Treatment. Panel A 
shows the mutational load (number of nonsynonymous mutations per exome) in the discovery and validation sets, 
according to status with respect to a clinical benefit from therapy. Panel B depicts the Kaplan–Meier curves for 
overall survival in the discovery set for patients with more than 100 nonsynonymous coding mutations per exome 
and patients with 100 or fewer mutations(39). 
 
Although the predictive markers research and discovery in cutaneous melanoma has made great 
progress, there is a paucity of data on the biomarkers predictive of response in patients with 
MCM and ALM receiving checkpoint inhibitor therapy and very little data in any patient 
population receiving combination CTLA4 and anti-PD1 inhibitor therapy. Furney et.al observed 
an average of twelve to sixty-six non-synonymous single nucleotide variants (SNV) in whole 
exome sequencing of both MCM and ALM (41, 42), at least 5-10 fold less mutational load than 
typically seen in cutaneous melanomas. Paucity of data from next-generation whole 
gnome/exome sequencing in MCM and ALM, with less than 10 study subjects from each 
disease, limits analytical power and is subjected to ascertainment bias. 
 
A preliminary study on whole exome sequencing of anorectal, nasopharyngeal and vulvovaginal 
MCM demonstrated unique mutational pattern between MCM and cutaneous melanoma. 
Additionally, analysis of each MCM subgroup could also distinguish each subset of MCM by 
different mutational profiles (Gorden et al; SMR 2015) 
 
1.8.3 Oncogenic Driver Mutation in MCM and ALM 


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The Cancer Genome Atlas project (TCGA) has described an important genomic landscape of 
cutaneous melanoma through next-generation analysis of DNA, RNA and proteins of 333 
primary and/or metastatic melanoma and categorized both genomic and transcriptomic landscape 
signature of cutaneous melanoma with distinct genomic and transcriptomic alterations. However, 
no patient with MCM or ALM was included in the study of 331 patients in the TCGA analysis. 
Previous report has suggested MUC2, MUC4, TNR, MED1, C17orf74, VN1R4 and ZNF717 as 
potential driver mutations in ALM, beside BRAF, TP53 and KIT, while authors did not detect 
new recurrent SNV besides KIT, PTEN and TPR in MCM cohort(41). 
 
1.8.4 Tumor Infiltrating Lymphocytes in Metastatic Melanoma 
Extensive studies on metastatic colorectal cancer has demonstrated that a new scoring system 
based 
on 
the 
two 
lymphocytes 
populations, 
CD3+/CD45RO+, 
CD3+/CD8+, 
or 
CD8+/CD45RO+, as well as density of immune cells infiltrates at the center of the tumor and its 
invasive margin, described as Immunoscore, could accurately separate a group of patients with 
high Immunoscore with improved DFS, and OS from those with low Immunoscore where the 
histopathological staging system cannot (43-45). Increased number of pre-treatment CD8+ T 
lymphocytes, and PD-L1 expression at the invasive tumor margin of advanced cutaneous 
melanoma metastases also predicted improved treatment outcome in patients receiving single 
agent therapy with the PD-1 inhibitor, pembrolizumab (46, 47). 
                             
 
Figure 2. CD8+-cell density at the invasive margin in samples from all responders (left; n = 13) 
and progressors (right; n = 12) who received a biopsy before and during treatment. Filled circle 
indicates complete response; open circle indicates partial response; triangle indicates delayed 
response(47). 
 
Further, there appeared to be an association between tumor response and clonality of the immune 
infiltrate based on ImmunoSEQ assays (Adaptive Biotechnologies). These findings suggested 
that response to single agent anti-PD1 therapy was related to the presence within the tumor of 
clones of tumor antigen-specific CD8 T cells primed to be unleashed by PD1/PDL1 blockade. 
 
1.8.5 Tumor’s Neoantigen Epitopes Discovery in Metastatic Melanoma 
Because antigen presentation via MHC class I molecule as well as cytotoxic T-cell recognition 


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and binding is essential for immune checkpoint inhibitors to function, computational analyses 
from whole exome sequencing have generated putative mutant and non-mutant peptides from 
each non-synonymous missense mutation. An algorhythmic computational method was able to 
predict that the somatic neoantigen epitope formed from mutant peptides, bound patient-specific 
MHC class 1 molecules with higher affinity (≤500nM) and strength.  
 
Further, a number of immunogenic peptides were discovered among patients who achieved long-
term clinical benefit. The number of 9- to 10- amino acid immunogenic neoantigens with 
(≤500nM binding affinity for HLA class I molecules (using patient-specific nonsynonymous 
mutation and HLA types) are also associated with clinical benefit to CTLA-4 monoclonal 
antibody therapy(39, 48). Interestingly, while both neoantigen loads and mutational burden are 
independently correlated with clinical benefit to CTLA-4 monoclonal antibody, both mutational 
load and neoantigen load were strongly correlated to each other (Spearman rho = 0.97, p<0.001) 
(48). Although prior study proposed a panel of shared neoantigen peptides among subjects with 
long-term clinical benefit from CTLA-4 monoclonal antibody (39), subsequent studies failed to 
identify recurrent or shared neoantigens or neoantigen epitopes that could predict response to 
CTLA-4 monoclonal antibody therapy. Instead, majority of neoantigen epitopes identified 
through exome sequencing appears to be private events without obvious shared recurrent 
domains among responders (48). 
 
Based on preliminary analysis of low mutational burden in MCM And ALM, it is important to 
assess whether MCM and ALM low mutational burden translate into low number of neoantigen 
loads, and whether predicted neoantigen peptides burden in variant melanomas have any 
clinically meaningful association with clinical benefits to combined immune-checkpoint 
inhibitor therapy or not. 
 
1.8.6 Gene Expression Profiling in Metastatic Melanoma 
High-throughput gene expression profiling has recently discovered several important findings in 
patients with melanoma.(49-53).  Next-generation sequencing (NGS) technology using RNA 
sequencing (RNA-Seq) provided unbiased determinations of RNA transcript enrichment and 
detection of novel transcripts with high sensitivity and specificity. It also gave broader dynamic 
range of detection and allowed detection of rare and low-abundance, yet potentially important, 
transcripts from the RNA samples. Information on gene expression profiling including the level 
of expression, landscape of transcript isoform variants, and the enrichment of specific RNA 
isoforms affect melanoma tumorigenesis, organ-specific metastasis, malignant transformation 
and inform intratumoral heterogeneity (54-56). This information is unobtainable by standard 
exome or genome sequencing alone.  Additionally, Van Allen demonstrated that prediction of 
neoantigen peptides based on WES alone overestimated 6320 neoantigens without RNA-
sequencing data, and missed 166 neoantigen that are expressed in the tumor, likely due to post-
transcriptional RNA processing. Moreover, the authors found that a large proportion of 
neoantigen predicted by WES are expressed at negligible levels in the patients’ tumors as 
determined by the TCGA database(48). 
 
While considerable information from RNA-Seq expression and methylation data are available 


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for cutaneous melanoma from the Cancer Genome Atlas (TCGA) database and other publicly 
available domains, including remarkable sub-classification of cutaneous melanoma according to 
their transcriptomic expression levels and patterns (57), there is no transcriptomic information on 
MCM and ALM gene expression available in literature.  
 
1.8.7 Gut Microbiota and Immune Checkpoint Inhibitor Therapy 
Recent breakthrough studies have demonstrated that, in mouse models, certain intestinal 
microbiomes, i.e. Bacteriodes fragilis and Bifidobacterium sp. altered the response of cutaneous 
melanoma in mouse model to the anti-PD-1 and anti-CTLA-4 monoclonal antibodies. Further 
studies into Bifidobacterium demonstrated that the bacteria alters dendritic cell activity, which 
led to improvement of tumor-specific CD8+ T cell activity, and delays in melanoma growth (58, 
59). It is not clear whether intestinal microbiomes in variant melanoma have similar or distinct 
immunomodulatory effects against tumor and affect the activity of immune checkpoint 
blockades. 
 
1.8.8 Circulating Cell-Free Tumor Mutant DNA in Metastatic Melanoma 
Successful techniques of circulating cell-free tumor-specific mutant DNA recovery have been 
established with various detection methods, from restriction enzyme digestion and selective 
priming of mutant alleles (60), BEAMing (Beads, Emulsion, Amplification, and Magnetics) (61), 
and droplet digital PCR (ddPCR) (62) in cutaneous melanoma with known driver BRAF, NRAS 
or KIT mutations. However, identifiable driver mutations are detected in only 10-30% of variant 
melanoma, identification of potential circulating tumor-specific mutant DNA as circulating 
biomarkers in variant melanoma whose tumors are often identified as BRAF/NRAS/KIT-wild 
type is an unmet need. 
 
1.9 
Study Rationale 
Immunotherapy with HD-IL-2 has produced durable benefit in 10% of patients with metastatic 
cutaneous melanoma. The antitumor activity of IL-2 has been limited at least in part by 
immunosuppressive and immune-regulatory forces within the tumor microenvironment. 
Antibodies against CTLA4 (e.g. ipilimumab), PD1 and its ligand (PD-L1) produced long-term 
benefit in approximately 20-40% of patients with advanced melanoma. In addition, PD1 pathway 
blockers have shown anti-tumor activity in a variety of advanced solid tumors, including renal 
cell carcinoma, non-small cell and small cell lung carcinoma, bladder cancer, head and neck 
cancer, Merkel cell carcinoma and a rapidly expanding list of other tumors.  In addition, the 
combination of ipilimumab with the anti-PD1 antibody, nivolumab, has shown tumor responses 
in up to 60% of patients with advanced melanoma (34). These findings have led to FDA approval 
of ipilimumab and nivolumab as an indication for treatment of patients with advanced melanoma 
and nivolumab for treatment of patients with advanced non-small cell lung carcinoma, with 
approvals of nivolumab and other PD1 pathway blockers either alone or in combination 
anticipated in other cancer types in the near future. While these data are exciting, few if any 
patients enrolled to this study had metastatic MCM or ALM. Only two retrospective analyses 
evaluated the efficacy of ipilimumab in patients with MCM with the overall objective response 
rate of 6-10% (35, 36), with no prospective immunotherapy studies conducted in MCM or ALM-
specific population. Therefore the activity of the ipilimumab + nivolumab combination in these 


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subsets or patients remains unknown 
 
Reliable predictive biomarkers for the use of immune checkpoint inhibitors are needed to 
identify pretreatment those patients most likely to respond and early on in treatment assays could 
help identify mechanisms of tumor response and resistance necessary to improve therapy. 
Although tumor PD-L1 expression in tumor confers higher treatment response rate, responses to 
nivolumab or nivolumab + ipilimumab alone were noted in 55% and 41% of patients, 
respectively, with PD-L1- tumors (63). Therefore, more reliable predictive biomarkers are 
needed. 
 
Recently, extensive studies on metastatic colorectal cancer have demonstrated that a new scoring 
system as well as density of immune cells infiltrates at the center of the tumor and its invasive 
margin, described as Immunoscore, could accurately separate a group of patients with high 
Immunoscore with improved DFS, and OS from those with low Immunoscore where the 
histopathological staging system cannot. A recent study has also demonstrated relationship 
between degree of pre-treatmentCD8+ tumor infiltrating lymphocytes (TILs) infiltration and PD-
L1 expression at the invasive margin of the advanced cutaneous melanoma and improved long-
term clinical benefits in patients with advanced melanoma who received Pembrolizumab 
monotherapy (47). Further, there appeared to be an association between tumor response and 
clonality of the immune infiltrate based on a next-generation sequencing method used to evaluate 
T-cell receptor rearrangement pre- and in response to checkpoint inhibitor therapy. Also, high 
mutational burden correlated with overall survival in patients with cutaneous melanoma treated 
with ipilimumab or lung cancer treated with anti-PD1. However, the biology of MCM and ALM 
are distinct from cutaneous melanoma at multiple levels as mentioned in Section 1.2.1 &1.2.2.  
Consequently, the utility of predictive biomarkers developed for cutaneous melanoma remains 
unknown 
 
1.10 
Research Hypothesis 
Treatment with nivolumab in combination with ipilimumab in patients with advanced MCM will 
result in an objective response rate of >25% with 90% confidence.  
2.0 
OBJECTIVES 
 
Primary Objectives 
2.1 To assess objective response rate (ORR, defined as complete response [CR] + partial 
response [PR] per investigator-assessed RECIST 1.1 criteria in patients with mucosal 
melanoma (MCM) 
 
Secondary Objectives  
2.2 
To assess objective response rate (ORR, defined as complete response [CR] + 
partial response [PR] per investigator-assessed RECIST 1.1 criteria in patients with acral 
lentiginous melanoma (ALM) 
2.3 
To determine progression-free survival (PFS), and overall survival (OS) in each 
cohort 


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2.4 
To determine whether pre-existing immune cells infiltrate, as well as Ki-67 and 
PD-L1 expressing cells at the invasive tumor margin correlate with clinical response to a 
combination of CTLA-4 and PD-1 blocking therapy 
 
2.5 
To evaluate subject’s cancer genomic landscape using whole exome sequence 
profiling, identify the mutational pattern, and the frequency of somatic mutations and 
their relationship to tumor response in MCM and ALM, as well as subject’s cancer 
genomic landscape using whole exome sequence profiling and utilize bioinformatic tools 
to translate mutations in exomes to identify specific driver mutations in MCM and ALM 
 
Exploratory Objective 
2.6 
To evaluate tumor’s putative neoantigen epitopes from le exome sequencing data 
and determine binding affinity to MHC class I molecules as a potential predictor of 
response to combined checkpoint inhibitors treatment 
2.7 
To determine clonal preservation, expansion, and selection of T-cell receptor 
rearrangement of tumor infiltrating lymphocytes before and after treatment with 
combined ipilimumab/nivolumab therapy as a correlative biomarker of treatment 
response 
2.8 
To identify MCM and ALM gene expression profiling pattern associated with 
treatment response or resistance to combined checkpoint inhibitors treatment 
2.9 
To identify relationship of specific gut microbiota in MCM and ALM with 
CD8+T cell function and anti-tumor immunity 
2.10 
To identify circulating cell-free nucleic acids and its pattern that might be 
associated with combined checkpoint inhibitor treatment response/resistance in order to 
further narrow the application of combined checkpoint inhibitors to those who will 
benefit the most 
 
 
3.0 
SELECTION OF PATIENTS 
 
3.1 
Eligibility Criteria 
 
3.1.1 Patients must have histologically confirmed MCM or ALM that is 
metastatic or unresectable. 
3.1.2 Patients must have measurable disease and be eligible to receive 
nivolumab in combination with ipilimumab treatment per institutional 
guidelines. 
3.1.3 Patients must have a tissue block (or 26 unstained slides) available with 
adequate tumor to perform multiplex immunohistochemistry and nucleic 
acids analyses (i.e. whole exome sequencing) Patients with only a 
previous fine-needle aspirate are ineligible for enrollment. 
3.1.4 Patients must be willing to donate a small amount of whole blood prior to 
treatment and during treatment for laboratory analysis. 


