Phase Ib/II Study of Autologous Dendritic Cell Therapy Delivered Intratumorally After Cryoablation in Combination With Pembrolizumab for Patients With Metastatic or Unresectable Melanoma
Phase: PHASE1
Conditions: Stage III Cutaneous Melanoma AJCC v7, Stage IIIA Cutaneous Melanoma AJCC v7, Stage IIIB Cutaneous Melanoma AJCC v7, Stage IIIC Cutaneous Melanoma AJCC v7, Stage IV Cutaneous Melanoma AJCC v6 and v7
Sponsor: Mayo Clinic

 
 
 
 
 
MC1771 / 17-001666 
 
 
 
Phase Ib/II Study of Autologous Dendritic Cell Therapy Delivered 
Intratumorally After Cryoablation in Combination With 
Pembrolizumab for Patients With Metastatic or Unresectable 
Melanoma 
 
 
NCT03325101 
 
 
Document Date: 07/03/2019 

MC1771 
Protocol version: 03Jul2019 
Mayo Clinic Cancer Center 
MC1771: Phase Ib/II Study of Autologous Dendritic Cell Therapy Delivered Intratumorally after 
Cryoablation in Combination with Pembrolizumab for Patients with Metastatic or Unresectable 
Melanoma 
Study Chairs: 
Matthew S Block MD PhD 
Mayo Clinic 
200 First Street SW 
Rochester, MN 55905 
507/284-2511 
 
Study Cochairs: 
Yi Lin MD PhD√ 
Svetomir N Markovic MD PhD 
Jesus Vera Aguilera MD√ 
Thomas D Atwell MD 
Grant D Schmit MD 
Haidong Dong MD PhD√ 
Allan B Dietz PhD√ 
  
Statistician: 
Vera J Suman PhD√ 
507/284-8803 
 
√Study contributor(s) not responsible for patient care 
 
Drug Availability 
Supplied Investigational Agents: 
Mayo Clinic: Autologous dendritic cells (IND#17522 ) 
Commercial Agent: Pembrolizumab (Anti-PD-1 antibody, MK-3475) 
Supplied Commercial Agent: Prevnar13® 
 
Document History 
(Effective Date) 
Activation 
15Nov2017 
MCCC Amendment 1 
29May2018 
MCCC Amendment 2 
05Feb2019 
MCCC Amendment 3 
pending 
 
 
 
 

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Protocol Resources 
Questions: 
Contact Name: 
Patient eligibility, test schedule, treatment 
delays/interruptions/adjustments, dose 
modifications, adverse events, 
forms completion and submission 
Rebecca (Becca) L Palmer  
Quality Assurance Specialist/Data Manager 
Phone: 507/538-4370 
Email: palmer.rebecca@mayo.edu  
Clinical Research Coordinator 
Renee K Bradshaw 
Clinical Research Coordinator 
Phone: 507/284-2041 
Email: bradshaw.renee@mayo.edu 
Clinical Data Coordinator 
Robbin A Peterson 
Clinical Data Coordinator 
Phone: 507/266-3234 
Email: peterson.robbin@mayo.edu 
Melanoma Program Coordinator 
Jill M Schimke MS  
Melanoma Program Coordinator 
Phone: 507/266-0743 
Email: schimke.jill@mayo.edu 
Protocol document, consent form, 
regulatory issues 
Lynn M Flickinger MA 
Senior Research Protocol Specialist 
Phone: 507/538-7034 
Email: flickinger.lynn@mayo.edu 
Mitchell (Mitch) G Perrizo 
Research Protocol Specialist 
Phone: 507/284-9104 
Email: perrizo.mitchell@mayo.edu  
Immune, Progenitor, and Cell Therapeutics 
(IMPACT) Lab 
Phone: 507/284-4010  
Pager: 127-00384 
Hilton 2-68 
Email: RSTIMPACTMFG@mayo.edu 
Cryoablation 
Thomas D Atwell MD 
Phone: 507/284-7482 
Email: atwell.thomas@mayo.edu 
Serious Adverse Event Reporting 
Patricia G McNamara 
SAE Coordinator 
Phone: 507/266-3028 
Email: mcnamara.patricia@mayo.edu 
*No waivers of eligibility 
 

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Table of Contents 
MC1771: Phase Ib/II Study of Autologous Dendritic Cell Therapy Delivered Intratumorally after 
Cryoablation in Combination with Pembrolizumab for Patients with Metastatic or Unresectable 
Melanoma ................................................................................................................................................... 1 
Protocol Resources ..................................................................................................................................... 2 
Table of Contents ........................................................................................................................................ 3 
Schema ........................................................................................................................................................ 4 
1.0 
Background ................................................................................................................................... 5 
2.0 
Goals ........................................................................................................................................... 12 
3.0 
Patient Eligibility ........................................................................................................................ 13 
4.0 
Study Schedules .......................................................................................................................... 16 
5.0 
Grouping Factor .......................................................................................................................... 17 
6.0 
Registration Procedures. ............................................................................................................. 18 
7.0 
Protocol Treatment ...................................................................................................................... 20 
8.0 
Dosage Modification Based on Adverse Events ......................................................................... 22 
9.0 
Ancillary Treatment/Supportive Care ......................................................................................... 23 
10.0 
Adverse Event (AE) Reporting and Monitoring ..................................................................... 28 
11.0 
Treatment Evaluation .............................................................................................................. 37 
12.0 
Descriptive Factors: None ....................................................................................................... 42 
13.0 
Treatment/Follow–up Decision at Evaluation of Patient ........................................................ 42 
14.0 
Body Fluid Biospecimens ....................................................................................................... 44 
15.0 
Drug Information .................................................................................................................... 46 
16.0 
Statistical Considerations and Methodology ........................................................................... 51 
17.0 
Pathology Considerations/Tissue Biospecimens: ................................................................... 56 
18.0 
Records and Data Collection Procedures ................................................................................ 56 
19.0  
Budget ..................................................................................................................................... 57 
20.0 
References ............................................................................................................................... 58 
Appendix I 
ECOG Performance Status .............................................................................................. 64 
Appendix II 
New York Heart Association Classification of Congestive Heart Failure ...................... 65 
 
 

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Schema  
Prior to discussing protocol entry with the patient, call the Mayo Clinic Cancer Center Registration 
Office (507-284-2753) to confirm study status and insure that a place on the protocol is currently 
available to the patient 
 
Registration 
 
 
↓ 
 
 
Cycle 1: Apheresis, pembrolizumab 
 
 
↓ 
 
 
Cycles 2-3: Pembrolizumab, biopsy, ablation, 
dendritic cells, and Prevnar13® 
 
 
↓ 
 
 
Cycle 4+: Pembrolizumab 
 
 
↓ 
 
 
At any time: 
Disease Progression 
Intolerable adverse effects 
Patient refusal 
 
 
↓ 
 
 
Event Monitoring 
 
 
Cycle length = 21 days 
Generic name: Dendritic cells 
Brand name: NA 
Mayo abbreviation: mDC  
Availability: Provided through 
Mayo Clinic Immune, 
Progenitor, and Cell 
Therapeutics (IMPACT) Lab 
Generic name: Pneumococcal 
13-valent Conjugate Vaccine 
(Diphtheria CRM197 Protein) 
Brand name: Prevnar13® 
Mayo abbreviation: 
PREVNAR13 
Availability: Provided through 
Mayo Clinic Pharmacy 
Generic name: Pembrolizumab, 
MK-3475 (anti-PD-1 antibody) 
Brand name: Keytruda® 
Mayo abbreviation: MK-3475 
Availability: Commercial supply 

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1.0 
Background 
1.1 
Immune checkpoint blockade responses in advanced melanoma 
Melanoma is the most malignant form of skin cancer, and the fifth most common cancer in men 
and sixth in women in the United States with its highest incidence in the Caucasian population.1-3 
New cases of melanoma in the USA would reach 73,870 in 2015 and that year, 9,940 patients 
were expected to die from the disease.4 Over the last decade, the understanding of the immune 
checkpoint pathway has paved the way for immunotherapy in metastatic melanoma.  
Immune checkpoints are crucial for maintenance of self-tolerance under normal physiological 
conditions. 5-8 However, such maintenance can be dysregulated in tumors as an important 
mechanism of tumor resistance to immune control.9,10 Accumulating evidence shows that 
antitumor immune responses can be unleashed by immune checkpoint blockade, and blockade of 
cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death (PD-1) receptor have shown 
durable anti-melanoma effects.11,12 
The PD-1 receptor-ligand interaction is a major pathway used by tumors to suppress immune 
control. PD-1 receptor (encoded by the gene Pdcd1) is an Ig superfamily member related to CD28 
and CTLA-4. It is expressed on the cell surface of activated T-cells under normal conditions.6 By 
binding to its ligand (PD-L1 and PD-L2), PD-1 down-regulates T-cell activation and therefore 
dampens unwarranted and excessive immune responses, including autoimmunity. The interaction 
between PD-L1 expressed on tumor and stromal cells and PD-1 on T cells can trigger inhibitory 
signaling pathways that reduce effector cell functions and T-cell-killing capacity. Blocking the 
PD-1/PD-L1 interaction has been shown to potentiate tumor-specific CD8+ T-cell infiltration and 
effector T-cell activation that promote tumor rejection.13,14 
Anti-PD1/PD-L1 therapy is, to date, one of the most effective single-agent therapies used in the 
treatment of melanoma, however, it has been shown that as many as 60 % of patients who receive 
it display primary resistance, and, approximately 25 % of melanoma patients who had objective 
response to anti-PD1 therapy developed acquired resistance, as characterized by disease 
progression at a median follow-up of 21 months.15-17 
Several studies of advanced melanoma patients suggest that combining therapies that target tumor 
mechanisms of immune evasion (e.g., removing suppressive cell types via PD1/PD-L1 
neutralization) with activation of normal immune cell functionality (i.e. T cell activation) may 
provide optimal benefits for patients. 18,19 
 
1.2 
Pembrolizumab for advanced melanoma 
Pembrolizumab is a human IgG4 PD-1 blocking antibody that is recently approved by the FDA 
for the treatment of patients with unresectable or metastatic melanoma and disease, including 
progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor. 
Pembrolizumab showed an unprecedented rate of durable clinical responses, with an overall 
response rate of 26% and median progression-free survival of 22 weeks (95% CI: 12-36 weeks).12 
Treatment has generally been well-tolerated, and the most common drug-related adverse events 
(AEs) of any grade were fatigue (33%), pruritus (26%), and rash (18%). Patients responding to 
treatment showed proliferation of CD8 T cells in their tumors.14 
Despite the promising data associated with pembrolizumab treatment, the majority of melanoma 
patients do not respond to pembrolizumab. Several possible mechanisms for pembrolizumab (and 
other PD-1 blockade) resistance exist, including additional mechanisms of immunosuppression in 
the tumor microenvironment and derangements in systemic immune competence.20,21 The 
evaluation of melanoma gene expression revealed that tumors infiltrated with CD8+ T cells also 

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showed expression of a type I interferon transcriptional signature. 22,23 Interferon gamma (IFNγ) 
has the capacity to increase MHC-dependent antigen presentation in a variety of cell populations, 
including transformed melanocytes both in vitro and in vivo24, and is produced in response to T 
cell recognition of tumor-associated antigens25. IFNγ also causes many tumor cells to upregulate 
PD-L1, suggesting that an ongoing T cell response in which IFNγ is produced may prime tumors 
to benefit from anti-PD-1 therapy. Conversely, tumors with few tumor-related T cells have 
minimal IFNγ and are unlikely to benefit from PD-1-based therapies. 
The failure of the immune system to recognize and eradicate cancer cells may partly be a result of 
insufficient immunological activation. One potential hurdle to benefit from anti-PD-1 therapy is a 
lack expanded tumor-related T cells. As such, there is strong rationale for combining a strategy to 
expand melanoma-specific tumor-infiltrating lymphocytes (TILs) with pembrolizumab. 
 