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3.1.5 Patients must give informed consent prior to initiation of therapy. 
3.1.6 Patients must be ambulatory with good performance status (ECOG 0 or 1) 
 
 
3.2 
Ineligibility Criteria  
 
3.21 
Patients who do not have available tissue for immunohistochemistry and 
nucleic acids analyses. 
3.22 
Patients who have received prior immunotherapy for unresectable or 
metastatic disease.  
3.23 
Patients with evidence of active brain metastases, or active leptomeningeal 
disease are ineligible. Patients with a history of brain metastases must 
have completed treatment (i.e. surgery or radiation) prior to enrollment..  
3.24 
Patients with inadequate tissue for analysis. 
 
4.0 
REGISTRATION PROCEDURES 
 
All patients must be registered with the Georgetown CRMO (Clinical Research Management 
Office) before enrollment to study.  Prior to registration, eligibility criteria must be confirmed 
with the Georgetown University Multi-Site Study Coordinator.   
 
All study documents will be redacted by the local site to remove Protected Health Information as 
defined by HIPAA, and will be labeled with subject initials and number. These materials will be 
uploaded by the local site to a secure password-protected cloud-based Georgetown Box portal. 
Access to Box will be provided by Georgetown-LCCC staff, who will receive and review the 
source documents and confirm eligibility. The local site will receive written notification from 
Georgetown-LCCC regarding eligibility. 
 
To register a patient, the patient registration form should be completed by the research nurse or 
data manager and faxed or emailed to the Coordinating Center Attn:Multi- Site coordinator:  fax-
202-687-9361; email-LCCCQAO@georgetown.edu along with the source documentation for the 
eligibility review. 
 
5.0 
STUDY DESIGN 
 
5.1 
Protocol Summary 
 
Eligible patients will receive treatment with nivolumab in combination with 
ipilimumab followed by nivolumab monotherapy following the FDA approved 
schedule. Patients will have a diagnosis of mucosal or acral lentiginous melanoma 
confirmed by a pathologist at each participating institution. Baseline unstained 
tumor slides will be collected. Some patient will have optional fresh biopsies at 
week#6. Pretreatment blood will be drawn, Peripheral blood mononuclear cells 
(PBMCs), and serum,will be separated.. Stool will be collected at baseline and at 


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3 weeks into treatment. All tissues, peripheral blood, and stool samples will be 
stored separately in frozen storage units within the MRFBC Virtual Tissue 
Repository until shipped to GU-LCCC. 
     
 
5.2 
Duration of Therapy and Response Assessment  
 
All patients will receive nivolumab administered IV combined with ipilimumab 
administered IV every 3 weeks for 4 treatment cycles per standard of care 
(Induction) then continue with nivolumab administered IV per standard of care 
until progression, intolerable toxicity, or a maximum of 48 doses, whichever 
comes first (Maintenance). Patients exhibiting complete response (CR) should 
continue nivolumab monotherapy at least 12 weeks beyond documentation of CR, 
if possible.  Patients will be treated within participating sites within the MRFBC. 
Commercial drugs will be used in this protocol and will not be supplied by the 
study. 
  
Patients will be evaluated for response at approximately 12 weeks following the 
initiation of treatment and then approximately every 12 weeks. Response will be 
evaluated by the study investigators using the Response Evaluation Criteria in 
Solid Tumors (RECIST) criteria ,v1.1. (See appendix C). Following completion 
of therapy, patients with evidence of SD, PR or CR will have CT scans every 3 
months for 2 years, then every 6 months during year 3 and then yearly thereafter 
for years 4 and 5.  Attention will be paid to the time of disease progression.  Both 
responding and non-responding patients will be followed until death.  
 
6.0  
PATIENT MONITORING AND DATA SAFETY MONITORING 
 
As this is a tissue collection protocol in patients receiving standard of care therapy, 
patient management will occur through the standard operating procedures of the 
individual participating institution in general accordance with the FDA approved 
regimen.  
 
Case Report forms documenting 1) patient demographic and tumor characteristics, 2) 
extent of treatment, 3) SAEs, 4) efficacy parameters and 5) specimen collection forms 
will be filled out for each patient.   
 
The Principal Investigator and the Co-Investigators will discuss accrual, unusual 
toxicities and any other protocol issues including safety monitoring at their regular 
institution-based 
disease 
group 
meetings 
and 
on 
monthly 
investigator 
teleconferences. organized by the MRFBC,.   
 
The Georgetown Lombardi Comprehensive Cancer Center will be responsible for the 
data and safety monitoring of this multi-site trial.  As this study is an investigator initiated 
study utilizing FDA approved drugs for which the PI does not hold the IND it is 


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considered a moderate risk study which requires real-time monitoring by the PI and study 
team and semi-annual reviews by the LCCC Data and Safety Monitoring Committee 
(DSMC) 
All Severe Adverse Events (SAEs) are required to be reported to the Multi-Site 
Coordinator Office at G-LCCC for review by the DSMC. (see below) 
 
In addition, all SAEs are required to be reported to the local IRB per IRB policy. Based 
on SAEs, the IRB retains the authority to suspend further accrual pending more detailed 
reporting and/or modifications to further reduce risk and maximize the safety of 
participating patients. 
 
Additionally, all SAEs will be reported to BMS Worldwide Safety using BMS SAE 
forms. Please refer to additional details in Appendix E. The sponsor/investigators are 
required to reconcile SAEs reported in the clinical database with SAE cases transmitted 
to BMS Global Pharmacovigilance (GPV&E); worldwide.safety@bms.com. BMS 
requests this to be done quarterly and prior to the database lock or final data summary.  
A summary of the process for the sponsor/investigator is listed below:  
 Sponsor/Investigator sends a request to BMS GPV&E for a “GPV&E reconciliation 
report”. Requests for reconciliation should be sent to aepbusinessprocess@bms.com. 
The request should provide the BMS protocol ID, study title and PI, and 
sponsor/investigator protocol ID.  
 BMS will send a report back to the sponsor/investigator. The data elements listed on 
the GPV&E reconciliation report will contain information the investigator can use for 
individual case identification. Cases on the list from BMS GPV&E should be 
compared to the SAE cases in the clinical database.  
If the sponsor/investigator determines a SAE case was not transmitted to BMS GPV&E, 
the case should be sent immediately to BMS 
  
Progress on the trial and the toxicities experienced will be reviewed by the G-LCCC Data 
and Safety Monitoring Committee every 6 months from the time the first patient is 
enrolled on the study. Results of the DSMC meetings will be forwarded to the G-LCCC 
IRB with recommendations regarding need for study closure. 
   
A written copy of the DSMC recommendations will be given to the trial PI and the 
Georgetown IRB.  If the DSMC recommends a study change for patient safety or efficacy 
reasons the trial PI must act to implement the change as expeditiously as possible.  In the 
unlikely event that the trial PI does not concur with the DSMC recommendations, then 
the G-LCCC Associate Director (AD) of Clinical Research must be informed of the 
reason for the disagreement.  The trial PI, DSMC Chair, and the LCCC AD for Clinical 
Research will be responsible for reaching a mutually acceptable decision about the study 
and providing details of that decision to the IRB.  Confidentiality must be preserved 
during these discussions.  However, in some cases, relevant data may be shared with 


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other selected trial investigators and staff to seek advice to assist in reaching a mutually 
acceptable decision. 
  
If a recommendation is made to change a trial for reasons other than patient safety or 
efficacy, the DSMC will provide an adequate rationale for its decision. If the DSMC 
recommends that the trial be closed for any reason, the recommendation will be reviewed 
by the Associate Director for Clinical Research at G-LCCC.  Authority to close a trial for 
safety reasons lies with the IRB, with the above described input from DSMC and the AD 
for Clinical Research.  
 
 
7.0  
EVALUATION AND MANAGEMENT OF TOXICITY 
 
As this is a tissue collection protocol in patients receiving standard of care therapy, 
formal documentation of physician evaluation and management of toxicity is not 
necessary. However, it is suggested that patients with life-threatening or persistent, severe 
toxic reactions to either checkpoint inhibitors receive no further treatment with either 
agent.   
 
8.0 
CRITERIA FOR REMOVAL FROM STUDY 
 
 
As this is a tissue collection protocol, there is no specific criteria for removal from the 
study with one notable exception; if the patient withdraws consent.  Otherwise, patients 
will continue to be followed clinically and information such as date of progression and 
date of death will be documented. 
 
9.0     LABORATORY STUDIES   
 
The goal of this protocol are to acquire pretreatment tissue and blood from patients 
receiving standard of care nivolumab/ipilimumab combination for advanced MCM or 
ALM.  Studies will be performed on both blood and tissue samples.  Samples will be 
processed and stored at the Virtual Tissue Repository at each site for later batch shipping 
to the specified lab for analysis. Please refer to the Laboratory Manual in Appendix H for 
a complete instructions for specimen collection, processing, and storage.  The tissue, 
blood and stool specimen shipment form can be found in Appendices A and B. 
 
9.1 
The materials required for this protocol are: 
 
Reports and Forms Required 
 
 MRFBC Pathology Material Submission Form  
 A copy of the surgical pathology report. 
 
Biological Material Submission 


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1. Available hematoxillin and eosin stained slides for pathology review 
2. Tumor tissues from a primary melanoma excisional biopsy specimen or 
from a metastatic lesion 
a. A paraffin-fixed, formalin-embedded tumor block, or 
b. 16 unstained slides at 10micron and 10 unstained slides at 5 
micron 
3. Blood samples (see Section 9.3) 
4. Stool samples (see Section 9.4) 
5. (Optional) On-treatment fresh tumor biopsies at baseline and at 6-8 weeks 
of therapy 
NOTE: An informed consent MUST be signed prior to the submission of any material 
for any correlative study, including mandatory diagnostic reviews.  
NOTE: Appendix A and B must be submitted with each specimen submission at the end 
of the study. All samples, sample treatments, and collection time points are to be 
indicated on the form(s). Appendix A and B should accompany the samples to the 
central laboratory. Appendix C must be submitted with on-treatment biopsy 
specimens. 
9.2 
Studies to be performed on tissue 
 
All participants will have either formalin-fixed, paraffin embedded (FFPE) tissue or fresh tumor 
biopsy collected at baseline. Fresh tumor biopsies will be obtained from safely accessible 
locations. Preferred biopsies include excisional or punch biopsy; 14-16 gauge core needle 
biopsies are acceptable if these are not feasible.  
 
Please refer to Laboratory Manual for detailed instructions on specimen collection, processing, 
storage and shipment. 
 
Baseline tumor specimen from each patient will be divided for three different biomarker analyses 
(i) Tumor Whole Exome Sequencing (WES) for analysis of mutational burden, mutational 
pattern, prediction of tumor neoantigen peptides that react to patient’s Major Histocompatibility 
(MHC) class I molecules requires 8 unstained slides at 10microns thickness.  (ii) 
Immunohistochemistry study and quantification of immune cells and PD-L1+ cells infiltration at 
the invasive tumor margin requires 10 unstained slides at 5 microns thickness. (iii) Baseline 
assessment of T-cell receptor (TCR) rearrangement of TILs using next-gen sequencing technique 
requires 8 unstained slides at 10-micron thickness. After the initial analysis, tumor blocks from 
each participant will be stored at the site in MRFBC Tissue Bank for potential later investigation. 
 
Approximately 25% of patients enrolled in this protocol, n=15 (10 patients from MCM cohort 
and 5 patients from ALM cohort) are anticipated to have on-treatment fresh biopsies from safely 
accessible location during weeks 6, 7 or 8 into treatment. Each on-treatment biopsy will be 


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evaluated for CD8+, PD-L1+, and Ki-67+ cells at the invasive margin as a predictive biomarker 
of response (ii) TCR rearrangement in response to a combined immune checkpoint inhibitor 
therapy. 
 
9.2.1 Pathology Review 
 
 
A total of 26 unstained FFPE tumor tissue will be stored at each institution after enrollment. At 
the end of the study, all samples, including on-treatment biopsy samples, at participating 
institution will be batched and shipped to the Histopathology Shared Resource at the G-LCCC. 
Pathologic diagnosis will undergo central review by study dermatopathologist. A provided 
Haematoxillin & Eosin stained slide will be used to confirm physical characteristics of tumor 
samples and eligibility for the correlative studies. The following criteria are required for 
molecular analyses based on the TCGA Biospecimen Qualification Criteria: 
1. Tumor nuclei are presented in ≥60% of the slide 
2. Tumor necrosis is ≤ 20% 
3. In the case of frozen tissue submission, the samples must be continuously frozen with less than 
30 minutes between devascularization and freezing. 
 
 
9.2.2 Tumor Whole Exome Sequencing 
 
 
 
At the end of the study, unstained slides will be batched and shipped to Dr. William Robinson at 
University of Colorado where exome sequencing will be performed. 
 
9.2.3 Assessment of Tumor’s Mutational Burden and Driver Mutations 
 
Analysis will utilize matched paired of (i) tumor, and (ii) normal adjacent stromal tissue from 
pre-treatment samples. Tissue dissection will be done by means of macrodissection. Paired 
normal tissue will serve as control and reference sequence for each patient’s tumor and 
differentiation of germline mutations/polymorphisms from true somatic mutations. Tumor and 
normal stromal tissue will undergo DNA extraction according to standard FFPE DNA extraction 
protocol. DNA fragmentation, DNA library preparation, exome enrichment, automated cluster 
generation, paired-end sequencing, and base calling will be performed at University of Colorado 
at the end of the study. 
 
Human exome sequencing from gDNA will include initial DNA sample QC using NanoDrop 
and Qubit. Exome sequences will be captured using the Agilent SureSelect Exome enrichment 
kit, along with associated QC, and libraries constructed using standard Illumina library 
preparation. Samples will be sequenced on an Illumina HiSeq2000 using PE100-125bp read 
length chemistry, designated 400x average coverage for the cancer and 200x for normal exomes 
with approximately 9-11 Gb output. Standard exome bioinformatic analysis will include 
mapping and alignment (BAM file), QC, coverage, sequence variation (SNPs/Indel report (.VCF 
file)), VCF annotations, functional prediction, and population frequency. Deliverable will 
include raw data FASTq and analysis files (.VCF) 
 


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Data cleanup, variant discovery, annotation, and evaluation will be performed at University of 
Colorado. Mutational load will be quantified as the number of non-synonymous somatic 
mutations per megabase (MB). Raw and annotated exome sequencing data will be available at 
MRFBC Cloud library for future data mining and comparison.  
 
9.2.4 Assessment of MCM and ALM Gene Expression Profiling 
Analysis will utilized matched paired of (i) tumor, and (ii) normal adjacent stromal tissue from 
pre-treatment and (optional) on-treatment samples. Tissue dissection will be done by means of 
macrodissection. Paired normal tissue will serve as internal control for each patient’s tumor and 
tumor and normal stromal tissue will undergo RNA extraction according to standard FFPE 
DNA&RNA extraction protocol. This process will be performed simultaneously with DNA 
extraction using the same starting FFPE or frozen tissue material. Poly(A) enrichment, RNA 
fragmentation, library preparation, automated cluster generation, paired-end sequencing, base 
calling and mapping will be performed. Following RNA-sequencing, raw data will undergo 
standard bioinformatic analysis. BAM files will be converted to merged, demultiplexed 
FASTQs. Paired-end reads will be mapped to the UCSC hg19 human transcriptome using 
specific algorithm to allow alignment of sequence with single base changes due to point 
mutations. Expression level of genes will be quantified using Li et. al algorithm or equivalent 
method (Ei,j=log2(TPMi,j/10+1), where TPMi,j refers to transcript-per-million (TPM) for gene i 
in sample j, as calculated by RSEM(64) v1.2.3 in paired-end mode) Gene expression profiling 
analysis will be performed at MD Anderson Cancer Center under a direction of Drs. Michael 
Davies and Alexander Lazar at the end of the study.  
 
 
9.2.5 Analysis of putative tumor neoantigen epitopes 
Sequence annotation and variant data from each patient at pre-treatment baseline will be used for 
computational analysis to validate neoantigen peptides-MHC class I binding prediction 
algorithm. The prediction will generate a set of candidate T-cell epitopes that could bind to 
patient’s MHC class I alleles, and determine binding affinity between patient’s specific 
neoantigen epitopes and patient’s MHC class I alleles in silico.  
 
The analysis will be performed by Dr. Ming T. Tan at department of Biostatistics & 
Bioinformatics, Georgetown University-Lombardi Comprehensive Cancer Center. 
 
Human Leukocyte Antigen Testing 
High-resolution Human leukocyte antigen (HLA) typing information from each participant will 
be extracted from whole exome sequencing data obtained from whole blood (genomic DNA). 
The analysis will be performed at University of Colorado (Dr. William Robinson) at the end of 
the study. 
 
Please refer to Laboratory Manual for detailed instruction on specimen collection, processing, 
storage and shipment. 
 
 
9.2.6 Assessment of Tumor Immune Cells Infiltrates and PD1+ Cells at Invasive Margin 


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25 
 
Planned analyses include immunohistochemistry (IHC) for PD-1, PD-L1+, CD28, CD3, CD4, 
CD8, CD45RO, FoxP3, Granzyme B, CD14, CD20, DAPI, Pan Melanoma Cocktail (HMB45, 
MART-1, Tyrosinase), and Ki-67+ expression, and quantification of immune cells infiltrates, 
and co-inhibitory/co-stimulatory marker-positive cells at the invasive tumor margin by multiplex 
immunohistochemistry and digital pathology system.  The study will be performed at G-LCCC 
under a direction of Drs. Suthee Rapisuwon and Deborah Berry. Additional IHC markers may be 
added based on available information from MRFBC Consensus information.  
 
Please refer to Laboratory Manual for detailed instruction on specimen collection, processing, 
storage and shipment. 
 
9.2.7 Assessment and Monitoring of T-Cell Receptor Rearrangement 
Planned analyses include assessment of TILs T-cell receptor β-chain genes using ImmunoSeq® 
platform. The analyses will assess the baseline TCR recombination (unique TCR sequences, 
clonal frequency distribution, and TCR diversity) between variable (V), joining (J), and diversity 
(D) segments that result in unique sequences in the antigen-binding site, as well as the effect of 
combined immune checkpoint inhibitor therapy on V(D)J rearrangement of TCR. Specimens for 
TCR rearrangement assay will be batched and sent from G-LCCC to Adaptive Biotechnology for 
analyses at the end of the study. Data analysis will be performed by Dr. Ming Tan at G-LCCC.  
 
Please refer to Laboratory Manual for detailed instruction on specimen collection, processing, 
storage and shipment. 
 
 
9.2.8 Shipping Procedures 
 
At the end of study, fax the tissue specimen shipment form (Appendix A and Appendix C) in 
advance to attn: Deborah Berry  All tissue samples are to be shipped overnight to: 
G-LCCC Histopathology Shared Resource (HTSR)-MRFBC Mucosal & Acral Melanoma Tissue 
Bank 
c/o  Deborah Berry, Ph.D 
Georgetown-Lombardi Comprehensive Cancer Center 
3900 Reservoir Road N.W. 
Pre-Clinical Science Bldg., LR-10 
Washington, DC 20007 
Phone: (202) 687-1924 
Fax: (202) 687-0485 
 
Materials are to be shipped SUNDAY THROUGH THURSDAY only.  Do not ship samples the 
day before a Holiday. 
 
9.3   Studies to be performed on blood 
 


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26 
Peripheral blood samples will be collected on all patients prior to initiation of checkpoint 
inhibitors therapy and during treatment. 
 
Serum will be used in circulating nucleic acids analyses. One additional 10ml tube of blood at 
each specimen collection step has been incorporated into the protocol to exclusively store as a 
part of MRFBC Virtual Repository and for future research.  
  
At the conclusion of the therapy for each patient, specimens should be batched and transported to 
G-LCCC-Histopathology Shared Resource (HTSR) c/o Deborah Berry at the provided address 
for assessment of circulating cell-free nucleic acids. Circulating nucleic acid analyses will be 
done under the direction of Dr. Suthee Rapisuwon at GU-LCCC.  
 
Please refer to Laboratory Manual for detailed instruction on specimen collection, processing, 
storage and shipment. 
 
 
  9.3.1 Assessment of Pretreatment and Serial Levels of Circulating Cell-Free Nucleic 
Acids 
All patients will have plasma and serum collected at pre-treatment, every 6 weeks on-treatment, 
and at PD. Guidelines for isolation of plasma and serum are outlined in Section 9.3. Each sample 
will be linked to the corresponding tumor and labeled with non-identifiable coding system 
designated for each tumor. 
 
Information on mutant DNA enriched in each tumor will be extracted from WES analyses. The 
analysis will assess circulating cell-free mutant DNA from the top most enriched mutations in 
each patient’s tumor, in addition to a panel of cancer related genes. Circulating cell-free mutant 
DNA analysis will be performed on Illumina HiSeq system with TruSeq Nano Sample 
Preparation Kit (or equivalent system) to detect and quantify the circulating mutation load per ml 
of serum. The frequency of circulating mutant DNA will be assessed at baseline, at 6 weeks 
interval, and at RECIST-defined disease progression. The kinetics of circulating cell-free mutant 
DNA compared to baseline will be compared with radiographic response (per RECIST v.1.1) to 
establish the relationship between circulating mutant DNA burden and response to combined 
immune-checkpoint blockade. 
 
Serum samples from each patient may be used to analyze for circulating non-coding RNA (i.e. 
miRNA) and exosomal substrates (i.e. protein, miRNA, snRNA) 
 We will attempt to establish predictive circulatory biomarker that could separate responders 
from non-responders in MCM and ALM cohorts 
 
 
 
9.3.2 Sample Submission Schedule 
 
- Blood samples will be taken  7 days prior to treatment of checkpoint 
inhibitors. 
- Two 10ml Cell-Free DNA BCT® tubes (Streck™; Omaha, NE), two 10ml CPT™ 
Mononuclear cell preparation tube and one 10ml serum separators (red-top 


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27 
SST) will be drawn at pre-treatment. 
- Two 10ml Cell-Free DNA BCT® tube (Streck™; Omaha, NE) One 10ml CPT™ 
Mononuclear cell preparation tube and One 10ml serum separators (red-top 
SST) will be drawn every 6 weeks during treatment and at the time of disease 
progression. 
 
9.3.3 Sample Preparation Guidelines 
 
A batch of Cell-Free DNA BCT® tubes will be distributed to each participating site as the 
patients are being registered to the study. Each site will provide their own CPT™ Mononuclear 
cell preparation tube and serum separators (red-top SST) tubes. All samples will be processed 
locally at each participating institution according to the laboratory manual (see Section 9.3). 
PBMCs, serum and plasma will be stored at -80°C freezer at each participating institution. 
Specimen will be batched and shipped on dry ice overnight to G-LCCC HTSR (attn.Deborah 
Berry) at the end of the study. Specimen will be stored under MRFBC Mucosal & Acral 
Melanoma Tissue Bank. 
 
9.3.4 Shipping Procedures 
 
Fax the blood  and stool specimen shipment form (Appendix B) in advance to 202-687-1924 
attn. Deborah Berry.  
 
At the conclusion of the treatment, all samples are to be batched and shipped on dry ice 
overnight to G-LCCC-HTSR attn. Deborah Berry. All samples are to be shipped overnight to: 
 
G-LCCC Histopathology Shared Resource (HTSR)-MRFBC Mucosal & Acral Melanoma Tissue 
Bank 
c/o  Deborah Berry, Ph.D 
Georgetown-Lombardi Comprehensive Cancer Center 
3900 Reservoir Road N.W. 
Pre-Clinical Science Bldg., LR-10 
Washington, DC 20007 
Phone: (202) 687-1924 
Fax: (202) 687-0485 
 
  
 
 
Materials are to be shipped SUNDAY THROUGH THURSDAY only.  Do not ship samples the 
day before a Holiday. 
 
9.3.5 Collection 
 
All blood samples will be stored in the MRFBC Virtual Specimen Repository and then batch 
shipped to G-LCCC.   
 


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28 
9.4 Study to be performed on Stool 
In this study, collection of stool will occur at baseline (within 7 days prior to starting treatment 
up to within 24 hours of the first treatment), at 3weeks into the treatment (+/- 5 days), and at the 
time of disease progression or study conclusion (+/- 7 days), as indicated on Appendix E.  
 
At the conclusion of the therapy for each patient, specimens should be batched and transported to 
GU-LCCC-Tissue Culture Shared Resource (TCSR) c/o Dionyssia Clagett at the provided 
address in Appendix B. Gut microbiota analyses will be done under the direction of Drs. Jennifer 
Wargo and Michael Davies at MD Anderson Cancer Center.  
 
Please refer to Laboratory Manual for detailed instruction on specimen collection, processing, 
storage and shipment. 
 
 
9.4.1 Effects of Gut Microbiota and Anti-Tumor Activities and Effects of Combined 
Immune Checkpoint Inhibitor Therapy on Gut Microbiota in MCM and ALM 
 
At the end of the study, genomic DNA will be isolated from fecal samples using QIAamp DNA 
Stool Mini Kit (Qiagen), or equivalent brand, following the manufacturer’s instruction. Targeted 
qPCR system will be applied using either Taqman technology (for systems targeting all bacteria 
domain and Bacteroidetes/Prevotella group) or SYBRGreen (for different Bacteroides species) 
Quantitative –PCR will be performed using ABI PRISM 7300 qPCR system or equivalent.  
 