1.3 
Evidence for dendritic cell vaccine immunotherapy in cancer.  
Dendritic cells (DCs) are the professional antigen-presenting cells that induce and regulate both 
adaptive and innate immune responses.26,27 DCs have been used in the past by exposing these 
cells to some form of tumor antigen in vitro, and then returning antigen-loaded DCs to the patient 
to stimulate anti-tumor immunity.28 Clinical trials of DC immunotherapy have suggested that this 
approach can result in significant stimulation of the immune response against many different 
forms of cancer.29  
The use of DCs as vaccines showed beneficial effects in an autologous setting and led to the first 
FDA approved immunotherapy.30,31 DC immunotherapy consists of exposing dendritic cells to 
tumor antigen and using the antigen-loaded DCs as a vaccine to stimulate anti-tumor 
response.32,33 
Since 1996, there have been several clinical studies investigating tumor antigen-loaded DC-based 
vaccines, mainly in metastatic melanoma patients. 34,35 Over the years, many parameters in DC 
vaccination have been optimized in clinical studies; DC vaccination has only minimal side effects 
and thus provides a well-tolerable treatment. 36  
 
1.31 
Dendritic Cell generation for vaccine therapy 
Results of this immunotherapy method against many different forms of cancer have been 
encouraging in animal models but have had mixed results in clinical trials 37 Available evidence 
suggests that the quality of the DC preparation used in a vaccination protocol has a substantial 
impact on the immune response elicited. 38 35 39 Specifically, the functional status of DCs depends 
on their state of activation at the time they are delivered, completely activated DC (mature DCs or 
mDCs) are able to migrate to lymph nodes and initiate stimulation of the immune system in vivo, 
while immature DCs can inhibit effector T cell function and induce tolerance.40 Unfortunately, 
many clinical trials were initiated before this distinction came to light and patients received 
immature DCs; inhibiting the immune response instead of stimulating it. 37 Thus, the lack of 
potent immune stimulation and unsatisfactory clinical response demonstrated in earlier trials of 
DC immunotherapy may at least in part be attributable to the functional state of the DCs 
employed. 40 
 
1.32 
 Ex vivo-generated DC vaccines.  
DCs that are generated ex vivo by culturing hematopoietic progenitor cells or monocytes with 
cytokine combinations have been tested as therapeutic vaccines in cancer patients for more than a 

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decade. 29 41 Treatment of metastatic prostate cancer with sipuleucel-T (also known as APC 
8015), approved by the US Food and Drug Administration (FDA) for the treatment of metastatic 
prostate cancer, consists of a cellular product based on enriched blood antigen-presenting cells 
(APCs) that are briefly cultured with a fusion protein of prostatic acid phosphatase (PAP) and 
GM-CSF; this resulted in an approximately 4-month-prolonged median survival in Phase III 
trials. 42 29 
A Phase III trial in metastatic melanoma that tested peptide vaccine in combination with high-
dose IL-2 versus IL-2 alone showed significant improvement in overall response rate and 
progression-free survival in patients who received the vaccine.43 Also, a Phase III trial in patients 
with follicular lymphoma (FL) showed that an idiotype vaccine therapy significantly prolongs the 
duration of chemotherapy-induced remission 44 (Table 1). 
Table 1 Examples of clinical trials testing vaccination with ex vivo DCs 65 
Vaccine and antigen 
Indication 
Key observations 
Refs 
GM-CSF–IL-4 DCs with or without 
HLA-A*0201-restricted peptides or 
peptides alone 
Metastatic prostate 
cancer 
One of the first studies that tested the 
immunogenicity of DCs 
45
 
GM-CSF–IL-4 DCs with peptides, 
tumour lysates or autologous tumour-
eluted peptides 
Stage IV melanoma, 
renal cell carcinoma 
and malignant glioma 
• Loading DCs with complex antigen 
preparations 
• Objective clinical responses 
46-48
 
Blood DCs and idiotype antigens 
Multiple myeloma 
• Immunogenicity of DCs 
• Tumour regression 
49,50 
Mature GM-CSF–IL-4 DCs and 
peptides 
Stage IV melanoma 
• Well-controlled and validated vaccine 
manufacture process 
• Testing mature DCs 
• Immunogenicity 
• Objective clinical responses 
51
 
CD34+ HPC-derived DCs and peptides 
Stage IV melanoma 
• One of the first studies to test CD34+ 
HPC-derived DCs 
• Loading vaccines with a mixture of well-
defined peptides 
• Durable immune responses in long-term 
survivors 
• Objective clinical responses 
52,53 
FLT3 ligand-expanded blood DCs and 
altered peptides 
Advanced CEA+ 
cancer 
• Immunogenicity 
• Objective clinical responses 
54
 
Immature GM-CSF–IL-4 DCs 
Healthy volunteers 
Antigen-specific inhibition of effector T 
cell function after injection of immature 
DCs 
40
 
GM-CSF–IL-4 DCs and tumour lysates 
Refractory pediatric 
solid tumors 
• Immunogenicity 
• Objective clinical responses 
55
 
Mature cryopreserved GM-CSF–IL-4 
DCs 
Stage IV melanoma 
Immunogenicity 
56
 
DCs loaded with autologous tumour 
RNA 
Colon cancer 
• Feasibility 
• Immunogenicity 
57
 
DCs loaded with killed allogeneic 
tumour cells 
Stage IV melanoma 
• Immunogenicity 
• Durable objective clinical responses 
• Long-term survival 
58,59 
Monocyte-derived DCs loaded with the 
NK T cell ligand α-galactosylceramide 
Advanced cancer 
Adjuvant effect of NK cell activation on 
CD8+ T cell-mediated immune response 
60
 
Monocyte-derived DCs 
Melanoma 
In vivo identification of antigen-specific 
immune response by PET imaging in 
patients 
61
 
  
  
Route of DC administration affects T cell 
activation, with intra-dermal 
administration showing better responses 
than intra-nodal administration 
62
 

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Vaccine and antigen 
Indication 
Key observations 
Refs 
Comparative study of CD34+ HPC-
derived Langerhans cells versus 
monocyte-derived DCs 
Melanoma 
Langerhans cell-based vaccines stimulated 
significantly greater tyrosinase-HLA-
A*0201 tetramer reactivity than the 
monocyte-derived DC vaccines 
63
 
Type 1-polarized monocyte-derived 
DCs 
Glioma 
Combination of DC vaccination with 
polyICLC to trigger systemic 
inflammation driven by type I interferon 
family members 
 64 
CEA, carcinoembryonic antigen; DC, dendritic cell; IL-4, interleukin-4; GM-CSF, granulocyte–macrophage colony-
stimulating factor; HLA, human leukocyte antigen; HPC, haematopoietic progenitor cell; NK cell, natural killer cell; 
PET, positron emission tomography; polyICLC, polyinosinic–polycytidylic acid stabilized with poly-L-lysine and 
carboxymethylcellulose.  
 
 
1.33 
In situ DC tumor antigen loading for vaccine therapy 
Differences in loading of DC with antigen in vitro may induce immune tolerance rather than 
stimulation.27,39 The optimal method for preparation of antigen and antigen loading to DC 
remains to be identified. 35 Optimal tumor antigen delivery is one of the most important factors 
for DC-based immunotherapy success.66 For that reason, autologous tumor cell lysate, whole 
tumor cells, and mRNA have been tested as antigen providers for DCs. 67-69 Furthermore, 
allogeneic melanoma cell lysates also constitute a valuable alternative (Table 1).58,66 
Route of DC delivery also critically impacts efficacy of DC to stimulate an anti- tumor 
response. Inconsistent generation of systemic anti-tumor immune responses with intravenous 
and subcutaneous administration has prompted evaluation of novel delivery approach. 70 
Intratumor DC delivery was shown to generate anti-tumor immunity and tumor regression in 
animal models.71 Also, clinical trials to date have shown this approach to be safe with promising 
response72  
Antigen can be generated in vivo using conventional therapies including chemical, radiation or 
even cryoablation.73-75 Recently Kolstad et al reported promising results in FL patients treated 
with radiation, and intra- tumor injection of rituximab and DC.75 Direct injection in vivo offers 
two advantages: delivery of mature DCs into a milieu of dying tumor cells, resulting in 
maximum exposure of tumor cell antigens; and avoidance of the suppressive effects of blood 
monocytes.76 This combined approach was found to be feasible and safe in prior studies 
performed by our group in Lymphoma (Lin, et. al., unpublished observations). We will evaluate 
this method in addition to pembrolizumab treatment to enhance clinical efficacy against 
melanoma.  
 
1.34 
 Safety considerations from DC therapy 
DC vaccine therapy clinical trials in melanoma and other types of have thus far demonstrated no 
significant adverse events or autoimmunity; DC immunotherapy has been used for many 
different tumors types where adverse events (such as induced autoimmunity) were monitored. 
Importantly, very few significant adverse effects of DC vaccination have been reported and DC 
immunotherapy clinical trials performed to date have shown no evidence of resultant generation 
of auto-immunity. (Table 1). To date, 15 patients with lymphoma, 19 patients with ovarian 
cancer, and 30 patients with glioblastoma have been treated with DC therapy at Mayo Clinic 
without any serious drug related toxicity reported. The most common adverse event reported 
was injection site pain, more often at the time of cryoablation, that usually resolved within 2 
days of treatments. 

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1.4 
Rationale for pneumococcal conjugate vaccine (PCV) therapy 
 
PCV (Prevnar®) is approved by the US Food and Drug Administration for vaccination against 
pneumococcal infections in children and works by activating helper T cells to interact with B 
cells for antibody production. 77 Pneumococcal vaccination has been shown to be safe and 
recommended for cancer patients that will or have undergone chemotherapy. Thus, the use of 
pneumococcal vaccines presents little if any additional risk to the patient. 78 The use of these 
vaccines does allow determination of the state of the immune system and its response to the DC 
based vaccines. Prevnar has been used in two clinical trials for multiple myeloma patients in this 
manner. These trials included one with adoptive T cell transfer after stem cell transplant79 and 
another with lenalidomide. 80 Both trials reported minimal toxicity. In addition there may be a 
bystander effect on immunity with enhanced lymphocyte trafficking and the response to one 
antigen leading to enhanced response to anti-tumor antigens. 81 
 
Preliminary studies performed by Yin and colleagues showed that Prevnar13 enhanced mature 
DC stimulated CD4 T cell proliferation by approximately 1.75 fold compared to mature DC 
stimulation alone (Figure 1). Importantly, a review of the results of DC therapy in melanoma 
suggests that addition of adjuvant capable of T cell help significantly improved overall clinical 
response. 82 81 This vaccine will allow us to monitor the immune response prior to and after our 
intervention. The vaccine will also allow us to quantify any potential in vivo bystander effect 
due to the combination of DC and pneumococcal vaccine. In the LS1081 study (Vaccine 
Therapy With or Without Cryosurgery in Treating Patients With Residual, Relapsed, or 
Refractory B-Cell Non-Hodgkin Lymphoma) no adverse events were seen with Prevnar 
administrations. 
 
Figure 1. CD4+ T cell proliferation is stimulated with mature dendritic cells 
pulsed with Prevnar. Monocyte-depleted PBMNC from healthy donors were labeled 
with CFSE and cultured for 7 days without stimulation or with Prevnar, autologous 
mature dendritic cells (mDC), or autologous mDC pulsed with Prevnar. At the end of 
culture, cells were stained with 7-AAD, anti-CD3 and anti-CD4 and analyzed by flow 
cytometry. Percent of live, CD4 T cells that proliferated are shown here for 3 donors. 
CD4 T cell proliferation was the greatest in co-culture with Prevnar-pulsed mDC. 
 
1.41 
Safety considerations for the use of 13-valent pneumococcal conjugate vaccine 
(PCV) therapy 
PCV (Prevnar®) is approved by the US Food and Drug Administration for vaccination against 
pneumococcal infections in children and works by activating helper T cells to interact with B 
cells for antibody production.77 Pneumococcal vaccination is considered safe for cancer patients 
undergoing chemotherapy with mild side effects including soreness, low-grade fever and 
induration at the site of vaccination lasting 1-3 days.78 It has been used in two clinical trials for 
multiple myeloma patients, one with adoptive T cell transfer after stem cell transplant 79 and 
another with lenalidomide.80 Both trials reported minimal toxicity and some evidence of 

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improved immune response. As in Rapoport et al, we will use this pneumococcal vaccine to 
evaluate and monitor the developing immune response. The proposed Prevnar13 (Prevnar) 
dosing schedule in this study is similar to the Prevnar schedule in LS1081 study. No significant 
toxicities were observed in 15 patients who received Prevnar along with their DC vaccines. We 
will use the dose administration and timing as approved for use (Prevnar product insert: 
http://www.pfizerpro.com/content/showlabeling.asp?id=501). 
 
1.5  
Cryoablation therapy 
1.51  
Cryoablation background 
Percutaneous cryoablation systems are currently available from two manufacturers including 
HealthTronics, Incorporated (Austin, TX) and Galil Medical (St. Paul, MN); each are FDA 
510K cleared for treatment of soft-tissue tumors. Since 2003, investigators in this study have 
used cryoablation for the treatment of renal, lung, soft tissue, and bone tumors, including 
treatment of patients enrolled in the LS1081 study. Probes may be placed percutaneously using 
US or CT image guidance. 
The mechanism of cryoablation is based on delivery of argon gas through a segmentally 
insulated probe with expansion of the gas within the probe lumen, resulting in rapid cooling (via 
Joule Thompson effect). With expansion of the argon gas within the probe, rapid and extreme 
temperature drops are produced along the distal, uninsulated probe shaft reaching –100 ºC 
within a few seconds. Adjacent tissues are quickly frozen, resulting in the formation of an 
iceball. Thawing of the iceball is achieved with either active instillation of helium gas into the 
cryoprobe instead of argon gas or activation of a small heating element in the distal probe shaft. 
Cell death is due to two primary causes. First, rapid freezing immediately adjacent to the probe 
results in intracellular ice formation, organelle disruption, and subsequent cell destruction. At a 
further distance from the probe, relative gradual cooling causes osmotic differences across the 
cell membrane with secondary cellular dehydration and death. Subsequently, further cell death 
occurs due to thrombosis of affected blood vessels and resultant ischemic necrosis. 
The size of the iceball varies depending on the length of uninsulated tip, with commonly used 
cryoprobes generating an iceball of about 3 cm in diameter and 5 cm in longitudinal length 
along the probe shaft. The growth and size of the iceball can be controlled by the delivery 
system through the relative amount of argon passed through the cryoprobe. Importantly, the 
margins of the iceball are visible with CT, MRI and ultrasound, allowing monitoring of 
treatment. Given relative cellular tolerance to freezing temperatures, cell death occurs within 
about 3-5 mm internal to the iceball margin 83, 84. Following planned cryoprobe placement(s), a 
target lesion is typically treated with curative intent using two freezing cycles of approximately 
10 minutes each, separated by a 5-minute thaw period. 
 
1.52  
Safety considerations for cryoablation 
Outside of the liver, complications due to cryoablation are uncommon. A large experience 
exists regarding percutaneous cryoablation of renal tumors, showing a major complication rate 
of 5-8%. 85,86. Similar to radiofrequency ablation (RFA), the majority of complications are 
related to local tissue trauma, including hemorrhage in about 3% of patients. Allowing for a 
more limited published experience, complications following bone and soft tissue ablation are 
even more infrequent, ranging from 0- 2% 87 88 89. Unique to cryoablation, compared to RFA, 
is the ability to visualize the cytotoxic ice develop using CT imaging, allow the user to 
precisely control the volume of tissue treated and minimize adjacent tissue injury.  

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Of particular note, a systemic phenomenon called “cryoshock” has been observed primarily in 
the cryoablation of liver tumors. Cryoshock is clinically manifested by severe coagulopathy, 
disseminated intravascular coagulation, and multiorgan failure, possibly related to a systemic 
inflammatory response syndrome. Historically, this syndrome has been seen in about 1% of 
cases of hepatic cryotherapy and only 0.04% of prostate cryoablations.90 More recent 
experience describing percutaneous cryoablation of hepatic tumors in selected patients showed 
grade 3 or greater complications in 6% of patients, including cryoshock in 3 of 342 procedures 
(0.9%) (Littrup et al. Abd Rad 2016; 41:767-780). In these 3 patients, tumors measuring larger 
than 3cm in size were ablated; cryoshock did not occur in the treatment of smaller hepatic 
lesions. 
 
1.6  
Rationale for the proposed study and research hypothesis 
Among patients with advanced melanoma, pembrolizumab administration is associated with an 
overall objective response rate of 33%, 12-month progression-free survival rate of 35%, and 
median overall survival of 23 months; 91 approximately 25 % of melanoma patients who 
demonstrated an objective response to pembrolizumab therapy developed disease progression; 
results are similar to those reported for nivolumab. Moreover, many patients’ tumors primarily 
fail to respond to pembrolizumab. 
Agents capable of stimulating infiltration and functional activation of both T cells and APCs in 
such tumors could synergize with PD-1 blockade to increase the frequency of responding patients 
and expand the range of tumors treatable with PD-1 inhibitors.92 Such stimulatory agents have the 
potential to enhance the extent or durability of responses even in patients who have failed to 
respond to pembrolizumab monotherapy. 
Pembrolizumab (MK-3475) is a highly selective, humanized monoclonal IgG4-κ isotype antibody 
against PD-1 that is approved around the world for the treatment of patients with unresectable or 
metastatic melanoma with disease progression following ipilimumab and, if BRAFV600 (NCBI 
accession number NM_004324.2) mutated, a BRAF inhibitor. Pembrolizumab is well-tolerated at 
200mg/3 weeks with an acceptable adverse effects profile. This therapy in combination with an 
anti-melanoma vaccine capable of expanding melanoma-specific T cells might dramatically 
improve response rated to Anti-PD-1 therapy.  
We will conduct a Phase II clinical trial with a safety run-in phase for patients with unresectable 
or metastatic melanoma to test the hypothesis that PD-1 blockade combined with cryotherapy and 
a DC vaccine will have an acceptable adverse event profile and will enhance mDC priming of T 
cells with anti-tumor antigen and activate T cell functions. Combined immunotherapy with 
pembrolizumab and cryoablation followed by intra-tumoral injection of mDCs will generate anti-
tumor immunity and clinical regression of melanoma in patients who have not responded 
optimally to PD-1 axis-targeting therapy. 
 
 

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2.0 
Goals 
2.1 
Primary 
To determine the objective response rate (ORR) of pembrolizumab combined with 
cryoablation and intratumoral mDCs in patients with metastatic melanoma that has 
failed to respond or has stopped responding to initial therapy with a PD-1 axis-
blocking monoclonal antibody. 
2.2 
Secondary 
2.21 
To assess the safety profile of pembrolizumab combined with cryoablation 
and intratumoral mDCs in patients with metastatic melanoma that have failed 
to respond or have stopped responding to initial therapy with a PD-1 axis-
blocking monoclonal antibody. 
2.22 
To determine median progression-free survival (PFS) obtained with this 
approach in this patient population. 
2.23 
To determine median overall survival (OS) obtained with this approach in 
this patient population. 
 
2.3 
Translational  
2.31 
To quantitate tumor infiltrating lymphocytes (TILs) in tumor biopsies prior 
to and following cryoablation and intratumoral mDCs. 
 
2.32 
To measure PD-L1 levels in tumor biopsies and blood biopsies prior to and 
following cryoablation and to assess whether a change in PD-L1 levels 
differ among those patients who met the criteria for clinical benefit 
(progression-free and on study for at least 6 months) and those who do not. 
 
2.33 
To measure peripheral blood mononuclear cells (PBMC) proliferation and 
function after coculture with frozen tumor before and after intratumoral 
mDC injection.  
 
 

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3.0 
Patient Eligibility 
3.1 
Inclusion Criteria 
3.11  
Age ≥18 years of age on the day of registration. 
3.12 
Histological or cytologically confirmed diagnosis of unresectable stage III or 
metastatic melanoma (stage IV) not amenable to curative local therapy. 
3.13 
Lack of response to therapy with a PD-1- or PD-L1-targeting monoclonal 
antibody (pembrolizumab, nivolumab, etc.) after at least 18 weeks of therapy 
OR documented progression of disease at any time after initiation of therapy 
with a PD-1- or PD-L1-targeting monoclonal antibody.  
NOTE: This treatment could have been at any time prior to registration. If 
given in the adjuvant setting, progression must have been ≤26 weeks after the 
last dose of therapy. 
3.14 
ECOG Performance Status (PS) 0 or 1 (Appendix I) 
3.15 
Minimum of 3 radiographically apparent lesions such that there is:  
(1) Minimum of one lesion in areas that have not been previously irradiated that 
is considered measurable by RECIST 1.1 criteria  
AND 
(2) Minimum of two lesions in areas that have not been previously irradiated 
that are determined by Interventional Radiology to be of a size and in a 
location that a single probe could ablate at least 75% of the lesion.  
Note: Hepatic lesions measuring ≤3cm may be treated, as determined by 
Interventional Radiology. 
Note: Brain metastases are not acceptable as lesions defining measurable disease, 
nor are they candidate lesions for cryoablation. 
3.16 
Adequate venous access for apheresis as assessed by apheresis team. 
NOTE: If a central venous catheter is required for apheresis, the patient is not 
eligible. 
3.17 
The following laboratory values obtained ≤14 days prior to registration. 
• 
Absolute neutrophil count (ANC) ≥1000/mm3 
• 
Absolute lymphocyte count ≥500/mm3 
• 
Platelet count ≥100,000/mm3 
• 
Hemoglobin ≥10 g/dL  
• 
Total bilirubin ≤1.5 x upper limit of normal (ULN), unless due to 
Gilbert’s disease 
• 
Aspartate transaminase (AST/SGOT) and alanine transaminase 
(ALT/SGPT) ≤2.5 x ULN 
• 
Creatinine ≤1.5 x ULN or calculated creatinine clearance ≥60 mL/min 
for subject with creatinine˃1.5 x institutional ULN 
3.18 
Negative serum pregnancy test for persons of childbearing potential ≤7 days 
prior to registration. 
3.19a 
Provide written informed consent. 
3.19b Willing to return to the enrolling institution for follow-up (during active 

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treatment and active monitoring phase of the study). 
3.19c 
Willing to provide tissue and blood samples for research purposes (see Sections 
6.0, 14.0 and 17.0). 
3.19d Willing to use adequate contraception while on the study and until 120 days after 
the last dose of study drug. 
3.2 
Exclusion Criteria 
3.21 
Choroidal melanoma. 
3.22 
Any of the following because this study involves an investigational agent whose 
genotoxic, mutagenic and teratogenic effects on the developing fetus and 
newborn are unknown: 
● 
Pregnant persons 
● 
Nursing persons 
3.23 
History of HIV, hepatitis B, or hepatitis C,  
3.24 
Active tuberculosis or active, non-infectious pneumonitis 
3.25 
Evidence of interstitial lung disease 
3.26 
Active infection requiring the use of systemic antibiotics 
3.27 
Symptomatic congestive heart failure (New York Heart Association 
Classification III or IV cardiovascular disease (Appendix II)),  
myocardial infarction ≤6 months prior to registration ,  
unstable angina pectoris or cardiac arrhythmia ≤3 months prior to registration, or 
cardiac arrhythmia. 
3.28 
Currently receiving or have received any other investigational agent considered 
as a treatment for the primary neoplasm ≤21 days prior to registration. 
3.29a 
History of other primary malignancy requiring systemic treatment ≤3 years prior 
to registration. Patients must not be receiving chemotherapy or immunotherapy 
for another cancer. Patients must not have another active malignancy requiring 
active treatment.  
EXCEPTIONS: Non-melanotic skin cancer or carcinoma-in-situ of the cervix. 
3.29b Failure to recover from prior side effects of immune checkpoint inhibitor therapy 
to ≤Grade 1. 
NOTE: Patients will not be excluded for adrenal insufficiency or hypothyroidism 
secondary to immunotherapy provided they are receiving hormonal replacement 
3.29c 
Major surgery ≤4 weeks prior to registration.  
3.29d Prior chemotherapy, targeted therapy, or radiation therapy ≤2 weeks prior to 
registration or who has not recovered (i.e. to ≤Grade 1 or baseline) from an 
adverse event due to the previously administered therapy. 
3.29e 
History of hypersensitivity and anaphylactoid reactions to pneumococcal vaccine 
or any component of the formulation, including diphtheria toxoid. 
 
 

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3.29f 
Active autoimmune disease such as Crohn’s disease, rheumatoid arthritis, 
Sjögren’s disease, systemic lupus erythematosus, or similar conditions requiring 
systemic treatment within the past 3 months or a documented history of clinically 
severe autoimmune disease/syndrome difficult to control in the past. 
EXCEPTIONS (the following are allowed): 
• 
Vitiligo or resolved childhood asthma/atopy 
• 
Intermittent use of bronchodilators or local steroid injections 
• 
Non-immunosuppressive maintenance treatments in the setting of clinically 
asymptomatic disease (e.g., sulfasalazine for ulcerative colitis) 
• 
Hypothyroidism stable on hormone replacement, 
• 
Diabetes stable with current management 
• 
History of positive Coombs test but no evidence of hemolysis 
• 
Psoriasis not requiring systemic treatment 
• 
Conditions not expected to recur in the absence of an external trigger 
• 
Secondary adrenal insufficiency from previous hypophysitis, currently on 
physiologic replacement steroid dosing only. 
3.29g Coagulopathy, including the use of therapeutic anticoagulants that cannot be 
discontinued for the cryoablation procedure.  
NOTE: Heparin for line patency without detectable lab abnormalities for 
coagulation will be allowed. 
3.29h Corticosteroid use ≤14 days prior to registration. 
NOTE: Patients must be off systemic corticosteroids for at least 2 weeks prior to 
registration. This includes oral or IV route of administration. Patients on chronic 
corticosteroids for adrenal insufficiency or other reasons may enroll if they 
receive less than 10 mg/day of prednisone (or equivalent). Patients receiving 
inhaled or intranasal or intra-articular steroids are not excluded. 
3.29i 
Active CNS metastasis. 
NOTE: Patients with prior brain metastases that are asymptomatic without 
corticosteroid use and stable or improved ≥90 days after treatment with surgery 
or radiation are not excluded. 
3.29j 
Receipt of a live vaccine ≤30 days prior to registration. 
 