 
 
9.4.2 Sample Submission Schedule 
 
Stool samples will be collected at the following time points 
- Less than 5 days prior to treatment with combined checkpoint inhibitors. 
- Three weeks or C1W4 (+/- 5 days) of therapy 
- At the time of disease progression or at the end of the study 
 
9.3.3 Sample Preparation Guidelines 
 
All samples will be processed locally at each participating institution according to the laboratory 
manual (see Section 9.4). Stool samples in Omnigene®-Gut kits will be stored at -80°C freezer 
at each participating institution. Specimen will be batched and shipped on dry ice overnight to 
GU-LCCC HTSR (attn. Deborah Berry) at the end of the study first, then batched & shipped to 
Dr. Jennifer Wargo at MD Anderson Cancer Center. 
 
9.3.4 Shipping Procedures 
 
Fax the blood and stool specimen shipment form (Appendix B) in advance to 202-687-1924 attn. 
Deborah Berry.  
 
At the conclusion of the treatment, all samples are to be batched and shipped on dry ice 
overnight to G-LCCC-HTSR attn. Deborah Berry. All samples are to be shipped overnight to: 


Protocol Number: CA209763 
Version dated: January 31, 2019 
 
29 
 
G-LCCC Histopathology Shared Resource (HTSR)-MRFBC Mucosal & Acral Melanoma Tissue 
Bank 
c/o  Deborah Berry, Ph.D 
Georgetown-Lombardi Comprehensive Cancer Center 
3900 Reservoir Road N.W. 
Pre-Clinical Science Bldg., LR-10 
Washington, DC 20007 
Phone: (202) 687-1924 
Fax: (202) 687-0485 
 
  
 
 
Materials are to be shipped SUNDAY THROUGH THURSDAY only.  Do not ship samples the 
day before a Holiday. 
 
10.0  STATISTICAL CONSIDERATIONS  
 
10.1 
Design and Objectives 
 
All patients who meet eligibility criteria and enroll in the trial will receive the same nivolumab in 
combination with ipilimumab followed by nivolumab monotherapy regimen following the FDA 
approved schedule.  Patients whose tissue block is not evaluable, either due to failure to be 
received by the GU-LCCC MRFBC Mucosal and Acral Lentiginous Melanoma Tissue Bank or 
due to insufficient tumor on the block, will be replaced. 
 
The primary objective of this trial is to determine if treatment with nivolumab in combination 
with ipilimumab would produce ORR greater than historical 23% response rate in patients with 
MCM who received anti-PD1 monotherapy. Secondary objectives are to determine ORR, PFS 
and OS in each cohort, evaluate MCM and ALM genomic landscape by using whole exome 
sequence profiling and identify frequency of somatic mutations and relationship to tumor 
response, identify specific driver mutation in MCM and ALM. Exploratory objectives are to 
evaluate whether pre-existing immune cell infiltrates and tumors at the invasive tumor margin 
could predict clinical response to the combination of nivolumab and ipilimumab, as previously 
suggested in cutaneous melanoma, evaluate tumor putative neoantigen epitopes and determine 
binding affinity to MHC class I molecules as a potential predictor of response to combined 
checkpoint inhibitors treatment. The study will also explore relationship of specific local 
microbiota in MCM and ALM and response or resistance to the combination therapy. The final 
exploratory objective is to determine clonal preservation, expansion, and selection of TCR 
rearrangement of TILs before and during treatment with combined immune checkpoint inhibitors 
therapy as a predictive biomarker for treatment response. 
 
The primary endpoint is best overall response (complete or partial response as defined in Section 
10.3.1); patients will be followed for progression-free and overall survival. 
 


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30 
Patients who enroll in this trial but do not receive at least one dose of nivolumab in combination 
with ipilimumab, or those for whom tissue is not submitted (see section 9.2) and therefore cannot 
be classified into a predictive factor group, will be deemed unevaluable for the primary endpoint 
of the study; available information on these patients will be described in the final report.  
 
10.2 
Sample Size Considerations 
 
The primary endpoint is objective response rate (CR+PR) as defined earlier in the metastatic 
MCM population. ORR in the ALM populations will be exploratory. The response rate with 
current standard therapy is approximately 23-25%(65). Secondary clinical endpoints include, 
median PFS and OS, 6 month landmark PFS and 1 year landmark OS. Biomarker exploratory 
endpoints will include: 1) the relationship of CD8 T cell score at the invasive margin and tumor 
response for each malignancy and 2) the relationship with tumor mutational frequency using 
whole exome sequencing and response. Bioinformatic tools will be used in an attempt to identify 
recurring driver mutations in MCM (as well as ALM). 
 
The sample size is based on the primary endpoint objective response rate (ORR). The null 
hypothesis that the ORR is at most 25% will be tested against a one-sided alternative. If the ORR 
is less than 25%, we will consider this therapy not worth pursuing. If it is more than 25%, the 
therapy is considered worth pursuing. With 39 patients, the study would have 90% power at a 
significance level of 10% if the true ORR is 45%. To take into consideration a potential non-
evaluable or dropout rate of 20%, 49 patients with MCM (cohort A) will need to be enrolled into 
this study. The exact binomial calculation is used. 
Given the less frequent occurrence of metastatic melanoma involving ALM subtypes, we will 
plan to enroll only 24 patients with ALM (cohort B). This should allow the accrual to both 
cohorts to finish at roughly the same time. Analysis of activity Cohorts B and the biomarker data 
will be primarily descriptive and exploratory.  
 
Patients who enroll in this trial but do not receive the immune checkpoint inhibitor combination, 
or those for whom tissue is not submitted (see section 9.0) and therefore cannot be classified into 
a predictive factor group, will be deemed unevaluable for the primary endpoint of the study; 
available information on these patients will be described in the final report.  
 
The overall accrual rate is anticipated to be 2-4 patients per month on each cohort, based on past 
experience among each institution in the Melanoma Research Foundation Breakthrough 
Consortium , resulting in expected accrual duration of about 20-24 months. The trial’s primary 
analyses are planned to occur at 6 months after each patient has received the treatment. 
  
 
10.3 
Statistical Analysis for Correlative Studies 
 
10.3.1 Best Overall Response 
 


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31 
The proportion of patients achieving a response (best overall response; complete or partial 
response as per RECIST v1.1 criteria (outlined in appendix C) and exact binomial 95% CI will 
be calculated. Objective response rates (complete response + partial response) will be calculated 
separately for each cohort.  
 
Among all enrolled patients, additional hypothesis-generating analyses will be undertaken to 
explore whether somatic mutational loads, difference in driver mutation profiles, level of pre-
treatment CD8+ TIL and PD-L1 expressing cells at the invasive tumor margin, presence of 
tumor specific neoantigen, TCR clonality, gene expression profiles and enrichment of specific 
gut microbiota might be associated with responsiveness in order to further refine the optimal 
population for treatment with ipilimumab and nivolumab combination in MCM and ALM.  
 
Categorical factors and continuous factors will be compared between responders and non-
responders using Fisher's exact tests and two-sample Wilcoxon rank sum test respectively. 
 
 
 
 
10.3.2 Time to Event 
 
Progression-free survival, response interval and overall survival will be summarized using the 
Kaplan-Meier method.  
 
Progression-free survival is defined as the time from the date of treatment initiation until the date 
that disease progression criteria are met or the date death without progression, or is censored at 
the date of last disease assessment without evidence of progression.  
 
Response interval is defined among patients achieving a complete or partial response, from the 
date that response is achieved until the date that disease progression criteria are met or death 
without progression, or is censored at the date of last disease assessment without evidence of 
progression. 
 
Overall survival is calculated from the date of treatment initiation to the date of death, or 
censored at date of last contact.  
 
The association between somatic mutational load and time to event outcome in MCM and ALM 
can be assessed using log rank tests. Kaplan-Meier curves by mutation status will be presented.  
 
 
10.3.3 Toxicity 
 
The toxicity of nivolumab in combination with ipilimumab is well established in the melanoma 
patient population (9-11). As this treatment represents standard of care for this patient 
population, a formal analysis of toxicity is not considered as an endpoint for this study.  As such, 
only the incidence of severe adverse events (immune related AEs) will be collected at the end of 
each patient’s treatment course. A single page form will be provided to collect this summary 


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32 
information. (see Section 7). 
 
 
 
11.0 
RECORDS TO BE KEPT 
 
The original signed consent form will be kept with the patient's other study documentation (e.g., 
the hospital or clinic chart, the case report form).  
 
A data manager will be assigned to this protocol to ensure that data is collected and reviewed on 
a continuous basis.  
The following forms will be submitted to the electronic data capture (eDC) system: 
Forms:  
 
 
 
 
   
 
 
 
 
 
 
 
1) Patient Registration Form 
2) On-Study Form (within 2 weeks after initiation of treatment) 
3) Measurement Form (at baseline and then every 12 weeks) until treatment 
completion.  For patients with persistent SD or response at time of treatment 
discontinuation, subsequent imaging will be every 3 months until 2years from 
treatment initiation, then every 6 months during year 3, and then yearly thereafter for 
years 4 and 5 per standard of care) 
4) Treatment Form (within 4 weeks of decision to stop treatment for each subject) 
Documenting the number of doses of ipilimumab and nivolumab combination 
and nivo monotherapy  
5) Toxicity Form listing Grade 3, 4  SAEs and Grade 2-4 iRAEs on each subject 
(within 12 weeks of decision to stop treatment) 
6) Follow-up Form (every 3 months) 
7) Pathology (submitted along with pathology specimens at end of patient treatment)  
8) Off-Treatment Form (within 2 weeks after treatment terminates) 
 
12.0 
PATIENT CONSENT AND PEER JUDGMENT 
 
All institutional, NCI, FDA, state, and Federal regulations concerning informed consent 
and peer judgment will be fulfilled. 
13.0  PUBLICATION PLAN 
We anticipate that upon completion of this trial, the subsequent manuscript reporting the 
results will be published in the Journal of Clinical Oncology, or similar publication, 
within 12 months of the completion of the study analysis.  Correlative science may be 
reported in a separate manuscript. 


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33 
14.0 
REFERENCES  
1. 
Diepgen TL, Mahler V. The epidemiology of skin cancer. The British journal of 
dermatology. 2002;146 Suppl 61:1-6. 
2. 
Chang AE, Karnell LH, Menck HR. The National Cancer Data Base report on cutaneous 
and noncutaneous melanoma: a summary of 84,836 cases from the past decade. The American 
College of Surgeons Commission on Cancer and the American Cancer Society. Cancer. 
1998;83(8):1664-78. 
3. 
Patrick RJ, Fenske NA, Messina JL. Primary mucosal melanoma. Journal of the 
American Academy of Dermatology. 2007;56(5):828-34. 
4. 
Beadling C, Jacobson-Dunlop E, Hodi FS, Le C, Warrick A, Patterson J, et al. KIT gene 
mutations and copy number in melanoma subtypes. Clinical cancer research : an official journal 
of the American Association for Cancer Research. 2008;14(21):6821-8. 
5. 
Carvajal RD, Antonescu CR, Wolchok JD, Chapman PB, Roman RA, Teitcher J, et al. 
KIT as a therapeutic target in metastatic melanoma. Jama. 2011;305(22):2327-34. 
6. 
Curtin JA, Busam K, Pinkel D, Bastian BC. Somatic activation of KIT in distinct 
subtypes of melanoma. Journal of clinical oncology : official journal of the American Society of 
Clinical Oncology. 2006;24(26):4340-6. 
7. 
Kong Y, Si L, Zhu Y, Xu X, Corless CL, Flaherty KT, et al. Large-scale analysis of KIT 
aberrations in Chinese patients with melanoma. Clinical cancer research : an official journal of 
the American Association for Cancer Research. 2011;17(7):1684-91. 
8. 
Cress RD, Holly EA. Incidence of cutaneous melanoma among non-Hispanic whites, 
Hispanics, Asians, and blacks: an analysis of california cancer registry data, 1988-93. Cancer 
Causes Control. 1997;8(2):246-52. 
9. 
Chang JW, Yeh KY, Wang CH, Yang TS, Chiang HF, Wei FC, et al. Malignant 
melanoma in Taiwan: a prognostic study of 181 cases. Melanoma Res. 2004;14(6):537-41. 
10. 
Hudson DA, Krige JE. Melanoma in black South Africans. J Am Coll Surg. 
1995;180(1):65-71. 
11. 
Yamazaki N, Tanaka R, Tsutsumida A, Namikawa K, Eguchi H, Omata W, et al. BRAF 
V600 mutations and pathological features in Japanese melanoma patients. Melanoma Res. 
2015;25(1):9-14. 
12. 
Zebary A, Omholt K, Vassilaki I, Hoiom V, Linden D, Viberg L, et al. KIT, NRAS, 
BRAF and PTEN mutations in a sample of Swedish patients with acral lentiginous melanoma. J 
Dermatol Sci. 2013;72(3):284-9. 
13. 
Puig-Butille JA, Badenas C, Ogbah Z, Carrera C, Aguilera P, Malvehy J, et al. Genetic 
alterations in RAS-regulated pathway in acral lentiginous melanoma. Exp Dermatol. 
2013;22(2):148-50. 
14. 
Guo J, Si L, Kong Y, Flaherty KT, Xu X, Zhu Y, et al. Phase II, open-label, single-arm 
trial of imatinib mesylate in patients with metastatic melanoma harboring c-Kit mutation or 
amplification. Journal of clinical oncology : official journal of the American Society of Clinical 
Oncology. 2011;29(21):2904-9. 
15. 
Hodi FS, Corless CL, Giobbie-Hurder A, Fletcher JA, Zhu M, Marino-Enriquez A, et al. 
Imatinib for melanomas harboring mutationally activated or amplified KIT arising on mucosal, 
acral, and chronically sun-damaged skin. Journal of clinical oncology : official journal of the 
American Society of Clinical Oncology. 2013;31(26):3182-90. 