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4.0 
Study Schedules 
4.1 
Test schedule for Melanoma 
Tests and Procedures1 
≤14 days prior to 
Registration 
After 
Reg 
Cycle 1 
Day 1 
Cycles  
2-3 
At completion of  
each cycle (Cycle 3+) 
End of 
Tx 
window 
 
 
±1 day 
±3 days 
±3 days2 
±7 days 
Physical exam, weight, ECOG performance status 
X 
 
X 
X 
X 
X 
Height 
X 
 
 
 
 
 
Serum pregnancy test 
X3 
 
 
 
 
 
Hematology (WBC with differential, Hgb, PLT) 
X 
 
X4 
 
X 
X 
Chemistry profile (creatinine, AST, ALT, alkaline phosphatase, T bili, 
direct bilirubin, sodium, potassium, random glucose) 
X 
 
 
 
X 
X 
Thyroid function cascade (TSH, reflex per Mayo standard if abnormal) 
X 
 
 
X 
X 
X 
LDH 
X 
 
 
 
X 
X 
Coagulation (INR) 
X 
 
 
X5 
X6 
 
Adverse event assessment 
X 
 
 
 
X 
X 
Radiologic evaluations (CT scan, PET/CT, or MRI) 
X7 
 
 
 
X8 
X 
Venous Access Assessment 
X9 
 
 
 
 
 
Apheresis 
 
 
X 
 
 
 
Research blood for immune monitoringR (See Section 14.0) 10 
 
X 
 
X11 
X 
X 
Research specimen submission: Tumor biopsiesR (See Section 17.0) 
 
 
 
X12 
X13 
 
 
1  Cycle = 21 days; All tests and procedures are as clinically indicated, unless noted with an R to indicate funding by research. Hematology, chemistry profile, thyroid function 
cascade, coagulation tests, and imaging may be performed more frequently at physician discretion. 
2  Window of 3 days applies to Cycles 2-5. After Cycle 5, window is ±7 days. 
3  For persons of childbearing potential only. Must be done ≤7 days prior to registration. 
4  Repeat hematology tests on Cycle 1, Day 1 only if most recent tests were performed >7 days prior to Cycle 1, Day 1. 
5  Coagulation tests should be repeated at the completion of Cycles 1, 2, and 4. If treatment is delayed, coagulation tests should be repeated ≤48 hours prior to tumor biopsy. 
6  Coagulation tests should be repeated at the completion of Cycles 1, 2, and 4. If treatment is delayed, coagulation tests should be repeated ≤48 hours prior to tumor biopsy. 
7  Baseline radiology ≤28 days prior to registration 
8  Imaging is performed at the end of Cycle 4 and then at the end of every fourth cycle of treatment (end of Cycle 8, 12, 16, etc.). Exception: if a patient has PD yet continues 
protocol treatment, the next imaging should take place two cycles after the imaging showing PD. If that patient continues on protocol treatment, imaging should then take place 
every 4 cycles. 
9  Assessment of venous access must be done ≥3 days prior to apheresis. 
10  Blood draw for immune monitoring will be collected prior to the start of Cycles 1-5 and prior to the start of each post-imaging cycle thereafter (prior to Cycle 9, 13, 17, etc.) 
11  In addition to the research blood collection on Day 1, research blood will be collected as per Section 14 on Cycle 2, Days 2-5. The collection on Cycle 2, Day 2 should take 
place 2-4 hours after completion of cryoablation and mDC therapy. 
12 Tumor biopsies will be performed immediately prior to cryoablation on Cycle 2 Day 1 or 2 and Cycle 3 Day 1 or 2 
13  Tumor biopsies will be performed at the end of Cycle 4 after radiologic evaluation 

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4.2 
Event Monitoring/Survival Follow-up 
 
Event Monitoring Phase1 
q. 3 months 
until PD 
At PD 
After PD  
q. 6 months 
Death 
New Primary 
Event Monitoring 
X 
X 
X 
X 
At each occurrence 
1. If a patient is still alive 5 years after registration, no further follow-up is required. 
 
5.0 
Grouping Factor 
Cohort: Run-in Phase Ib Dose level 1 vs. Run-in Phase Ib Dose level -1 vs. Continuation Phase (Phase II) 
 
 

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6.0 
Registration Procedures 
Prior to discussing protocol entry with the patient, call the Mayo Clinic Cancer Center 
Registration Office (507-284-2753) to confirm study status and insure that a place on the 
protocol is currently available to the patient 
6.1 
Registering a patient 
To register a patient, access the Mayo Clinic Cancer Center (MCCC) web page and enter 
the remote registration/randomization application. The registration/randomization 
application is available 24 hours a day, 7 days a week. Back up and/or system support 
contact information is available on the Web site. If unable to access the Web site, call the 
MCCC Registration Office at (507) 284-2753 between the hours of 8 a.m. and 4:30 p.m. 
Central Time (Monday through Friday). 
The instructions for the registration/randomization application are available on the 
MCCC web page (http://hsrwww.mayo.edu/ccs/training) and detail the process for 
completing and confirming patient registration. Prior to initiation of protocol treatment, 
this process must be completed in its entirety and a MCCC subject ID number must be 
available as noted in the instructions. It is the responsibility of the individual and 
institution registering the patient to confirm the process has been successfully completed 
prior to release of the study agent. Patient registration via the registration/randomization 
application can be confirmed in any of the following ways: 
• 
Contact the MCCC Registration Office (507) 284-2753. If the patient was fully 
registered, the MCCC Registration Office staff can access the information from the 
centralized database and confirm the registration. 
• 
Refer to “Instructions for Remote Registration” in section “Finding/Displaying 
Information about A Registered Subject.” 
6.2 
Verification 
Prior to accepting the registration, registration/randomization application will verify the 
following: 
• 
IRB approval at the registering institution 
• 
Patient eligibility 
• 
Existence of a signed consent form 
• 
Existence of a signed authorization for use and disclosure of protected health 
information  
6.3 
Documentation of IRB approval 
Documentation of IRB approval must be on file in the Registration Office before an 
investigator may register any patients.  
In addition to submitting initial IRB approval documents, ongoing IRB approval 
documentation must be on file (no less than annually) at the Registration Office (fax: 
507-284-0885). If the necessary documentation is not submitted in advance of attempting 
patient registration, the registration will not be accepted and the patient may not be 
enrolled in the protocol until the situation is resolved. 
When the study has been permanently closed to patient enrollment, submission of annual 
IRB approvals to the Registration Office is no longer necessary. 
6.4 
Correlative studies 
A mandatory translational research component is part of this study; the patient will be 
automatically registered onto this component (Sections 3.19b, 14.0, 17.0). 

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At the time of registration, the following will be recorded: 
• 
Patient has/has not given permission to store and use his/her sample(s) for 
future research of cancer at Mayo. 
• 
Patient has/has not given permission to store and use his/her sample(s) for 
future research to learn, prevent, or treat other health problems. 
• 
Patient has/has not given permission for MCCC to give his/her sample(s) to 
researchers at other institutions. 
6.5 
Treatment on protocol 
Treatment on this protocol must commence at Mayo Clinic in Rochester, Minnesota 
under the supervision of a medical oncologist. 
6.6 
Treatment start 
Treatment cannot begin prior to registration and must begin ≤7 days after registration. 
6.7 
Pretreatment 
Pretreatment tests/procedures (see Section 4.0) must be completed within the guidelines 
specified on the test schedule. 
6.8 
Baseline symptoms 
All required baseline symptoms (see Section 10.6) must be documented and graded. 
6.9a 
Study Conduct 
The clinical trial will be conducted in compliance with regulations (21 CFR 312, 50, and 
56), guidelines for Good Clinical Practice (ICH Guidance E6), and in accordance with 
general ethical principles outlined in the Declaration of Helsinki; informed consent will 
be obtained from all participating patients; the protocol and any amendments will be 
subject to approval by the designated IRB prior to implementation, in accordance with 21 
CFR 56.103(a); and subject records will be stored in a secure location and subject 
confidentiality will be maintained. The investigator will be thoroughly familiar with the 
appropriate use of the study drug as described in the protocol and Investigator’s 
Brochure. Essential clinical documents will be maintained to demonstrate the validity of 
the study and the integrity of the data collected. Master files should be established at the 
beginning of the study, maintained for the duration of the study and retained according to 
the appropriate regulations.  
 

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7.0 
Protocol Treatment 
Use actual weight or estimated dry weight if fluid retention. 
Cycle length is 21 days with the exception of Cycle 1 which may be extended an additional 
7 days to allow for dendritic cell production.  
Dose-limiting toxicity (DLT) is defined as one of the following events occurring during the first 
two cycles of protocol therapy and attributed as possibly, probably, or definitely related to 
protocol therapy. 
- 
Any Grade 3+ toxicity 
- 
Grade 2 infusion reaction, acute or chronic kidney disease, or pneumonitis that does not 
resolve to Grade 0-1 within 3 weeks. 
7.1 
Drugs used in this study 
Pembrolizumab at FDA approved dose for metastatic melanoma of 200mg IV every 21 
days 
Autologous mature dendritic cells (mDCs) injected into the tumor site 
Prevnar13® injected into the tumor site with the DCs 
7.2 
Run-in Phase Ib 
7.21 
Schedule 1 (6 patients): 
Cycle 1, Day 1: Apheresis (see Section 7.3) and pembrolizumab 200mg IV 
Cycles 2 and 3, Day 1: Pembrolizumab 200mg IV 
Cycles 2 and 3, either Day 1 or Day 2: cryoablation, injection of 30-60 x106 
mDCs, and injection of 0.5 ml Prevnar13, per Section 7.4 
Cycle 4 Day 1 and all subsequent cycles: pembrolizumab 200mg IV 
If at most one of the 6 patients enrolled onto Schedule 1 develops a DLT during 
the first 3 cycles of treatment then Schedule 1 will be carried forward to the 
continuation phase. 
7.22 
Schedule -1: 
If two or more patients enrolled onto Schedule 1 develop a DLT during the first 
3 cycles of treatment, then an additional 6 patients will be treated as follows: 
Cycle 1, Day 1: Apheresis and pembrolizumab 200mg IV 
Cycles 2 and 3, either Day 1 or Day 2: Cryoablation, injection of 30-60 x106 
mDCs, and injection of 0.5 ml Prevnar13, per Section 7.5. 
Note: No pembrolizumab will be given in Cycles 2 and 3 on this schedule. 
Cycle 4 Day 1 and all subsequent cycles: pembrolizumab 200mg IV 
- If at most one of the 6 patients enrolled onto Schedule -1 develops a DLT 
during the first 3 cycles of treatment then Schedule -1 will be carried 
forward to the continuation phase. 
- If two or more of the patients enrolled onto Schedule -1 develop a DLT 
during the first 3 cycles of treatment, enrollment will be discontinued for 
the study team to review the safety data and consider amending the 
protocol.  

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7.3 
Phase II 
Schedule of pembrolizumab, cryoablation, mDCs, and Prevnar13 will be based on the 
tolerability findings of the run-in phase 
7.4 
Apheresis 
Patients will undergo apheresis on Cycle 1, Day 1, from which monocytes will be 
collected by immunomagnetic isolation and cultured in vitro under GMP conditions to 
generate mature DCs (mDCs). The generated mDCs will be frozen and stored until 
ready for use. Autologous mature dendritic cells (mDCs) will be prepared for 
administration into the cryoablated tumor. At a minimum, mDCs necessary for 
2 injections (30-60 x106 per injection) will be manufactured. Additional mDCs and 
lysate will be stored for potential retreatment and immune monitoring assays. If 
insufficient mDCs are available for treatment, the total mDCs should be split into two 
doses, and the treating physician may determine whether to continue with protocol 
treatment or take the patient off study. 
NOTE: If first mDC production attempt does not yield any releasable mDCs, then one 
additional apheresis may be attempted. 
7.5 
Cryoablation, mDC administration, and Prevnar13 administration 
Each patient will undergo percutaneous cryoablation within 36 hours of pembrolizumab 
treatment on Cycles 2 and 3. (Generally, this will take place on Cycle 2, Day 2, and 
Cycle 3, Day 2. However, it is acceptable that cryoablation could be performed on Cycle 
2, Day 1, and Cycle 3, Day 1.) Two distinct metastatic lesions will be treated with 
cryoablation, one during Cycle 2, and the other during Cycle 3. Our general cryoablation 
technique has been previously described (McMenomy et al. JVIR 2013; 24:207-213). 
Due to the extended length of the procedure, requiring the patient to lie still (expected 
duration 45 minutes), sedation will be managed by the Department of Anesthesia. Using 
ultrasound and/or CT guidance, a cryoprobe will be placed in the index melanoma lesion. 
Research biopsies (6 18-gauge cores) will be obtained immediately prior to freezing the 
tumor. The lesion will be treated using a conventional freeze-thaw- freeze cycle with the 
endpoint being the generation of an iceball that measures at least 75% of the diameter of 
the index tumor but may encompass the entire tumor with a margin of normal tissue.  
Following the freezing procedure, the cryoprobe will be actively warmed and then 
withdrawn. After the lesion has thawed, a 22 G needle will be placed under ultrasound or 
CT-guidance into the ablated portion of the lesion. The mDC will be administered 
through this needle over different areas of the cryoablated region and then flushed with 
1ml of sterile saline. Prevnar13® will be injected into the ablated portion of the lesion 
after administration of mDCs. The patient will be transferred to the recovery area and 
monitored for 2 hours following cryoablation and DC injections for acute toxicity. Given 
the elevated risk of solid organ cryoablation, patients undergoing ablation of such tumors 
will be admitted to the hospital for overnight observation, including hematologic 
assessment prior to discharge. 
7.6 
Duration of therapy 
Patients may remain on pembrolizumab treatment for up to two years. After two 
years, patients go to event monitoring and may receive pembrolizumab off protocol 
at the discretion of the treating provider. 
 

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8.0 
Dosage Modification Based on Adverse Events 
8.1 
Treatment schedule modifications in patients based on adverse events 
Patients will be evaluated by the study team prior to each dose of pembrolizumab for an 
adverse event check. Determination will be made if the adverse event is treatment-related 
(possible, probable or definite), i.e. a toxicity (see Section 10; Adverse event monitoring 
and reporting). If any adverse event has occurred, then the treatment schedule will be 
modified as in Section 8.2. If an adverse event occurs during Cycle 2 that requires 
treatment to be held, then omit pembrolizumab for Cycle 3 and resume pembrolizumab 
with Cycle 4. 
8.2 
Treatment schedule modifications for pembrolizumab. 
Adverse events (both non-serious and serious) associated with pembrolizumab exposure 
may represent an immunologic etiology. These adverse events may occur shortly after the 
first dose or several months after the last dose of treatment. Pembrolizumab must be 
withheld for drug-related toxicities and severe or life-threatening AEs per Table 8.21 
below. See Section 9.0 for supportive care guidelines, including use of corticosteroids. 
Table 8.21 
Dose Modification Guidelines for Drug-Related Adverse Events  
CTCAE 
System/Organ/Class 
(SOC) 
Adverse Event 
Grade 
Action 
General disorders and 
administration site 
conditions 
Infusion related reaction 
2 
Hold all protocol treatment until Grade 0-1 
If AE does not resolve to Grade 0-1 within 
3 weeks, then permanently discontinue all 
protocol treatment 
3-4 
Permanently discontinue all protocol treatment 
Respiratory, thoracic 
and mediastinal 
disorders 
Pneumonitis 
2 
Hold all protocol treatment until Grade 0-1 
If AE does not resolve to Grade 0-1 within 
3 weeks, then permanently discontinue all 
protocol treatment 
3-4 
Permanently discontinue all protocol treatment 
Renal and urinary 
disorders 
Acute kidney injury or 
Chronic kidney disease 
2 
Hold all protocol treatment until Grade 0-1 
If AE does not resolve to Grade 0-1 within 
3 weeks, then permanently discontinue all 
protocol treatment 
3-4 
Permanently discontinue all protocol treatment 
 
All Other Nonhematologic 
Events 
3 
Hold all protocol treatment until Grade 0-1 
If AE does not resolve to Grade 0-1 within 
3 weeks, then permanently discontinue all 
protocol treatment 
4 
Permanently discontinue all protocol treatment 

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9.0 
Ancillary Treatment/Supportive Care 
9.1 
Full Supportive Care 
Patients should receive full supportive care while on this study. This includes blood 
product support, antibiotic treatment, and treatment of other newly diagnosed or 
concurrent medical conditions. All blood products and concomitant medications such as 
antidiarrheals, analgesics, and/or antiemetics received from within 28 days before the 
study treatment administration until 30 days after the final dose will be recorded in the 
medical records.  
9.11 
Antiemetics 
Antiemetics may be used at the discretion of the treating physician. 
9.12 
Non-Steroidal Anti-Inflammatory Drugs (NSAIDS) 
NSAIDs use limited to standard non-prescription doses. 
9.13 
Blood Products and Growth Factors 
Blood products and growth factors should be utilized as clinically warranted and 
following institutional policies and recommendations. The use of growth factors 
should follow published guidelines of the American Society of Clinical 
Oncology: Recommendations for the Use of WBC Growth Factors: American 
Society of Clinical Oncology Practice Guidelines Update. Journal of Clinical 
Oncology 2015;33:3199-3212.. 
9.2 
Suggested supportive care measures for the management of adverse events with 
potential immunologic etiology 
Subjects should receive appropriate supportive care measures as deemed necessary by the 
treating investigator. Suggested supportive care measures for the management of adverse 
events with potential immunologic etiology are outlined below. Where appropriate, these 
guidelines include the use of oral or intravenous treatment with corticosteroids as well as 
additional anti-inflammatory agents if symptoms do not improve with administration of 
corticosteroids. Note that several courses of steroid tapering may be necessary as 
symptoms may worsen when the steroid dose is decreased. For each disorder, attempts 
should be made to rule out other causes such as metastatic disease or bacterial or viral 
infection, which might require additional supportive care. The treatment guidelines are 
intended to be applied when the investigator determines the events to be related to 
pembrolizumab. 
Note: if after the evaluation the event is determined not to be related, the investigator 
does not need to follow the treatment guidance (as outlined below). Refer to Section 8.0 
for dose modification. 
It may be necessary to perform additional procedures such as bronchoscopy, endoscopy, 
or skin photography as part of evaluation of the event. 
• 
Pneumonitis: 
o For Grade 2 events, treat with systemic corticosteroids at 0.5 -1 mg/kg of 
prednisone or equivalent. Monitor O2 saturations. When symptoms improve to 
Grade 1 or less, steroid taper should be started and continued over no less than 4 
weeks. 
o For Grade 3 events. Rule out infectious events, but do not hold steroids while 
doing so. Consider hospitalization, start steroids at 2 mg/kg of prednisone or 
equivalent. Monitor O2 and administer oxygen as needed. Once symptoms 

MC1771 
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Amendment 3 
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improve to grade 1 or less, steroid taper should be started and continued over no 
less than 4 weeks. Consider bronchoscopy, to evaluate for infection. 
o For Grade 4 events, patients should be hospitalized in an intensive care unit, and 
started on high dose IV steroids at 1 gram of methylprednisolone. Rule out 
infectious etiology, but do not hold steroids while doing so. Consult 
Pulmonology and patients should undergo bronchoscopy. Administer additional 
anti-inflammatory measures, as needed. 
o Add prophylactic antibiotics for opportunistic infections in the case of prolonged 
steroid administration. 
• 
Diarrhea/Colitis: 
Subjects should be carefully monitored for signs and symptoms of enterocolitis (such 
as diarrhea, abdominal pain, blood or mucus in stool, with or without fever) and of 
bowel perforation (such as peritoneal signs and ileus). Any patients with these 
symptoms should immediately undergo CT of the abdomen to rule out bowel 
perforation. 
o All subjects who experience diarrhea/colitis should be advised to drink liberal 
quantities of clear fluids. If sufficient oral fluid intake is not feasible, fluid and 
electrolytes should be substituted via IV infusion. For Grade 2 or higher diarrhea, 
consider GI consultation and endoscopy to confirm or rule out colitis. 
o For Grade 2 diarrhea/colitis, lasting >one week patients should be started on 
budesonide 12 mg QD. Additionally infectious etiologies should be ruled out. Do 
not hold steroids while ruling out infection. For patients who do not respond to 
budesonide or have progressive diarrhea, these patients should be started on 
systemic steroids at 0.5-1 mg/kg of prednisone or equivalent. 
o For Grade 3 diarrhea/colitis, patients should be started on budesonide at 12 mg 
daily (if not already started) and 2 mg/kg of prednisone or equivalent. IF no 
response within 3 days, patient should be hospitalized and started on IV steroids 
(methylprednisolone 500 mg-1000mg/daily). Beware of rebound diarrhea. 
o For Grade 4 diarrhea/colitis patients should be hospitalized and started on 1 gram 
of methylprednisolone daily, and 12 mg budesonide QD, for up to 3 days (then 
reduce to 2 mg/kg of prednisone or equivalent) and consult with GI immediately. 
Patients who do not respond to steroids, may consider infliximab (under direction 
of GI). Patients who respond initially to high dose IV steroids can be transitioned 
to high dose oral steroids (2mg/kg). Beware of rebound diarrhea. 
o For all patients, when symptoms improve to Grade 1 or less, steroid taper should 
be started and continued over no less than 4 weeks. 
• 
Type 1 diabetes mellitus (if new onset, including diabetic ketoacidosis [DKA]) or 
≥Grade 3 Hyperglycemia. 
• 
For T1DM or Grade 3-4 Hyperglycemia 
o Insulin replacement therapy is recommended for Type I diabetes mellitus, 
treat based on grade of hyperglycemia as below. 
o For Grade 3 Hyperglycemia- Consider hospitalization, insulin therapy is 
required. Monitor glucose closely, and refer to Endocrinology for further 
management. 
o For Grade 4 hyperglycemia associated with metabolic acidosis or ketonuria. 
Hospitalize patient (consider admission to an ICU), immediate insulin 
therapy is necessary, and refer to endocrinology for further management. 

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Amendment 3 
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o Evaluate patients with serum glucose and a metabolic panel, urine ketones, 
glycosylated hemoglobin, and C-peptide. 
• 
Hypophysitis: 
o For Grade 2 events, treat with corticosteroids 0.5mg/kg. When symptoms 
improve to Grade 1 or less, steroid taper should be started and be rapidly tapered 
to physiologic steroid dosing provided patient remains asymptomatic (some 
patients may require a slower taper). Replacement of appropriate hormones may 
be required as the steroid dose is tapered. Refer to Endocrinology for further 
management. 
o For Grade 3-4 events, treat with high dose (1-2 mg/kg) of prednisone or 
equivalent). When symptoms improve to Grade 1 or less, rapidly taper to 
physiologic steroid dosing provided patients remains asymptomatic (some 
patients may require a slower taper). Replacement of appropriate hormones may 
be required as the steroid dose is tapered. Refer to endocrinology for further 
management. 
• 
Hyperthyroidism or Hypothyroidism: 
Thyroid disorders can occur at any time during treatment. Monitor patients for 
changes in thyroid function (at the start of treatment, periodically during treatment, 
and as indicated based on clinical evaluation) and for clinical signs and symptoms of 
thyroid disorders. 
• 
Grade 2 hyperthyroidism events: 
o Non-selective beta-blockers (e.g. propranolol) are suggested as initial 
therapy. 
• 
Grade 3-4 hyperthyroidism 
o As above- Additionally patients should be hospitalized and may need 
possible thyroid suppression therapy. Patients should be immediately 
referred to an endocrinologist for treatment. 
• 
Hypothyroidism (TSH <10) 
o Asymptomatic- no intervention needed, continue to monitor TFTs 
o Symptomatic- thyroid replacement indicated.  
• 
Hypothyroidism (TSH >10) 
o Thyroid replacement indicated. 
• 
Hepatic: 
o For Grade 2 events, monitor liver function tests twice weekly until returned to 
Grade 1 and/or baseline values. 
 