Protocol Number: CA209763 
Version dated: January 31, 2019 
 
34 
16. 
Atkins MB, Hsu J, Lee S, Cohen GI, Flaherty LE, Sosman JA, et al. Phase III trial 
comparing concurrent biochemotherapy with cisplatin, vinblastine, dacarbazine, interleukin-2, 
and interferon alfa-2b with cisplatin, vinblastine, and dacarbazine alone in patients with 
metastatic malignant melanoma (E3695): a trial coordinated by the Eastern Cooperative 
Oncology Group. Journal of clinical oncology : official journal of the American Society of 
Clinical Oncology. 2008;26(35):5748-54. 
17. 
Hauschild A, Agarwala SS, Trefzer U, Hogg D, Robert C, Hersey P, et al. Results of a 
phase III, randomized, placebo-controlled study of sorafenib in combination with carboplatin and 
paclitaxel as second-line treatment in patients with unresectable stage III or stage IV melanoma. 
Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 
2009;27(17):2823-30. 
18. 
Kim KB, Sosman JA, Fruehauf JP, Linette GP, Markovic SN, McDermott DF, et al. 
BEAM: a randomized phase II study evaluating the activity of bevacizumab in combination with 
carboplatin plus paclitaxel in patients with previously untreated advanced melanoma. Journal of 
clinical oncology : official journal of the American Society of Clinical Oncology. 2012;30(1):34-
41. 
19. 
Flaherty KT, Infante JR, Daud A, Gonzalez R, Kefford RF, Sosman J, et al. Combined 
BRAF and MEK inhibition in melanoma with BRAF V600 mutations. The New England journal 
of medicine. 2012;367(18):1694-703. 
20. 
Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. 
Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. The New England 
journal of medicine. 2012;366(26):2443-54. 
21. 
McDermott D, Drake CG, Sznol M, Choueiri TK, Powderly J, Smith DC, et al. Clinical 
activity and safety of antiprogrammed death-1 (PD-1) (BMS-936558/MDX-1106/ONO-4538) in 
patients (pts) with previously treated, metastatic renal cell carcinoma (mRCC): An updated 
analysis. Journal of clinical oncology : official journal of the American Society of Clinical 
Oncology. 2013;31(2013):(suppl 6; abstr 351). 
22. 
Gettinger SN, Shepherd FA, Antonia SJ, Brahmer JR, Chow L, Juergen RA, et al. First-
line nivolumab (anti-PD-1; BMS-936558, ONO-4538) monotherapy in advanced NSCLC: 
Safety, efficacy, and correlation of outcomes with PD-L1 status. Journal of clinical oncology : 
official journal of the American Society of Clinical Oncology. 2014;32:5s((suppl; abstr 8024)). 
23. 
Hamid O, Robert C, Daud A, Hodi FS, Hwu WJ, Kefford R, et al. Safety and tumor 
responses with lambrolizumab (anti-PD-1) in melanoma. The New England journal of medicine. 
2013;369(2):134-44. 
24. 
Topalian SL, Sznol M, McDermott DF, Kluger HM, Carvajal RD, Sharfman WH, et al. 
Survival, durable tumor remission, and long-term safety in patients with advanced melanoma 
receiving nivolumab. Journal of clinical oncology : official journal of the American Society of 
Clinical Oncology. 2014;32(10):1020-30. 
25. 
Weber JS, D'Angelo SP, Minor D, Hodi FS, Gutzmer R, Neyns B, et al. Nivolumab 
versus chemotherapy in patients with advanced melanoma who progressed after anti-CTLA-4 
treatment (CheckMate 037): a randomised, controlled, open-label, phase 3 trial. The Lancet 
Oncology. 2015;16(4):375-84. 
26. 
Ribas A, Puzanov I, Dummer R, Schadendorf D, Hamid O, Robert C, et al. 
Pembrolizumab versus investigator-choice chemotherapy for ipilimumab-refractory melanoma 


Protocol Number: CA209763 
Version dated: January 31, 2019 
 
35 
(KEYNOTE-002): a randomised, controlled, phase 2 trial. The Lancet Oncology. 
2015;16(8):908-18. 
27. 
Curran MA, Montalvo W, Yagita H, Allison JP. PD-1 and CTLA-4 combination 
blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 
melanoma tumors. Proc Natl Acad Sci U S A. 2010;107(9):4275-80. 
28. 
Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved 
survival with ipilimumab in patients with metastatic melanoma. The New England journal of 
medicine. 2010;363(8):711-23. 
29. 
Robert C, Thomas L, Bondarenko I, O'Day S, Weber J, Garbe C, et al. Ipilimumab plus 
dacarbazine for previously untreated metastatic melanoma. The New England journal of 
medicine. 2011;364(26):2517-26. 
30. 
McDermott D, Haanen J, Chen TT, Lorigan P, O'Day S, Investigators MDX. Efficacy 
and safety of ipilimumab in metastatic melanoma patients surviving more than 2 years following 
treatment in a phase III trial (MDX010-20). Annals of oncology : official journal of the European 
Society for Medical Oncology / ESMO. 2013;24(10):2694-8. 
31. 
Prieto PA, Yang JC, Sherry RM, Hughes MS, Kammula US, White DE, et al. CTLA-4 
blockade with ipilimumab: long-term follow-up of 177 patients with metastatic melanoma. 
Clinical cancer research : an official journal of the American Association for Cancer Research. 
2012;18(7):2039-47. 
32. 
Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM, et al. 
Nivolumab plus ipilimumab in advanced melanoma. The New England journal of medicine. 
2013;369(2):122-33. 
33. 
Postow MA, Chesney J, Pavlick AC, Robert C, Grossmann K, McDermott D, et al. 
Nivolumab and Ipilimumab versus Ipilimumab in Untreated Melanoma. The New England 
journal of medicine. 2015. 
34. 
Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, et al. Combined 
Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. The New England journal 
of medicine. 2015. 
35. 
Postow MA, Luke JJ, Bluth MJ, Ramaiya N, Panageas KS, Lawrence DP, et al. 
Ipilimumab for patients with advanced mucosal melanoma. The oncologist. 2013;18(6):726-32. 
36. 
Del Vecchio M, Di Guardo L, Ascierto PA, Grimaldi AM, Sileni VC, Pigozzo J, et al. 
Efficacy and safety of ipilimumab 3mg/kg in patients with pretreated, metastatic, mucosal 
melanoma. Eur J Cancer. 2014;50(1):121-7. 
37. 
Alexandrov LB, Nik-Zainal S, Wedge DC, Campbell PJ, Stratton MR. Deciphering 
signatures of mutational processes operative in human cancer. Cell reports. 2013;3(1):246-59. 
38. 
Berger MF, Levin JZ, Vijayendran K, Sivachenko A, Adiconis X, Maguire J, et al. 
Integrative analysis of the melanoma transcriptome. Genome Res. 2010;20(4):413-27. 
39. 
Snyder A, Makarov V, Merghoub T, Yuan J, Zaretsky JM, Desrichard A, et al. Genetic 
basis for clinical response to CTLA-4 blockade in melanoma. The New England journal of 
medicine. 2014;371(23):2189-99. 
40. 
Rizvi NA, Mazieres J, Planchard D, Stinchcombe TE, Dy GK, Antonia SJ, et al. Activity 
and safety of nivolumab, an anti-PD-1 immune checkpoint inhibitor, for patients with advanced, 
refractory squamous non-small-cell lung cancer (CheckMate 063): a phase 2, single-arm trial. 
The Lancet Oncology. 2015;16(3):257-65. 


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Version dated: January 31, 2019 
 
36 
41. 
Furney SJ, Turajlic S, Stamp G, Nohadani M, Carlisle A, Thomas JM, et al. Genome 
sequencing of mucosal melanomas reveals that they are driven by distinct mechanisms from 
cutaneous melanoma. J Pathol. 2013;230(3):261-9. 
42. 
Furney SJ, Turajlic S, Stamp G, Thomas JM, Hayes A, Strauss D, et al. The mutational 
burden of acral melanoma revealed by whole-genome sequencing and comparative analysis. 
Pigment Cell Melanoma Res. 2014;27(5):835-8. 
43. 
Angell H, Galon J. From the immune contexture to the Immunoscore: the role of 
prognostic and predictive immune markers in cancer. Curr Opin Immunol. 2013;25(2):261-7. 
44. 
Galon J, Mlecnik B, Bindea G, Angell HK, Berger A, Lagorce C, et al. Towards the 
introduction of the 'Immunoscore' in the classification of malignant tumours. J Pathol. 
2014;232(2):199-209. 
45. 
Pages F, Kirilovsky A, Mlecnik B, Asslaber M, Tosolini M, Bindea G, et al. In situ 
cytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancer. 
Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 
2009;27(35):5944-51. 
46. 
Messina JL, Fenstermacher DA, Eschrich S, Qu X, Berglund AE, Lloyd MC, et al. 12-
Chemokine gene signature identifies lymph node-like structures in melanoma: potential for 
patient selection for immunotherapy? Sci Rep. 2012;2:765. 
47. 
Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, et al. PD-1 
blockade induces responses by inhibiting adaptive immune resistance. Nature. 
2014;515(7528):568-71. 
48. 
Van Allen EM, Miao D, Schilling B, Shukla SA, Blank C, Zimmer L, et al. Genomic 
correlates of response to CTLA-4 blockade in metastatic melanoma. Science. 
2015;350(6257):207-11. 
49. 
Mandruzzato S, Callegaro A, Turcatel G, Francescato S, Montesco MC, Chiarion-Sileni 
V, et al. A gene expression signature associated with survival in metastatic melanoma. J Transl 
Med. 2006;4:50. 
50. 
Hoek K, Rimm DL, Williams KR, Zhao H, Ariyan S, Lin A, et al. Expression profiling 
reveals novel pathways in the transformation of melanocytes to melanomas. Cancer Res. 
2004;64(15):5270-82. 
51. 
Gallagher PG, Bao Y, Prorock A, Zigrino P, Nischt R, Politi V, et al. Gene expression 
profiling reveals cross-talk between melanoma and fibroblasts: implications for host-tumor 
interactions in metastasis. Cancer Res. 2005;65(10):4134-46. 
52. 
Becker B, Roesch A, Hafner C, Stolz W, Dugas M, Landthaler M, et al. Discrimination 
of melanocytic tumors by cDNA array hybridization of tissues prepared by laser pressure 
catapulting. J Invest Dermatol. 2004;122(2):361-8. 
53. 
Alonso SR, Tracey L, Ortiz P, Perez-Gomez B, Palacios J, Pollan M, et al. A high-
throughput study in melanoma identifies epithelial-mesenchymal transition as a major 
determinant of metastasis. Cancer Res. 2007;67(7):3450-60. 
54. 
Marzese DM, Liu M, Huynh JL, Hirose H, Donovan NC, Huynh KT, et al. Brain 
metastasis is predetermined in early stages of cutaneous melanoma by CD44v6 expression 
through epigenetic regulation of the spliceosome. Pigment Cell Melanoma Res. 2015;28(1):82-
93. 
55. 
Tirosh I, Izar B, Prakadan SM, Wadsworth MH, 2nd, Treacy D, Trombetta JJ, et al. 


Protocol Number: CA209763 
Version dated: January 31, 2019 
 
37 
Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science. 
2016;352(6282):189-96. 
56. 
Yao J, Caballero OL, Huang Y, Lin C, Rimoldi D, Behren A, et al. Altered Expression 
and Splicing of ESRP1 in Malignant Melanoma Correlates with Epithelial-Mesenchymal Status 
and Tumor-Associated Immune Cytolytic Activity. Cancer Immunol Res. 2016;4(6):552-61. 
57. 
Cancer Genome Atlas N. Genomic Classification of Cutaneous Melanoma. Cell. 
2015;161(7):1681-96. 
58. 
Sivan A, Corrales L, Hubert N, Williams JB, Aquino-Michaels K, Earley ZM, et al. 
Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. 
Science. 2015;350(6264):1084-9. 
59. 
Vetizou M, Pitt JM, Daillere R, Lepage P, Waldschmitt N, Flament C, et al. Anticancer 
immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science. 
2015;350(6264):1079-84. 
60. 
Panka DJ, Mier JW, Sullivan RJ. Assaying for BRAF V600E in tissue and blood in 
melanoma. Methods Mol Biol. 2014;1102:117-36. 
61. 
Santiago-Walker A, Gagnon R, Mazumdar J, Casey M, Long GV, Schadendorf D, et al. 
Correlation of BRAF Mutation Status in Circulating-Free DNA and Tumor and Association with 
Clinical Outcome across Four BRAFi and MEKi Clinical Trials. Clinical cancer research : an 
official journal of the American Association for Cancer Research. 2015. 
62. 
Sanmamed MF, Fernandez-Landazuri S, Rodriguez C, Zarate R, Lozano MD, Zubiri L, et 
al. Quantitative cell-free circulating BRAFV600E mutation analysis by use of droplet digital 
PCR in the follow-up of patients with melanoma being treated with BRAF inhibitors. Clin Chem. 
2015;61(1):297-304. 
63. 
Mahoney KM, Atkins MB. Prognostic and predictive markers for the new 
immunotherapies. Oncology (Williston Park). 2014;28 Suppl 3:39-48. 
64. 
Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or 
without a reference genome. BMC Bioinformatics. 2011;12:323. 
65. 
Shoushtari AN, Munhoz RR, Kuk D, Ott PA, Johnson DB, Tsai KK, et al. The efficacy 
of anti-PD-1 agents in acral and mucosal melanoma. Cancer. 2016;122(21):3354-62. 
 
 
 
 
 
   
 
 
 
 
 
 
 
 
 


Protocol Number: CA209763 
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38 
 
 
Appendix A 
MRFBC MUCOSAL AND ACRAL MELANOMA 
TISSUE SPECIMEN SHIPMENT FORM 
Instructions: Send overnight Sunday through Thursday to: 
G-LCCC HTSR-MRFBC Mucosal & Acral Melanoma Tissue Bank 
c/o  Deborah Berry, Ph.D 
Georgetown-Lombardi Comprehensive Cancer Center 
3900 Reservoir Road N.W. 
Pre-Clinical Science Bldg., LR-10 
Washington, DC 20007 
Phone: (202) 687-1924 
Fax: (202) 687-0485 
 
PROTOCOL:   
 
 
 
 
 
 
Case No.:  00__  __  __ 
 
 
 
 
 
01 – GU LCCC 
 
Subject Initials __  __  __ (F,M,L) 
 
Site #:_______ 
 
 
 
 
 
Contact Name:   
 
 
______________________ 
 
Contact Phone:   
 
 
______________________ 
 
Date of Shipment: 
 
 
______________________ 
 
 
Shipping Conditions:  
 
______________________ 
(indicate dry ice, room temp, etc) 
Sample Sent:   
 
 
______________________ 
(indicate total amount: i.e. one core biopsy) 
Site of Biopsy:  
 
 
______________________ 
(indicate primary vs. metastasis and location) 
 
 
 
Surgical Path Report Enclosed: 
 
YES 
 
NO 
 
Formalin-Fixed Paraffin Embedded Biopsy: YES 
 
NO    
 
 
Frozen Tissue Biopsy (on dry ice):   
YES 
 
NO     
 
 
Shipping Notes: 
Please fax form in advance to Bridget Haley  
Please include a copy of the form in the shipping container. 


Protocol Number: CA209763 
Version dated: January 31, 2019 
 
39 
Specimen Received by:  
_______________________________ 
Date/Time Received: _______________________________ 
 
Appendix B 
MRFBC MCM and ALM Protocol 
BLOOD & STOOL SPECIMEN SHIPMENT FORM 
Instructions: Send overnight Sunday through Thursday to: 
G-LCCC HTSR-MRFBC Mucosal & Acral Melanoma Tissue Bank 
c/o  Deborah Berry, Ph.D 
Georgetown-Lombardi Comprehensive Cancer Center 
3900 Reservoir Road N.W. 
Pre-Clinical Science Bldg., LR-10 
Washington, DC 20007 
Phone: (202) 687-1924 
Fax: (202) 687-0485 
 
PROTOCOL:   
 
 
 
 
 
 
Case No.:  00__  __  __ 
 
 
 
 
 
01 – GU_LCCC 
 
Subject Initials __  __  __ (F,M,L) 
 
Site #:_______ 
 
 
 
 
 
Contact Name:   
 
______________________ 
 
Contact Phone:   
 
______________________ 
 
Date of Shipment: 
 
______________________ 
 
Type of Specimen   
 
BLOOD               STOOL 
(circle one) 
Time point on study:  
______________________  
(E.g. Screening, C2D1, etc) 
Shipping Conditions: Dry Ice_____ 
Sample Sent:   
 
 
 
 
 
YES 
 
NO 
 
Shipping Notes: 
Please fax form in advance to 202 687-3821, ATTN:  Bridget L. Haley, RN 
Please include a copy of the form in the shipping container. 
 
Specimen Received By: 
__________________________ 
 
Date/Time Received:  
__________________________ 


Protocol Number: CA209763 
Version dated: January 31, 2019 
 
40 
 
 
Appendix C 
(OPTIONAL) MRFBC MUCOSAL AND ACRAL MELANOMA 
ON-TREATMENT  
TISSUE SPECIMEN SHIPMENT FORM 
Instructions: Send overnight Sunday through Thursday to: 
G-LCCC Histopathology Shared Resource (HTSR)-MRFBC Mucosal & Acral Melanoma Tissue 
Bank 
c/o  Deborah Berry, Ph.D 
Georgetown-Lombardi Comprehensive Cancer Center 
3900 Reservoir Road N.W. 
Pre-Clinical Science Bldg., LR-10 
Washington, DC 20007 
Phone: (202) 687-1924 
Fax: (202) 687-0485 
PROTOCOL:   
 
 
 
 
 
 
Case No.:  00__  __  __ 
 
 
 
 
 
01 – GU LCCC 
 
Subject Initials __  __  __ (F,M,L) 
 
Site #:_______ 
 
 
 
 
 
Contact Name:   
 
 
______________________ 
 
Contact Phone:   
 
 
______________________ 
 
Date of Shipment: 
 
 
______________________ 
 
 
Shipping Conditions:  
 
______________________ 
(indicate dry ice, room temp, etc) 
Date of Biopsy: 
 
 
______________________ 
 
Sample Sent:   
 
 
______________________ 
(indicate total amount: i.e. one core biopsy) 
Site of Biopsy:  
 
 
______________________ 
(indicate primary vs. metastasis and location) 
 
Surgical Path Report Enclosed: 
 
YES 
 
NO 
 
Formalin-Fixed Paraffin Embedded Biopsy: YES 
 
NO    
 
 
Frozen Tissue Biopsy (on dry ice):   
YES 
 
NO     
 
 
Shipping Notes: 
Please fax form in advance to Bridget Haley  


Protocol Number: CA209763 
Version dated: January 31, 2019 
 
41 
Please include a copy of the form in the shipping container. 
Specimen Received by:  
_______________________________ 
Date/Time Received: 
_______________________________ 
 
 
Appendix D 
RECIST v1.1 Response Criteria 
 
Definitions of measurable, non-measurable, target and non-target lesions and objective response 
criteria based on the REVISED (January, 2009) RECIST 1.1 criteria. This Appendix is for 
reference only. The complete RECIST 1.1 criteria are found at: www.recist.com.  
 
1. Definition of Measurable and Non-Measurable Lesions  
 
Measurable disease.  Measurable lesions are defined as those that can be accurately measured in 
at least one dimension (longest diameter to be recorded) as >20 mm by chest x-ray, as >10 mm 
with CT scan, or >10 mm with calipers by clinical exam.  All tumor measurements must be 
recorded in millimeters (or decimal fractions of centimeters). 
 
Note:  Tumor lesions that are situated in a previously irradiated area might or might not be 
considered measurable.  If the investigator thinks it appropriate to include them, the conditions 
under which such lesions should be considered must be defined in the protocol. 
 
Malignant lymph nodes.  To be considered pathologically enlarged and measurable, a lymph 
node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness 
recommended to be no greater than 5 mm).  At baseline and in follow-up, only the short axis will 
be measured and followed. 
 
Non-measurable disease.  All other lesions (or sites of disease), including small lesions (longest 
diameter <10 mm or pathological lymph nodes with ≥ 10 to <15 mm short axis), are considered 
non-measurable disease.  Bone lesions, leptomeningeal disease, ascites, pleural/pericardial 
effusions, lymphangitis cutis/pneumonitis, inflammatory breast disease, and abdominal masses 
(not followed by CT or MRI), are considered as non-measurable. 
 
Note:  Cystic lesions that meet the criteria for radiographically defined simple cysts should not 
be considered as malignant lesions (neither measurable nor non-measurable) since they are, by 
definition, simple cysts. 
 
‘Cystic lesions’ thought to represent cystic metastases can be considered as measurable lesions, 
if they meet the definition of measurability described above. However, if non-cystic lesions are 
present in the same patient, these are preferred for selection as target lesions. 
 
Target lesions.  All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in 
total, representative of all involved organs, should be identified as target lesions and recorded 


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42 
and measured at baseline.  Target lesions should be selected on the basis of their size (lesions 
with the longest diameter), be representative of all involved organs, but in addition should be 
those that lend themselves to reproducible repeated measurements.  It may be the case that, on 
occasion, the largest lesion does not lend itself to reproducible measurement in which 
circumstance the next largest lesion which can be measured reproducibly should be selected.  A 
sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target 
lesions will be calculated and reported as the baseline sum diameters.  If lymph nodes are to be 
included in the sum, then only the short axis is added into the sum.  The baseline sum diameters 
will be used as reference to further characterize any objective tumor regression in the measurable 
dimension of the disease. 
 
Non-target lesions.  All other lesions (or sites of disease) including any measurable lesions over 
and above the 5 target lesions should be identified as non-target lesions and should also be 
recorded at baseline.  Measurements of these lesions are not required, but the presence, absence, 
or in rare cases unequivocal progression of each should be noted throughout follow-up.  
  
2. Methods of measurements  
All measurements should be taken and recorded in metric notation using a ruler or calipers.  All 
baseline evaluations should be performed as closely as possible to the beginning of treatment and 
never more than 4 weeks before the beginning of the treatment. 
 
 
The same method of assessment and the same technique should be used to characterize each 
identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is 
preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be 
imaged but are assessable by clinical exam. 
 
Clinical lesions: Clinical lesions will only be considered measurable when they are superficial 
(e.g., skin nodules and palpable lymph nodes) and 10 mm diameter as assessed using calipers 
(e.g., skin nodules).  In the case of skin lesions, documentation by color photography, including a 
ruler to estimate the size of the lesion, is recommended.  
 
Chest x-ray: Lesions on chest x-ray are acceptable as measurable lesions when they are clearly 
defined and surrounded by aerated lung.  However, CT is preferable.  
 
Conventional CT and MRI: This guideline has defined measurability of lesions on CT scan based 
on the assumption that CT slice thickness is 5 mm or less.  If CT scans have slice thickness 
greater than 5 mm, the minimum size for a measurable lesion should be twice the slice thickness.  
MRI is also acceptable in certain situations (e.g. for body scans).   
 
Use of MRI remains a complex issue.  MRI has excellent contrast, spatial, and temporal 
resolution; however, there are many image acquisition variables involved in MRI, which greatly 
impact image quality, lesion conspicuity, and measurement.  Furthermore, the availability of 
MRI is variable globally.  As with CT, if an MRI is performed, the technical specifications of the 
scanning sequences used should be optimized for the evaluation of the type and site of disease.  


Protocol Number: CA209763 
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43 
Furthermore, as with CT, the modality used at follow-up should be the same as was used at 
baseline and the lesions should be measured/assessed on the same pulse sequence.  It is beyond 
the scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters for all 
scanners, body parts, and diseases.  Ideally, the same type of scanner should be used and the 
image acquisition protocol should be followed as closely as possible to prior scans.  Body 
scans should be performed with breath-hold scanning techniques, if possible. 
 
PET-CT: At present, the low dose or attenuation correction CT portion of a combined PET-CT is 
not always of optimal diagnostic CT quality for use with RECIST measurements.  However, if 
the site can document that the CT performed as part of a PET-CT is of identical diagnostic 
quality to a diagnostic CT (with IV and oral contrast), then the CT portion of the PET-CT can be 
used for RECIST measurements and can be used interchangeably with conventional CT in 
accurately measuring cancer lesions over time.  Note, however, that the PET portion of the CT 
introduces additional data which may bias an investigator if it is not routinely or serially 
performed.   
 
Ultrasound: Ultrasound is not useful in assessment of lesion size and should not be used as a 
method of measurement.  Ultrasound examinations cannot be reproduced in their entirety for 
independent review at a later date and, because they are operator dependent, it cannot be 
guaranteed that the same technique and measurements will be taken from one assessment to the 
next.  If new lesions are identified by ultrasound in the course of the study, confirmation by CT 
or MRI is advised.  If there is concern about radiation exposure at CT, MRI may be used instead 
of CT in selected instances. 
 
Endoscopy, Laparoscopy: The utilization of these techniques for objective tumor evaluation is 
not advised.  However, such techniques may be useful to confirm complete pathological 
response when biopsies are obtained or to determine relapse in trials where recurrence following 
complete response (CR) or surgical resection is an endpoint. 
 
Tumor markers: Tumor markers alone cannot be used to assess response.  If markers are initially 
above the upper normal limit, they must normalize for a patient to be considered in complete 
clinical response.  Specific guidelines for both CA-125 response (in recurrent ovarian cancer) 
and PSA response (in recurrent prostate cancer) have been published [JNCI 96:487-488, 2004; J 
Clin Oncol 17, 3461-3467, 1999; J Clin Oncol 26:1148-1159, 2008].  In addition, the 
Gynecologic Cancer Intergroup has developed CA-125 progression criteria which are to be 
integrated with objective tumor assessment for use in first-line trials in ovarian cancer [JNCI 
92:1534-1535, 2000]. 
 
Cytology, Histology: These techniques can be used to differentiate between partial responses 
(PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as 
germ cell tumors, where known residual benign tumors can remain). 
 