Treat with oral steroids at 1 mg/kg of prednisone or equivalent. Once levels 
return to grade 1 or less, begin steroid taper and taper over at least 4 weeks. 
o For Grade 3: 
 
Rule out other causes (i.e., progressive disease) 
 
Asymptomatic treat with 1-2 mg/kg of prednisone or equivalent  
 
Symptomatic- treat with IV steroids, 500 mg of methylprednisolone or 
equivalent, Consider hospitalization. Once resolved to grade 1 taper steroids 
over at least 4 weeks. 
o For Grade 4: 
 
Rule out other causes (i.e., progressive disease) 

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 
Hospitalize, and treat with intravenous corticosteroids (1 gram 
methylprednisolone) for 24 to 48 hours. Once stable, may switch to oral high 
dose steroids.  
o When symptoms improve to Grade 1 or less, a steroid taper should be started and 
continued over no less than 4 weeks. 
o For patients who are refractory to steroids, consider hepatology consult and 
starting mycophenolate. 
• 
Renal Failure or Nephritis: 
o For Grade 2 events, treat with steroids at 1 mg/kg prednisone or equivalent. Once 
resolved to Grade 1, may start taper over one month. 
o For Grade 3 events, consider hospitalization, consult nephrology, renal biopsy 
may be indicated. Administer steroids at 2 mg/kg prednisone or equivalent. 
o For Grade 4 - hospitalization, consult nephrology, renal biopsy as indicated, treat 
with IV systemic corticosteroids 1 gram of methylprednisolone. Dialysis may be 
indicated.  
o When symptoms improve to Grade 1 or less, steroid taper should be started and 
continued over no less than 4 weeks. 
• 
Rash 
o Grade 2 - Treat symptomatically with topical steroid cream, as well as 
antihistamines (i.e., loratidine, diphenhydramine, etc.). For patients that do not 
respond, consider starting steroids at 0.5 mg/kg prednisone or equivalent. 
o Grade 3 - As above for grade 2 and Start steroids at 1 mg/kg prednisone or 
equivalent.  
o If any signs of Steven Johnson Syndrome (SJS) or Toxic Epidermal Necrolysis 
(TENS), immediately hospitalize patient and treat with high dose IV steroids (1 
gram methylprednisolone) and consult dermatology. 
• 
Management of Infusion Reactions: 
o Signs and symptoms usually develop during or shortly after drug infusion and 
generally resolve completely within 24 hours of completion of infusion. 
o Table 9.21 below shows treatment guidelines for subjects who experience an 
infusion reaction associated with administration of pembrolizumab. 
 
 
 

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Table 9.21 
Infusion reaction treatment guidelines 
NCI CTCAE Grade 
Treatment 
Premedication at 
subsequent dosing 
Grade 1 
Mild reaction; infusion 
interruption not indicated; 
intervention not indicated 
Increase monitoring of vital signs as 
medically indicated until the subject is 
deemed medically stable in the opinion of 
the investigator. 
None 
Grade 2 
Requires infusion interruption 
but responds promptly to 
symptomatic treatment (e.g., 
antihistamines, NSAIDS, 
narcotics, IV fluids); 
prophylactic medications 
indicated for ≤24 hrs 
Stop Infusion and monitor symptoms. 
Additional appropriate medical therapy 
may include but is not limited to: 
IV fluids Antihistamines NSAIDS 
Acetaminophen Narcotics 
Increase monitoring of vital signs as 
medically indicated until the subject is 
deemed medically stable in the opinion of 
the investigator. 
If symptoms resolve within one hour of 
stopping drug infusion, the infusion may be 
restarted at 50% of the original infusion rate 
(e.g., from 100 mL/hr to 50 mL/hr). 
Otherwise dosing will be held until 
symptoms resolve and the subject should be 
premedicated for the next scheduled dose. 
Subjects who develop Grade 2 adverse 
events despite adequate premedication 
should be permanently discontinued 
from further trial treatment 
administration. 
Subject may be pre-
medicated 1.5h (±30 
minutes) prior to infusion of 
pembrolizumab with: 
Diphenhydramine 50 mg po 
(or equivalent dose of 
antihistamine) 
Acetaminophen 1000 mg po 
(or equivalent dose of 
antipyretic) 
Grades 3 or 4 Grade 3: 
Prolonged (i.e., not rapidly 
responsive to symptomatic 
medication and/or brief 
interruption of infusion); 
recurrence of symptoms 
following initial improvement; 
hospitalization indicated for 
other clinical sequelae (e.g., 
renal impairment, pulmonary 
infiltrates) 
Stop Infusion. 
Additional appropriate medical therapy 
may include but is not limited to: 
IV fluids 
Antihistamines 
NSAIDS 
Acetaminophen 
Narcotics 
Oxygen 
Pressors 
Corticosteroids 
Epinephrine 
No subsequent dosing 
Grade 4: 
Life-threatening; pressor or 
ventilatory support indicated 
Increase monitoring of vital signs as 
medically indicated until the subject is 
deemed medically stable in the opinion of 
the investigator. 
Hospitalization may be indicated. Subject 
is permanently discontinued from 
further trial treatment administration. 
 
Appropriate resuscitation equipment should be available in the room and a physician readily available during 
the period of drug administration. 

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10.0 
Adverse Event (AE) Reporting and Monitoring 
The site principal investigator is responsible for reporting any/all serious adverse events to the 
sponsor as described within the protocol, regardless of attribution to study agent or treatment 
procedure. 
The sponsor/sponsor-investigator is responsible for notifying FDA and all participating 
investigators in a written safety report of any of the following: 
• 
Any suspected adverse reaction that is both serious and unexpected. 
• 
Any findings from laboratory animal or in vitro testing that suggest a significant risk for 
human subjects, including reports of mutagenicity, teratogenicity, or carcinogenicity. 
• 
Any findings from epidemiological studies, pooled analysis of multiple studies, or 
clinical studies, whether or not conducted under an IND and whether or not conducted 
by the sponsor, that suggest a significant risk in humans exposed to the drug 
• 
Any clinically important increase in the rate of a serious suspected adverse 
reaction over the rate stated in the protocol or Investigator’s Brochure (IB). 
Summary of SAE Reporting for this study  
(please read entire section for specific instructions): 
WHO: 
WHAT form: 
WHERE to send: 
All sites 
Pregnancy Reporting 
http://ctep.cancer.gov/protocolDevelopment/electronic_
applications/docs/PregnancyReportFormUpdated.pdf 
Mayo Sites – attach to MCCC 
Electronic SAE Reporting Form 
Mayo Clinic 
Sites 
Mayo Clinic Cancer Center SAE Reporting Form:  
http://livecycle2.mayo.edu/workspace/?startEndpoint=
MC4158-56/Processes/MC4158-56-Process.MC4158-
56 
AND attach MedWatch 3500A: 
http://www.fda.gov/downloads/AboutFDA/Reports
ManualsForms/Forms/UCM048334.pdf 
Will automatically be sent to 
CANCERCROSAFETYIN@may
o.edu 
 
Definitions 
Adverse Event 
Any untoward medical occurrence associated with the use of a drug in humans, whether 
or not considered drug related. 
Suspected Adverse Reaction 
Any adverse event for which there is a reasonable possibility that the drug caused the 
adverse event. 
Expedited Reporting 
Events reported to sponsor within 24 hours, 5 days or 10 days of study team becoming 
aware of the event. 
Routine Reporting 
Events reported to sponsor via case report forms 
Events of Interest 
Events that would not typically be considered to meet the criteria for expedited reporting, 

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but that for a specific protocol are being reported via expedited means in order to 
facilitate the review of safety data (may be requested by the FDA or the sponsor). 
Unanticipated Adverse Device Event (UADE) 
Any serious adverse effect on health or safety or any life-threatening problem or death 
caused by, or associated with, a device, if that effect, problem, or death was not 
previously identified in nature, severity, or degree of incidence in the investigational plan 
or application (including a supplementary plan or application), or any other unanticipated 
serious problem associated with a device that relates to the rights, safety, or welfare of 
subjects 
10.1 
Adverse event characteristics 
CTCAE term (AE description) and grade: The descriptions and grading scales 
found in the revised NCI Common Terminology Criteria for Adverse Events 
(CTCAE) version 4.0 will be utilized for AE reporting. All appropriate treatment 
areas should have access to a copy of the CTCAE version 4.0. A copy of the 
CTCAE version 4.0 can be downloaded from the CTEP web site: 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm) 
a. Identify the grade and severity of the event using the CTCAE version 4.0. 
b. Determine whether the event is expected or unexpected (see Section 10.2). 
c. Determine if the adverse event is related to the study intervention (agent, 
treatment or procedure) (see Section 10.3). 
d. Determine whether the event must be reported as an expedited report. If yes, 
determine the timeframe/mechanism (see Section 10.4). 
e. Determine if other reporting is required (see Section 10.5). 
f. Note: All AEs reported via expedited mechanisms must also be reported via the 
routine data reporting mechanisms defined by the protocol (see Sections 10.6 
and 18.0). 
NOTE: A severe AE is NOT the same as a serious AE, which is defined in Section 
10.4. 
10.2 
Expected vs. unexpected events 
Expected events - are those described within the Section 15.0 of the protocol, the study 
specific consent form, package insert (if applicable), and/or the investigator brochure, (if 
an investigator brochure is not required, otherwise described in the general 
investigational plan). 
Unexpected adverse events or suspected adverse reactions are those not listed in Section 
15.0 of the protocol, the study specific consent form, package insert (if applicable), or in 
the investigator brochure (or are not listed at the specificity or severity that has been 
observed); if an investigator brochure is not required or available, is not consistent with 
the risk information described in the general investigational plan. 
Unexpected also refers to adverse events or suspected adverse reactions that are 
mentioned in the investigator brochure as occurring with a class of drugs but have not 
been observed with the drug under investigation. 
An investigational agent/intervention might exacerbate the expected AEs associated with 
a commercial agent. Therefore, if an expected AE (for the commercial agent) occurs with 
a higher degree of severity or specificity, expedited reporting is required. 

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NOTE: *The consent form may contain study specific information at the discretion of 
the Principal Investigator; it is possible that this information may NOT be included in 
the protocol or the investigator brochure. Refer to protocol or IB for reporting needs. 
10.3 
Attribution to agent(s) or procedure 
When assessing whether an adverse event (AE) is related to a medical agent(s) medical or 
procedure, the following attribution categories are utilized:  
Definite - The AE is clearly related to the agent(s)/procedure. 
Probable - The AE is likely related to the agent(s)/procedure. 
Possible - The AE may be related to the agent(s)/procedure. 
Unlikely - The AE is doubtfully related to the agent(s)/procedure. 
Unrelated - The AE is clearly NOT related to the agent(s)/procedure. 
10.31 EXPECTED Serious Adverse Events: Protocol Specific Exceptions to Expedited 
Reporting 
For this protocol only, the following Adverse Events/Grades are expected to 
occur within this population and do not require Expedited Reporting. These 
events must still be reported via Routine Reporting (see Section 10.6).* 
*Report any clinically important increase in the rate of a serious suspected 
adverse reaction (at your study site) over that which is listed in the protocol or 
investigator brochure as an expedited event. 
*Report an expected event that is greater in severity or specificity than expected 
as an expedited event. 
CTCAE 
System/Organ/Class 
Adverse Event 
CTCAE Grade at which the 
event will not be reported in an 
expedited manner1 
Blood and lymphatic 
system disorders 
Anemia 
≤Grade 4 
Investigations 
Lymphocyte count decreased 
≤Grade 4 
Neutrophil count decreased 
≤Grade 4 
Platelet count decreased 
≤Grade 4 
White blood cell decreased 
≤Grade 4 
1 These exceptions only apply if the adverse event does not result in hospitalization. If 
the adverse event results in hospitalization, then the standard expedited adverse 
events reporting requirements must be followed. 
Specific protocol exceptions to expedited reporting should be reported expeditiously 
by investigators ONLY if they exceed the expected grade of the event. 
 