The cytological confirmation of the neoplastic origin of any effusion that appears or worsens 
during treatment when the measurable tumor has met criteria for response or stable disease is 


Protocol Number: CA209763 
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44 
mandatory to differentiate between response or stable disease (an effusion may be a side effect of 
the treatment) and progressive disease. 
 
FDG-PET: While FDG-PET response assessments need additional study, it is sometimes 
reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in 
assessment of progression (particularly possible 'new' disease).  New lesions on the basis of 
FDG-PET imaging can be identified according to the following algorithm:  
a. Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of PD 
based on a new lesion. 
b. No FDG-PET at baseline and a positive FDG-PET at follow-up:  If the positive FDG-
PET at follow-up corresponds to a new site of disease confirmed by CT, this is PD.  If the 
positive FDG-PET at follow-up is not confirmed as a new site of disease on CT, 
additional follow-up CT  scans are needed to determine if there is truly progression 
occurring at that site (if so, the date of PD will be the date of the initial abnormal FDG-
PET scan).  If the positive FDG-PET at follow-up corresponds to a pre-existing site of 
disease on CT that is not progressing on the basis of the anatomic images, this is not PD. 
c. FDG-PET may be used to upgrade a response to a CR in a manner similar to a biopsy in 
cases where a residual radiographic abnormality is thought to represent fibrosis or 
scarring.  The use of FDG-PET in this circumstance should be prospectively described in 
the protocol and supported by disease-specific medical literature for the indication.  
However, it must be acknowledged that both approaches may lead to false positive CR 
due to limitations of FDG-PET and biopsy resolution/sensitivity. 
  
 Note:  A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake greater 
than twice that of the surrounding tissue on the attenuation corrected image. 
 
3. Tumor response evaluation  
 
3.1 Assessment of overall tumor burden and measurable disease  
To assess objective response, it is necessary to estimate the overall tumor burden at baseline and 
use this as a comparator for subsequent measurements. Only subjects with measurable disease 
(defined as the presence of at least 1 measurable lesion) at baseline should be included. If the 
measurable disease is restricted to a solitary lesion, its neoplastic nature should be confirmed by 
cytology/histology.  
 
3.2 Baseline documentation of “Target” and “Non-Target” lesions  
All measurable lesions up to a maximum of 5 lesions total (and maximum of 2 lesions per organ) 
representative of all involved organs should be identified as target lesions and will be recorded 
and measured at baseline. Target lesions should be selected on the basis of their size (lesions 
with the longest diameter) and their suitability for accurate repetitive measurements (either by 
imaging techniques or clinically). A sum of the longest diameter (LD) for all target lesions will 
be calculated and reported as the baseline sum LD. The baseline sum LD will be used as 
reference to further characterize the objective tumor response of the measurable dimension of the 
disease.  


Protocol Number: CA209763 
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45 
All other lesions (or sites of disease) including pathological lymph nodes should be identified as 
non-target lesions and should also be recorded at baseline. Measurements are not required and 
these lesions should be followed as “present,” “absent,” or “unequivocal progression.”  
 
4. Response Criteria 
 
4.1 Evaluation of target lesions  
CONFIDENTIAL  
Complete 
Response (CR):  
Disappearance of all target lesions. Any pathological lymph nodes (whether 
target or non-target) must have reduction in short axis to <10 mm.  
Partial Response 
(PR):  
At least a 30% decrease in the sum of LD of target lesions, taking as reference 
the baseline sum LD.  
Progressive 
Disease (PD):  
At least a 20% increase in the sum of LD of target lesions, taking as references 
the smallest sum of LD recorded since the treatment started or the appearance 
of 1 or more new lesions.  
Stable Disease 
(SD):  
Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify 
for PD, taking as references the smallest sum LD since treatment started.  
 
4.2 Evaluation of non-target lesions  
Complete Response (CR):  
Disappearance of all target lesions. Any pathological lymph nodes 
(whether target or non-target) must have reduction in short axis to 
<10 mm (the sum may not be “0” if there are target nodes)  
Disappearance of all non - target lesions and normalization of 
tumor marker level. All lymph nodes must be non-pathological in 
size (<10 mm short axis)  
Non-Complete Response 
(non-CR)/Non- Progressive 
Disease (non- PD):  
Persistence of 1 or more non-target lesion(s) and/or maintenance of 
tumor marker level above the normal limits  
Progressive Disease (PD):  
Unequivocal progression of existing non-target lesions defined as: 
Overall level of substantial worsening in non-target disease such 
that, even in presence of SD or PR in target disease, the overall 
tumor burden has increased sufficiently to merit discontinuation of 
therapy  
In the absence of measurable disease, change in non- measurable 
disease comparable in magnitude to the increase that would be 
required to declare PD for measurable disease.  
Examples include an increase in a pleural effusion from ‘trace’ to 
‘large’, an increase in lymphangitic disease from localized to 
widespread 
Target Lesions: >20% increase in the SLD taking as reference the 
smallest SLD recorded since the treatment started (nadir) and 
minimum 5 mm increase over the nadir  
Although a clear progression of “non target” lesions only is exceptional, in such circumstances, 


Protocol Number: CA209763 
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46 
the opinion of the treating physician should prevail and the progression status should be 
confirmed later by the review panel (or study chair).  
 
4.3 Evaluation of best overall response  
The best overall response is the best response recorded from the start of the treatment until 
disease progression/recurrence (taking as reference for progressive disease the smallest 
measurements recorded since the treatment started).  The patient's best response assignment will 
depend on the achievement of both measurement and confirmation criteria. 
 
        For Patients with Measurable Disease (i.e., Target Disease) 
 
Target 
Lesions 
Non-Target 
Lesions 
New 
Lesions 
Overall 
Response 
Best Overall Response 
when Confirmation is 
Required* 
CR 
CR 
No 
CR 
>4 wks. 
Confirmation** 
CR 
Non-
CR/Non-
PD 
No 
PR 
 
>4 wks. 
Confirmation** 
CR 
Not 
evaluated 
No 
PR 
PR 
Non-
CR/Non-
PD/not 
evaluated 
No 
PR 
SD 
Non-
CR/Non-
PD/not 
evaluated 
No 
SD 
documented at least 
once >4 wks. from 
baseline** 
PD 
Any 
Yes or 
No 
PD 
 
no prior SD, PR or CR 
Any 
PD*** 
Yes or 
No 
PD 
Any 
Any 
Yes 
PD 
*      See RECIST 1.1 manuscript for further details on what is evidence 
of a new lesion. 
**    Only for non-randomized trials with response as primary endpoint. 
***  In exceptional circumstances, unequivocal progression in non-
target lesions may be accepted as disease progression. 
 
Note: Patients with a global deterioration of health status requiring 
discontinuation of treatment without objective evidence of 
disease progression at that time should be reported as 
“symptomatic deterioration.”  Every effort should be made to 


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47 
document the objective progression even after discontinuation of 
treatment. 
 
 
        For Patients with Non-Measurable Disease (i.e., Non-Target Disease) 
Non-Target Lesions 
New Lesions 
Overall Response 
CR 
No 
CR 
Non-CR/non-PD 
No 
Non-CR/non-PD* 
Not all evaluated 
No 
not evaluated 
Unequivocal PD 
Yes or No 
PD 
Any 
Yes 
PD 
*  ‘Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease 
since SD is increasingly used as an endpoint for assessment of efficacy in 
some trials so to assign this category when no lesions can be measured is not 
advised 
 
5. Confirmatory measurement/Duration of response  
5.1 Confirmation  
The main goal of confirmation of objective response is to minimize the risk of overestimation of 
the response rate. This aspect of response evaluation is particularly important in non- randomized 
studies where response is the primary endpoint. In this setting, to be assigned a status of PR or 
CR, changes in tumor measurements must be confirmed by repeat studies that should be 
performed no less than 4 weeks after the criteria for response are first met. In the case of SD, 
follow-up measurements must have met the SD criteria at least once after study entry at a 
minimum interval of 6 weeks.  
 
6. Specifications for radiological imaging  
These notes are recommendations for use in clinical studies and as such these protocols for 
computed tomography (CT) and magnetic resonance imaging (MRI) scanning may differ from 
those employed in clinical practice at various institutions. The use of standardized protocols 
allows comparability both within and between different studies, irrespective of where the 
examination has been undertaken.  
 
6.1 Chest X-ray  
Measurement of lesions surrounded by pulmonary parenchyma is feasible, but not preferable as 
the measurement represents a summation of densities. Furthermore, there is poor identification of 
new lesions within the chest on X-ray as compared with CT. Therefore, measurements of 
pulmonary parenchymal lesions as well as mediastinal disease are optimally performed with CT 
of the chest. MRI of the chest should only be performed in extenuating circumstances. Even if IV 
contrast cannot be administered (for example, in the situation of allergy to contrast), a non-
contrast CT of the chest is still preferred over MRI or chest X-ray.  
 
6.2 CT  
CT scans of the thorax, abdomen and pelvis should be contiguous throughout the anatomical 


Protocol Number: CA209763 
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48 
region of interest. As a rule of thumb, the minimum size of the lesion should be no less than 
double the slice thickness. Lesions smaller than this are subject to significant “partial volume” 
effects and such a lesion may appear to have “responded” or “progressed” on subsequent 
examinations, when in fact they remain the same size. This minimum lesion size for a given slice 
thickness at baseline ensures that any lesion appearing smaller on subsequent examinations will 
truly be decreasing in size.  
The type of CT scanner is important regarding the slice thickness and minimum sized lesion. For 
spiral (helical) CT scanners, the minimum size of any given lesion at baseline may be 10mm, 
provided the images are reconstructed contiguously at 5 mm intervals. For conventional CT 
scanners, the minimum sized lesion should be 20 mm using a contiguous slice thickness of 10 
mm.  
 
The fundamental difference between spiral and conventional CT is that conventional CT acquires 
the information only for that particular slice thickness scanned, which is then expressed as a 2 
dimensional representation of that thickness or volume as a grey scale image. The next slice 
thickness needs to be scanned before it can be imaged and so on. Spiral CT acquires the data for 
the whole volume imaged, typically the whole of the thorax or upper abdomen in a single breath 
hold of about 20-30 seconds. To view the images, a suitable reconstruction algorithm is selected, 
by the machine, so the data are appropriately imaged. As suggested above, for spiral CT, 5 mm 
re-constructions can be made thereby allowing a minimum sized lesion of 10mm.  
Spiral CT is now the “standard” in most hospitals involved in cancer management in U.S., 
Europe, and Japan, so the comments related to spiral CT are pertinent. However, some 
institutions involved in clinical studies will have conventional CT, but the number of these 
scanners will decline as they are replaced by spiral CT.  
 
Other body parts, where CT scans are of different slice thickness, (such as the neck, which are 
typically of 5 mm thickness) or in the young pediatric population, where the slice thickness may 
be different, the minimum sized lesion allowable will be different. However, it should be double 
the slice thickness. The slice thickness and the minimum sized lesion should be specified in the 
study protocol.  
In subjects in whom the abdomen and pelvis have been imaged, oral contrast agents should be 
given to accentuate the bowel from other soft tissue masses. This is almost universally 
undertaken routinely.  
 
Intravenous contrast agents should also be given, unless contra-indicated for medical reasons, 
such as allergy. This is to accentuate vascular structures from adjacent lymph node masses and to 
help enhance liver and other visceral metastases. Although in clinical practice its use may add 
little, in the context of a clinical study where objective response rate based on measurable disease 
is the endpoint, unless an intravenous contrast agent is given, a significant number of otherwise 
measurable lesions will not be measurable. In subjects in whom the disease is apparently 
restricted to the periphery of the lungs, for example, the use of intravenous contrast agents 
appears unnecessary, but the aim of a clinical study is to ensure lesions are truly resolving, and 
there is no evidence of new disease at other sites scanned, e.g., small metastases in the liver.  
 


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49 
The method of administration of intravenous contrast agents is variable. Rather than try to 
institute rigid rules regarding methodology of administration of contrast agents and the volume 
injected, it is appropriate to suggest that an adequate volume of a suitable contrast agent should 
be given such that the metastases are demonstrated to best effect and a consistent method is used 
on subsequent examinations for any given subject.  
 
All images from each examination should be included and not “selected” images of the apparent 
lesion. This is to ensure that if a review is undertaken, the reviewer can satisfy him/herself that 
no other abnormalities coexist. All window settings should be included, particularly in the thorax 
where lung and soft tissue windows should be considered.  
 
When measuring lesions, lesions should be measured on the same window setting on each 
examination. It is not acceptable to measure a lesion on lung windows on 1 examination, then on 
soft tissue settings on the next. In the lung, it does not really matter whether lung or soft tissue 
windows are used for intra-parenchymal lesions, provided a thorough assessment of nodal and 
parenchymal disease has been undertaken and the target lesions are measured as appropriate 
using the same window settings for repeated examinations throughout the study.  
 
6.3 MRI  
MRI is a complex issue. MRI is entirely acceptable and capable of providing images in different 
anatomical planes. It is important therefore that when it is used lesions must be measured in the 
same anatomical plane using the same imaging sequences on subsequent examinations. MRI 
scanners vary in the images produced. Some of the factors involved include the magnet strength 
(high field magnets require shorter scan times, typically 2-5 minutes), the coil design and subject 
cooperation. Wherever possible, the same scanner should be used. For instance, the images 
provided by a 1.5T scanner will differ from those using a 0.5T scanner. Although a comparison 
can be made, it is not ideal. Moreover many subjects with advanced malignancy are in pain, so 
their ability to remain still for the duration of a scan sequence, in the order of 2-5 minutes is 
limited. Any movement during the scan time leads to motion artifacts, degradation of image 
quality such that the examination will probably be useless.  
 