The following hospitalizations are not considered to be SAEs because there is no 
“adverse event” (i.e., there is no untoward medical occurrence) associated with 
the hospitalization: 
● Hospitalizations for respite care 
● Planned hospitalizations required by the protocol 
● Hospitalization planned before informed consent (where the condition 
requiring the hospitalization has not changed post study drug administration) 
● Hospitalization for elective procedures unrelated to the current disease and/or 
treatment on this trial 
● Hospitalization for administration of study drug or insertion of access for 

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administration of study drug 
● Hospitalization for routine maintenance of a device (e.g., battery replacement) 
that was in place before study entry 
● Hospitalization, or other serious outcomes for signs and symptoms of 
progression of the cancer.] 
10.4 
Expedited Reporting Requirements for IND/IDE Agents 
10.41 Late Phase 2 and Phase 3 Studies: Expedited Reporting Requirements for 
Adverse Events that Occur on Studies under an IND/IDE within 30 Days of the 
Last Administration of the Investigational Agent/Intervention1, 2 
FDA REPORTING REQUIREMENTS FOR SERIOUS ADVERSE EVENTS (21 CFR Part 312) 
NOTE: Investigators MUST immediately report to the sponsor ANY Serious Adverse Events, whether or not they 
are considered related to the investigational agent(s)/intervention (21 CFR 312.64) 
 An adverse event is considered serious if it results in ANY of the following outcomes:  
1) 
Death 
2) 
A life-threatening adverse event  
3) 
An adverse event that results in inpatient hospitalization or prolongation of existing hospitalization for ≥ 24 
hours  
4) 
A persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions  
5) 
A congenital anomaly/birth defect.  
6) 
Important Medical Events (IME) that may not result in death, be life threatening, or require hospitalization 
may be considered serious when, based upon medical judgment, they may jeopardize the patient or 
subject and may require medical or surgical intervention to prevent one of the outcomes listed in this 
definition. (FDA, 21 CFR 312.32; ICH E2A and ICH E6). 
ALL SERIOUS adverse events that meet the above criteria MUST be immediately reported to the sponsor within 
the timeframes detailed in the table below. 
Hospitalization 
Grade 1 
Timeframes 
Grade 2 
Timeframes 
Grade 3 Timeframes 
Grade 4 & 5 
Timeframes 
Resulting in 
Hospitalization 
≥24 hrs 
7 Calendar Days 
24-Hour  
3 Calendar 
Days 
Not resulting in 
Hospitalization 
≥24 hrs 
Not required 
7 Calendar Days 
Expedited AE reporting timelines are defined as: 
o “24-Hour; 3 Calendar Days” - The AE must initially be reported within 24 hours of learning of the AE, 
followed by a complete expedited report within 3 calendar days of the initial 24-hour report. 
o “7 Calendar Days” - A complete expedited report on the AE must be submitted within 7 calendar days 
of learning of the AE. 
1Serious adverse events that occur more than 30 days after the last administration of investigational 
agent/intervention and have an attribution of possible, probable, or definite require reporting as follows:  
Expedited 24-hour notification followed by complete report within 3 calendar days for: 
• 
All Grade 4, and Grade 5 AEs 
Expedited 7 calendar day reports for: 
• Grade 2 adverse events resulting in hospitalization or prolongation of hospitalization  
• Grade 3 adverse events 
2 For studies using PET or SPECT IND agents, the AE reporting period is limited to 10 radioactive half-lives, 
rounded UP to the nearest whole day, after the agent/intervention was last administered. Footnote “1” above 
applies after this reporting period. 
Effective Date: May 5, 2011 

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NOTE: Refer to Section 10.31 for exceptions to Expedited Reporting 
10.42 General reporting instructions 
The Mayo IND Coordinator will assist the sponsor-investigator in the processing 
of expedited adverse events and forwarding of suspected unexpected serious 
adverse reactions (SUSARs) to the FDA and IRB. 
Use Mayo Expedited Event Report form 
http://livecycle2.mayo.edu/workspace/?startEndpoint=MC4158‐ 
56/Processes/MC4158‐56‐Process.MC4158‐56 for investigational agents or 
commercial/investigational agents on the same arm. 
10.43 Reporting of re-occurring SAEs 
ALL SERIOUS adverse events that meet the criteria outlined in table10.41 
MUST be immediately reported to the sponsor within the timeframes detailed in 
the corresponding table. This reporting includes, but is not limited to SAEs that 
re-occur again after resolution. 
10.5 
Other Required Reporting 
10.51 Unanticipated Problems Involving Risks to Subjects or Others (UPIRTSOS) 
Unanticipated Problems Involving Risks to Subjects or Others 
(UPIRTSOS) in general, include any incident, experience, or 
outcome that meets all of the following criteria: 
1. Unexpected (in terms of nature, severity, or frequency) given (a) the 
research procedures that are described in the protocol-related documents, 
such as the IRB-approved research protocol and informed consent 
document; and (b) the characteristics of the subject population being 
studied; 
2. Related or possibly related to participation in the research (in this 
guidance document, possibly related means there is a reasonable 
possibility that the incident, experience, or outcome may have been 
caused by the procedures involved in the research); and 
3. Suggests that the research places subjects or others at a greater risk of 
harm (including physical, psychological, economic, or social harm) than 
was previously known or recognized. 
Some unanticipated problems involve social or economic harm instead of the 
physical or psychological harm associated with adverse events. In other cases, 
unanticipated problems place subjects or others at increased risk of harm, but no 
harm occurs. 
Note: If there is no language in the protocol indicating that pregnancy is not 
considered an adverse experience for this trial, and if the consent form does not 
indicate that subjects should not get pregnant/impregnate others, then any 
pregnancy in a subject/patient or a male patient’s partner (spontaneously 
reported) which occurs during the study or within 120 days of completing the 
study should be reported as a UPIRTSO. 
Mayo Clinic Cancer Center (MCCC) Institutions: 
If the event meets the criteria for IRB submission as a Reportable 
Event/UPIRTSO, provide the Reportable Event coversheet and appropriate 

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documentation to CANCERCROSAFETYIN@mayo.edu. The Mayo 
Regulatory Affairs Office will review and process the submission to the 
Mayo Clinic IRB. 
10.52 Death 
Note: A death on study requires both routine and expedited reporting 
regardless of causality, unless as noted below. Attribution to treatment or 
other cause must be provided. 
Any death occurring within 30 days of the last dose, regardless of attribution to 
an agent/intervention under an IND/IDE requires expedited reporting within 
24-hours. 
Any death occurring greater than 30 days with an attribution of possible, 
probable, or definite to an agent/intervention under an IND/IDE requires 
expedited reporting within 24-hours. 
Reportable categories of Death  
• 
Death attributable to a CTCAE term. 
• 
Death Neonatal: A disorder characterized by cessation of life during the 
first 28 days of life.  
• 
Death NOS: A cessation of life that cannot be attributed to a CTCAE term 
associated with Grade 5. 
• 
Sudden death NOS: A sudden (defined as instant or within one hour of the 
onset of symptoms) or an unobserved cessation of life that cannot be 
attributed to a CTCAE term associated with Grade 5. 
• 
Death due to progressive disease should be reported as Grade 5 
“Neoplasms benign, malignant and unspecified (including cysts and 
polyps) – Other (Progressive Disease)” under the system organ class 
(SOC) of the same name. Evidence that the death was a manifestation of 
underlying disease (e.g., radiological changes suggesting tumor growth or 
progression: clinical deterioration associated with a disease process) 
should be submitted. 
Per NIH OBA Appendix M, should a patient die during the study or study 
follow-up, no matter what the cause, the study doctor will ask the patient’s 
family for permission to perform an autopsy. If permission is granted, a copy of 
the autopsy report will be sent to the sponsor after all identifying information has 
been removed. An autopsy will help the researchers learn more about the safety 
and efficacy of the treatment. Patients should advise their families about their 
wishes regarding autopsy. 
10.53 Secondary Malignancy 
• 
A secondary malignancy is a cancer caused by treatment for a previous 
malignancy (e.g., treatment with investigational agent/intervention, radiation 
or chemotherapy). A secondary malignancy is not considered a metastasis of 
the initial neoplasm. 
• 
All secondary malignancies that occur following treatment with an agent 
under an IND/IDE will be reported. Three options are available to describe 
the event: 
o Leukemia secondary to oncology chemotherapy (e.g., Acute Myeloctyic 
Leukemia [AML]) 

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o Myelodysplastic syndrome (MDS) 
o Treatment-related secondary malignancy 
• 
Any malignancy possibly related to cancer treatment (including AML/MDS) 
should also be reported via the routine reporting mechanisms outlined in each 
protocol. 
10.54 Second Malignancy 
A second malignancy is one unrelated to the treatment of a prior malignancy (and 
is NOT a metastasis from the initial malignancy). Second malignancies require 
ONLY routine reporting unless otherwise specified. 
10.55 Pregnancy, Fetal Death, and Death Neonatal 
If a female subject (or female partner of a male subject) taking investigational 
product becomes pregnant, the subject taking should notify the Investigator, and 
the pregnant female should be advised to call her healthcare provider 
immediately. The patient should have appropriate follow-up as deemed necessary 
by her physician. If the baby is born with a birth defect or anomaly, a second 
expedited report is required. 
Prior to obtaining private information about a pregnant woman and her infant, 
the investigator must obtain consent from the pregnant woman and the newborn 
infant’s parent or legal guardian before any data collection can occur. A consent 
form will need to be submitted to the IRB for these subjects if a pregnancy 
occurs. If informed consent is not obtained, no information may be collected. 
In cases of fetal death, miscarriage or abortion, the mother is the patient. In cases 
where the child/fetus experiences a serious adverse event other than fetal death, 
the child/fetus is the patient. 
NOTE: When submitting Mayo Expedited Adverse Event Report reports for 
“Pregnancy”, “Pregnancy loss”, or “Neonatal loss”, the potential risk of exposure 
of the fetus to the investigational agent(s) or chemotherapy agent(s) should be 
documented in the “Description of Event” section. Include any available medical 
documentation. Include this form: 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/Pregna
ncyReportFormUpdated.pdf 
10.551 
Pregnancy 
Pregnancy should be reported in an expedited manner as Grade 3 
“Pregnancy, puerperium and perinatal conditions - Other 
(pregnancy)” under the Pregnancy, puerperium and perinatal 
conditions SOC. Pregnancy should be followed until the outcome is 
known. 
10.552 
Fetal Death 
Fetal death is defined in CTCAE as “A disorder characterized by death 
in utero; failure of the product of conception to show evidence of 
respiration, heartbeat, or definite movement of a voluntary muscle after 
expulsion from the uterus, without possibility of resuscitation.” 
Any fetal death should be reported expeditiously, as Grade 4 
“Pregnancy, puerperium and perinatal conditions - Other 
(pregnancy loss)” under the Pregnancy, puerperium and perinatal 
conditions SOC. 

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10.553 
Death Neonatal 
Neonatal death, defined in CTCAE as “A disorder characterized by 
cessation of life occurring during the first 28 days of life” that is felt by 
the investigator to be at least possibly due to the investigational 
agent/intervention, should be reported expeditiously.  
A neonatal death should be reported expeditiously as Grade 4 
“General disorders and administration - Other (neonatal loss)” 
under the General disorders and administration SOC.  
10.6 
Required Routine Reporting 
10.61 Baseline and Adverse Events Evaluations 
Pretreatment symptoms/conditions to be graded at baseline and adverse events to 
be graded at each evaluation. 
Grading is per CTCAE v4.0 unless alternate grading is indicated in the table 
below: 
CTCAE 
System Organ Class (SOC) 
Adverse event/Symptoms 
Baseline 
Each 
evaluation 
Blood and lymphatic system 
disorders 
Anemia 
X 
X 
Endocrine disorders 
Hypothyroidism 
X 
X 
Gastrointestinal disorders 
Baseline # stools 
X 
 
Diarrhea 
 
X 
General disorders 
Fatigue 
X 
X 
Fever 
 
X 
Pain 
X 
X 
Immune system disorders 
Allergic reaction 
 
X 
Anaphylaxis 
 
X 
Investigations 
Alanine aminotransferase increased 
X 
X 
 
Aspartate aminotransferase increased 
X 
X 
 
Blood bilirubin increased 
X 
X 
 
Creatinine increased 
X 
X 
Metabolism and nutrition 
disorders 
Hyperglycemia 
X 
X 
Respiratory, thoracic and 
mediastinal disorders 
Dyspnea 
X 
X 
Skin and subcutaneous tissue 
disorders 
Rash, maculo-papular 
X 
X 
10.62 All other AEs 
Submit via appropriate MCCC Case Report Forms (i.e., paper or electronic, as 
applicable) the following AEs experienced by a patient and not specified in 
Section 10.6: 
10.621 Grade 2 AEs deemed possibly, probably, or definitely related to the study 
treatment or procedure. 
10.622 Grade 3 and 4 AEs regardless of attribution to the study treatment or 
procedure. 
 
 

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10.623 Grade 5 AEs (Deaths) 
10.6231 
Any death within 30 days of the patient’s last study 
treatment or procedure regardless of attribution to the study 
treatment or procedure. 
10.6232 
Any death more than 30 days after the patient’s last study 
treatment or procedure that is felt to be at least possibly 
treatment related must also be submitted as a Grade 5 AE, 
with a CTCAE type and attribution assigned. 
 
10.7 
Late Occurring Adverse Events 
Refer to the instructions in the Forms Packet (or electronic data entry screens, as 
applicable) regarding the submission of late occurring AEs following completion of the 
Active Monitoring Phase (i.e., compliance with Test Schedule in Section 4.0). 
 