For these reasons, CT is at this point in time the imaging modality of choice.  
The same imaging modality must be used throughout the study to measure disease. Different 
imaging techniques have differing sensitivities, so any given lesion may have different 
dimensions at any given time if measured with different modalities. It is therefore not acceptable 
to interchange different modalities throughout a study and use these measurements. It must be 
the same technique throughout.  
 
6.4. Ultrasound  
Ultrasound examinations should not be used in clinical studies to measure tumor regression or 
progression because the examination is necessarily subjective. It is not available for independent 
review at a later date, it has to be assumed that the hard copy films available represent a true and 
accurate reflection of events, which may or may not be the case. Furthermore, if, for example, 
the only measurable lesion is in the para-aortic region of the abdomen, and if gas in the bowel 


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50 
overlies the lesion, the lesion will not be detected because the ultrasound beam cannot penetrate 
the gas. This subject will then become a protocol violator.  
Appendix E. 
Bristol-Myer Squibb Worldwide Safety Adverse Event Reporting 
 
All Serious Adverse Events (SAEs) that occur following the subject’s written consent to 
participate in the study through 100 days of discontinuation of dosing must be reported to BMS 
Worldwide Safety 
 
If the BMS safety address is not included in the protocol document (e.g. multicenter studies 
where events are reported centrally), the procedure for safety reporting must be 
reviewed/approved by the BMS Protocol Manager.  Procedures for such reporting must be 
reviewed and approved by BMS prior to study activation. 
 
The BMS SAE form should be used to report SAEs.  If the BMS form cannot be used, another 
acceptable form (i.e CIOMS or Medwatch) must be reviewed and approved by BMS.  The BMS 
protocol ID number must be included on whatever form is submitted by the 
Sponsor/Investigators. 
 
Following the subject’s written consent to participate in the study, all SAEs, whether related or 
not related to study drug, are collected, including those thought to be associated with protocol-
specified procedures. The investigator should report any SAE occurring after these time periods 
that is believed to be related to study drug or protocol-specified procedure. 
 
For studies with long-term follow-up periods in which safety data are being reported, include the 
timing of SAE collection 
 
 In accordance with local regulations, BMS will notify investigators of all reported SAEs 
that are suspected (related to the investigational product) and unexpected (i.e., not 
previously described in the IB). In the European Union (EU), an event meeting these 
criteria is termed a Suspected, Unexpected Serious Adverse Reaction (SUSAR). 
Investigator notification of these events will be in the form of an expedited safety report 
(ESR).  
o Other important findings which may be reported by the as an ESR include: 
increased frequency of a clinically significant expected SAE, an SAE considered 
associated with study procedures that could modify the conduct of the study, lack 
of efficacy that poses significant hazard to study subjects, clinically significant 
safety finding from a nonclinical (eg, animal) study, important safety 
recommendations from a study data monitoring committee, or sponsor decision to 
end or temporarily halt a clinical study for safety reasons. 
o Upon receiving an ESR from BMS, the investigator must review and retain the 
ESR with the IB. Where required by local regulations or when there is a central 


Protocol Number: CA209763 
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51 
IRB/IEC for the study, the sponsor will submit the ESR to the appropriate 
IRB/IEC. The investigator and IRB/IEC will determine if the informed consent 
requires revision. The investigator should also comply with the IRB/IEC 
procedures for reporting any other safety information.  
In addition, suspected serious adverse reactions (whether expected or unexpected) shall be 
reported by BMS to the relevant competent health authorities in all concerned countries 
according to local regulations (either as expedited and/or in aggregate reports). 
 
Serious Adverse Event Collection and Reporting 
Following the subject’s written consent to participate in the study, all SAEs, whether related or 
not related to study drug, must be collected, including those thought to be associated with 
protocol-specified procedures. All SAEs must be collected that occur within 100 days of 
discontinuation of dosing.  
All SAEs must be collected that occur during the screening period. If applicable, SAEs must be 
collected that relate to any protocol-specified procedure (eg, a follow-up skin biopsy).  The 
investigator should report any SAE that occurs after these time periods that is believed to be 
related to study drug or protocol-specified procedure.  
SAEs, whether related or not related to study drug, and pregnancies must be reported to BMS 
within 24 hours. SAEs must be recorded on BMS or an approved form; pregnancies on a 
Pregnancy Surveillance Form. 
SAE Email Address:  Worldwide.Safety@BMS.com 
SAE Facsimile Number:  609-818-3804 
If only limited information is initially available, follow-up reports are required. (Note: Follow-up 
SAE reports should include the same investigator term(s) initially reported.)  
If an ongoing SAE changes in its intensity or relationship to study drug or if new information 
becomes available, a follow-up SAE report should be sent within 24 hours to the BMS (or 
designee) using the same procedure used for transmitting the initial SAE report.  
All SAEs should be followed to resolution or stabilization. 
The Sponsor/Investigator will ensure that all SAEs in the clinical database are reported to BMS 
and any applicable health authority during the conduct of the study. This reconciliation will 
occur at least quarterly and be initiated by the sponsor/investigator. Sponsor/investigator will 
request a reconciliation report from: aepbusinessprocess@bms.com. During reconciliation, any 
events 
found 
to 
not 
be 
reported 
previously 
to 
BMS 
must 
be 
sent 
to 
Worldwide.Safety@BMS.com. 
 


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52 
Appendix F 
Suggested Schedule of Events 
Assessment 
6 wks = 1 cycle 
 
Baseline 
Induc
tion 
 
 
Maintenance 
 
End of 
Treatment 
Disease 
Progression 
Follow-up/ Safety Follow-up 
Within 
4 weeks 
Cycle 
1 
Cycle 
1 
Cycle 
2 
Cycle 3 and 
after 
 
 
 
Day1 
Day 22 
Day 1 
Day 1 
Informed consent  
x 
 
 
 
 
 
 
 
Assignment 
identification numbers 
x 
 
 
 
 
 
 
 
Medical history and 
demographicse 
x 
 
 
 
 
 
 
 
Complete physical exam 
x 
 
 
 
 
 
 
 
Weight 
x 
 
 
 
 
 
 
 
Height 
x 
 
 
 
 
 
 
 
ECOG performance 
status 
x 
 
 
 
 
x 
x 
x 
Hematology&Chemistry 
labc,d 
x 
 
x 
x 
x 
x 
x 
 
Adverse events  
 
 
x 
x 
x 
x 
x 
x 
Imaging Studiesa 
xa 
 
 
 
xa 
 
 
xa 
Submission of Archival 
tumor for review 
x 
 
 
 
 
 
 
 
Fresh tumor biopsy 
 
 
 
xb 
 
 
 
 
Peripheral blood 
correlates  
x 
x 
 
x 
x 
 
 
 
Microbiota correlates 
 
x 
x 
 
 
 
x 
 
 
a. Non-CNS Imaging should include a chest, abdomen and pelvis CT. Imaging of other sites should be obtained when clinically indicated. If follow up scans 


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53 
indicate partial or complete response, a repeat confirmatory scan should be obtained within 12 weeks (but no sooner than 6 weeks). Baseline imaging should 
be done within 4 weeks prior to registration, then every 12 weeks if patient is < 2 years from study entry, then every 6 months (+/- 4 weeks) until 5 years 
from study entry 
b. Fresh tumor biopsy is expected in 25% of patients (N=25; 10 MCM and 5 ALM patients) to consent to on-treatment biopsies. Please refer to appendix G for 
detail. This can be collected within weeks 6-8. 
c. Hematology Labs should include CBC with differential (hemoglobin, hematocrit, white blood cells, platelets, neutrophils, lymphocytes, eosinophils and 
monocytes) 
d. Chemistry Labs should include albumin, amylase, lipase, BUN, creatinine, ALT, AST, LDH, serum alkaline phosphatase, direct and total bilirubin, glucose, 
total protein, sodium, potassium, chloride, HCO3, calcium, uric acid and CPK. Labs should also include TSH and free T4 if TSH is elevated.  Sites should 
follow their institutional guidelines for hematology and Chemistry labs.  
e. Updated history and limited physical examination are recommended to be performed every 3 weeks for the first two cycles, then on the first day of subsequent 
cycles.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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54 
 
 
Appendix G 
Specimen Submission Guidelines 
Baseline tumor: tumor block or 26 unstained FFPE slides from primary or met; preferred excisional or punch (if not possible 14-16 gauge core needle) 
- Each pt: at least 1 single tumor block or 16 unstained slides at 10 micron thickness plus 10 unstained slides at 5 micron thickness for 4 different baseline correlative studies 
- Fresh tumor will be split for processing as frozen and FFPE 
o 
For excisional & punch bx, approx 2/3 embedded in OCT, placed on dry ice and stored at -80c.  Remaining 1/3 processed for FFPE 
o 
For core needle bx, 3-4 14-16 gauge cores embedded in OCT and at least 2 14-16 gauge cores processed for FFPE 
Correlative Study 
Sample required at 
each time point 
Baseline 
6-8 wks 1 
after tx 
Every 6 wks 
during tx2 
Disease 
Progression 
Shipping/ Storage for Future Use3 
Baseline correlative Studies 
on Tissues 
Tumor block or 
16 unstained FFPE 
tumor tissue slides  at 
10-micron 
 
XX 
 
 
 
Ship to GU-LCCC-HTSR (Deborah Berry) at the 
end of the study. Leftover specimens (if any) 
stored at GU-LCCC-HTSR &  placed in 
MRFBC VR for future use 
Tumor Block or 10 
unstained FFPE tumor 
tissue slides at 5-
micron 
XX 
 
 
 
(Optional) On-treatment 
correlative Studies on 
Tissues 
1.Frozen tumor 
embedded in OCT at-
80c 
2. FFPE tumor block 
 
 
XX 
 
 
Ship to GU-LCCC-HTSR (Deborah Berry) at the 
end of the study. Leftover specimens (if any) 
stored at GU-LCCC-HTSR &  placed in 
MRFBC VR for future use 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                          
1  
Expect 25% of patients (N=15; 10 MCM and 5 ALM patients) to consent to on-treatment biopsies 
2  
Blood samples will be taken ≤ 7 days prior to treatment 
3  
Specimens should be batched shipped to GU-LCCC-GESR at the conclusion of therapy for each patient. (at the end of the trial)  


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55 
 
 
 
 
 
 
 
 
 
 
 
 
 
Correlative 
Study 
Sample required at 
each time point 
Baseline 
3 wks 4 
after tx 
Every 6 wks 
during tx5 
Disease 
Progression or 
at the end of 
study 
Shipping/ Storage for Future Use6 
High-resolution 
HLA typing 
CPT™ Mononuclear cell 
preparation tube blood 
XX; 10ml 
 
 
 
Ship to GU-LCCC-HTSR (Deborah Berry) at 
the end of the study. 
Blood Correlates 
 
Blood in Cell-Free 
DNA BCT® tubes 
(Streck™) 
-Red-top blood 
-CPT™ Mononuclear 
cell preparation tube 
blood 
XX7 
20ml blood in Cell-
Free DNA BCT® 
tubes; 10ml red-top 
blood, 10ml CPT™ 
tube blood 
(collect within 7 
days prior to 
treatment) 
 
XX8 
20ml blood in Cell-
Free DNA BCT® 
tube; 10ml red-top 
blood; 10ml CPT™ 
tube blood 
 
XX9 
20ml blood in 
Cell-Free DNA 
BCT® tube; 10ml 
red-top blood; 
10ml CPT™ tube 
blood 
Ship to GU-LCCC-HTSR (Deborah Berry) at 
the end of the study.; Leftover specimens (if 
any) stored at GU-LCCC-GESR &  placed in 
MRFBC VR for future use 
Microbiota 
Correlates 
Stool in Omnigene.Gut 
collection tube 
XX 
(collect  7 days 
prior to Rx up to 
within 24 hours of 
receiving first dose) 
XX 
(collect  
5 days 
prior to 
Rx) 
 
XX (+/- 7 days) 
Ship to GU-LCCC-HTSR (Deborah Berry) at 
the end of the study. 
 
All samples should be shipped overnight to: 
GU-LCCC Histopathology Shared Resource (HTSR)-MRFBC Mucosal & Acral Melanoma Tissue Bank 
c/o  Deborah Berry, Ph.D 
Georgetown-Lombardi Comprehensive Cancer Center 
3900 Reservoir Road N.W. 
                                                          
4  
Expect 25% of patients (N=15; 10 MCM and 5 ALM patients) to consent to on-treatment biopsies 
5  
Blood samples will be taken ≤ 7 days prior to treatment 
6  
Specimens should be batched shipped to GU-LCCC-GESR at the conclusion of therapy for each patient. (at the end of the trial) 
7 -9 
Red-top tubes will be subsequently processed into serum, which will be aliquotted into 1ml aliquots & stored at -80C.   
Cell-free DNA BCT® tube (Streck™) will be subsequently processed into plasma (which will be aliquotted into 1ml aliquots & stored at -80C) and 
PBMC.  Please refer to the protocol guideline in blood component preparation 
 
 


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56 
Pre-Clinical Science Bldg., LR-10 
Washington, DC 20007 
Phone: (202) 687-1924 
Fax: (202) 687-0485 
Materials are to be shipped SUNDAY THROUGH THURSDAY only.  Do not ship samples the day before a Holiday