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11.0 
Treatment Evaluation 
11.1 
Treatment evaluation using RECIST 1.1 guidelines 
The first scan after treatment initiation will be performed at the completion of Cycle 4; 
scans will be then performed at the completion of every fourth cycle of treatment until 
disease progression is confirmed or withdrawn of consent. If a patient experiences PD yet 
continues protocol treatment, the next imaging should take place 2 cycles after the 
imaging showing PD. If that patient continues on protocol treatment, imaging should then 
take place every 4 cycles (See Section 4.0). 
Disease progression for this protocol is defined as meeting the RECIST criteria for 
disease progression on two consecutive evaluations at least 6 weeks apart.  
11.2 
Definitions of measurable and non-measurable disease 
11.21  Measurable disease 
11.211 
A non-nodal lesion is considered measurable if its longest diameter can 
be accurately measured as ≥1.0 cm with CT scan, CT component of a 
PET/CT, or MRI. 
11.212 
A malignant lymph node is considered measurable if its short axis is 
>1.5 cm when assessed by CT scan (CT scan slice thickness 
recommended to be no greater than 5 mm).  
NOTE: Tumor lesions in a previously irradiated area are not considered 
measurable disease. 
11.22 Non-measurable disease 
All other lesions (or sites of disease) are considered non-measurable disease, 
including pathological nodes (those with a short axis ≥1.0 to <1.5 cm). Bone 
lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, 
lymphangitis cutis/pulmonis, inflammatory breast disease, and abdominal masses 
(not followed by CT or MRI), are considered as non-measurable as well. 
NOTE: ‘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.  
Lymph nodes that have a short axis <1.0 cm are considered non-  pathological 
(i.e., normal) and should not be recorded or followed. 
11.3 
Guidelines for evaluation of measurable disease 
11.31 Measurement methods:  
• 
All measurements should be recorded in metric notation (i.e., decimal 
fractions of centimeters) using a ruler or calipers. 
• 
The same method of assessment and the same technique must be used to 
characterize each identified and reported lesion at baseline and during 
follow-up. For patients having only lesions measuring at least 1 cm to less 
than 2 cm must use CT imaging for both pre- and post-treatment tumor 
assessments. 
 
 

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11.32 Acceptable modalities for measurable disease: 
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. 
• 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. The lesions should be measured on the same pulse 
sequence. 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: 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. 
• 
FDG-PET: FDG-PET scanning is allowed to complement CT scanning in 
assessment of progressive disease [PD] and particularly possible 'new' 
disease. A ‘positive’ FDG-PET scanned lesion is defined as one which is 
FDG avid with an update greater than twice that of the surrounding tissue on 
the attenuation corrected image; otherwise, an FDG-PET scanned lesion is 
considered ‘negative.’ 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:  
i. 
If the positive FDG-PET at follow-up corresponds to a new site of 
disease confirmed by CT, this is PD.  
ii. If the positive FDG-PET at follow-up is not confirmed as a new site 
of disease on CT at the same evaluation, additional follow-up CT 
scans (i.e., additional follow-up scans at least 4 weeks later) are 
needed to determine if there is truly progression occurring at that 
site. In this situation, the date of PD will be the date of the initial 
abnormal PDG-PET scan.  
iii 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, it is not classified as PD. 
• 
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 mandatory to differentiate between 
response or stable disease (an effusion may be a side effect of the treatment) 
and progressive disease. 

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• 
Cytologic and histologic techniques can be used to differentiate between PR 
and CR in rare cases (e.g., residual lesions in tumor types such as germ cell 
tumors, where known residual benign tumors can remain.) 
11.4 
Measurement of effect 
11.41 Target lesions & target lymph nodes 
• 
Measurable lesions (as defined in Section 11.21) up to a maximum of 5 
lesions, representative of all involved organs, should be identified as “Target 
Lesions” and recorded and measured at baseline. These lesions can be non-
nodal or nodal (as defined in 11.21), where no more than 2 lesions are from 
the same organ and no more than 2 malignant nodal lesions are selected.  
Note: If fewer than 5 target lesions and target lymph nodes are identified (as 
there often will be), there is no reason to perform additional studies beyond 
those specified in the protocol to discover new lesions.  
• 
Target lesions and target lymph nodes should be selected on the basis of their 
size, be representative of all involved sites of disease, but in addition should 
be those that lend themselves to reproducible repeated measurements. It may 
be the case that, on occasion, the largest lesion (or malignant lymph node) 
does not lend itself to reproducible measurements in which circumstance the 
next largest lesion (or malignant lymph node) which can be measured 
reproducibly should be selected.  
• 
Baseline Sum of Dimensions (BSD): A sum of the longest diameter for all 
target lesions plus the sum of the short axis of all the target lymph nodes will 
be calculated and reported as the baseline sum of dimensions (BSD). The 
BSD will be used as reference to further characterize any objective tumor 
response in the measurable dimension of the disease.  
• 
Post-Baseline Sum of the Dimensions (PBSD): A sum of the longest 
diameter for all target lesions plus the sum of the short axis of all the target 
lymph nodes will be calculated and reported as the post-baseline sum of 
dimensions (PBSD). If the radiologist is able to provide an actual measure 
for the target lesion (or target lymph node), that should be recorded, even if it 
is below 0.5 cm. If the target lesion (or target lymph node) is believed to be 
present and is faintly seen but too small to measure, a default value of 0.5 cm 
should be assigned. If it is the opinion of the radiologist that the target lesion 
or target lymph node has likely disappeared, the measurement should be 
recorded as 0 cm. 
• 
The minimum sum of the dimensions (MSD) is the minimum of the BSD and 
the PBSD. 
11.42 Non-target lesions & non-target lymph nodes 
Non-measurable sites of disease (Section 11.22) are classified as non- target 
lesions or non-target lymph nodes and should also be recorded at base line. These 
lesions and lymph nodes should be followed in accord with 11.433. 
11.43 Response criteria 
11.431 All target lesions and target lymph nodes followed by CT/MRI/PET-CT 
must be measured on re-evaluation at evaluation times specified in 
Section 11.1. Specifically, a change in objective status to either a PR or 

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CR cannot be done without re-measuring target lesions and target lymph 
nodes.  
Note: Non-target lesions and non-target lymph nodes should be 
evaluated at each assessment, especially in the case of first response or 
confirmation of response. In selected circumstances, certain non-target 
organs may be evaluated less frequently. For example, bone scans may 
need to be repeated only when complete response is identified in target 
disease or when progression in bone is suspected. 
11.432 
Evaluation of target lesions 
Complete Response (CR): 
All of the following must be true: 
a. Disappearance of all target lesions. 
b. Each target lymph node must have 
reduction in short axis to <1.0 cm. 
Partial Response (PR): 
At least a 30% decrease in PBSD (sum 
of the longest diameter for all target 
lesions plus the sum of the short axis of 
all the target lymph nodes at current 
evaluation) taking as reference the BSD 
(see Section 11.41). 
Progression (PD): 
At least one of the following must be 
true: 
a. At least one new malignant lesion, 
which also includes any lymph node 
that was normal at baseline (<1.0 
cm short axis) and increased to ≥1.0 
cm short axis during follow-up.  
b. At least a 20% increase in PBSD 
(sum of the longest diameter for all 
target lesions plus the sum of the 
short axis of all the target lymph 
nodes at current evaluation) taking 
as reference the MSD (Section 
11.41). In addition, the PBSD must 
also demonstrate an absolute 
increase of at least 0.5 cm from the 
MSD.  
c. See Section 11.32 for details in 
regards to the requirements for PD 
via FDG-PET imaging. 
Stable Disease (SD): 
Neither sufficient shrinkage to qualify 
for PR, nor sufficient increase to qualify 
for PD taking as reference the MSD. 
11.433 
Evaluation of Non-Target Lesions & Non-target Lymph Nodes 
Complete Response (CR): 
All of the following must be true: 
a. Disappearance of all non-target 
lesions.  

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b. Each non-target lymph node must 
have a reduction in short axis to 
<1.0 cm. 
Non-CR/Non-PD: 
Persistence of one or more non-target 
lesions or non-target lymph nodes. 
Progression (PD): 
At least one of the following must be 
true: 
a. At least one new malignant  lesion, 
which also includes any lymph node 
that was normal at baseline 
(<1.0 cm short axis) and increased 
to ≥1.0 cm short axis during follow-
up. 
b. Unequivocal progression of existing 
non-target lesions and non-target 
lymph nodes. (NOTE: Unequivocal 
progression should not normally 
trump target lesion and target lymph 
node status. It must be 
representative of overall disease 
status change.)  
c. See Section 11.32 for details in 
regards to the requirements for PD 
via FDG-PET imaging. 
11.44 Overall objective status 
The overall objective status for an evaluation is determined by combining the 
patient’s status on target lesions, target lymph nodes, non-target lesions, non-
target lymph nodes, and new disease as defined in the following tables:  
Target Lesions & 
Target Lymph Nodes 
Non-Target Lesions & 
Non-Target Lymph 
Nodes 
New 
Sites of Disease 
Overall Objective 
Status 
CR 
CR 
No 
CR 
CR 
Non-CR/Non-PD 
No 
PR 
PR 
CR 
Non-CR/Non-PD 
No 
PR 
CR/PR 
Not All Evaluated* 
No 
PR 
SD 
CR 
Non-CR/Non-PD 
Not All Evaluated* 
No 
SD 
Not all Evaluated 
CR 
Non-CR/Non-PD 
Not All Evaluated* 
No 
Not Evaluated (NE) 
PD 
Unequivocal PD 
CR 
Non-CR/Non-PD 
Not All Evaluated* 
Yes or No 
PD 

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Target Lesions & 
Target Lymph Nodes 
Non-Target Lesions & 
Non-Target Lymph 
Nodes 
New 
Sites of Disease 
Overall Objective 
Status 
CR/PR/SD/PD/Not all 
Evaluated 
Unequivocal PD 
Yes or No 
PD 
CR/PR/SD/PD/Not all 
Evaluated 
CR 
Non-CR/Non-PD 
Not All Evaluated* 
Yes 
PD 
*See Section 11.431 
 
11.45 Symptomatic Deterioration 
Patients with global deterioration of health status requiring discontinuation of 
treatment without objective evidence of disease progression at that time, and not 
either related to study treatment or other medical conditions, should be reported 
as PD due to “symptomatic deterioration.” Every effort should be made to 
document the objective progression even after discontinuation of treatment due to 
symptomatic deterioration. A patient is classified as having PD due to 
“symptomatic deterioration” if any of the following occur that are not either 
related to study treatment or other medical conditions: 
• 
Weight loss >10% of body weight. 
• 
Worsening of tumor-related symptoms. 
• 
Decline in performance status of >1 level on ECOG scale. 
 
12.0 
Descriptive Factors: None  
 
13.0 
Treatment/Follow–up Decision at Evaluation of Patient 
13.1 
Continuation of treatment 
Patients who have not had disease progression and have experienced acceptable toxicity 
are to continue treatment per protocol until confirmed PD, unacceptable toxicity or 
refusal. And then they will go to event monitoring until death or a maximum of 5 years 
post-registration.  
13.2 
Progressive Disease (PD) 
The first instance in which a patient’s disease status meets the RECIST criteria for PD 
will continue two additional cycles of treatment per protocol and then undergo disease 
revaluation. If the initial documented progression is not confirmed by this scan, patients 
will continue treatment per protocol. If the initial documented progression is confirmed 
by this scan, patients will go to the event-monitoring phase until death or a maximum of 
5 years post-registration. 
Exception: Patients who develop PD in the CNS should discontinue study treatment 
and go to Event Monitoring.  
13.3 
Criteria for discontinuation of protocol therapy include: 
• 
Disease progression: Where disease progression is: the development of a new 
metastatic lesion or an objective status of disease progression (as defined by RECIST 
criteria) on two consecutive evaluations at least 6 weeks apart (Section 13.2). 

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Exception: Patients who develop PD in the CNS should discontinue study 
treatment.  
• 
Request by patient to discontinue study treatment 
• 
Unacceptable toxicity 
• 
Intercurrent illness that would, in the judgment of the investigator, affect assessments 
of clinical status to a significant degree or require discontinuation of drug 
• 
Administration of radiotherapy, non-protocol chemotherapy, immunotherapy, 
biological agents, or an experimental drug during the trial 
• 
Development of new primary cancer 
• 
Ineligibility 
Patients who discontinue treatment due to progression or development of a second 
primary, desire for non-protocol treatment, intolerability, patient request, physician 
decision, or inter-current illness preventing further administration of protocol treatment 
will proceed to event monitoring phase of the trial where patient and disease status until 
death or a maximum of 5 years post-registration 
13.4 
Ineligible 
A patient is deemed ineligible if after registration, it is determined that at the time of 
registration, the patient did not satisfy each and every eligibility criteria for study entry.  
• 
If the patient received any protocol treatment, all data (except biospecimens) up until 
the point of confirmation of ineligibility must be submitted. Event monitoring will be 
required per Section 18.0 of the protocol.  
• 
If the patient never received any protocol treatment, on-study material (except 
biospecimens) and the End of Active Treatment/Cancel Notification Form must be 
submitted. No further No further data submission is necessary. 
13.5 
Withdrawn 
A patient who withdraws consent before any study treatment is given. On-study material 
(except biospecimens) and the End of Active Treatment/Cancel Notification Form must 
be submitted. No further data submission is necessary. 
13.6 
Subsequent Treatment 
Subsequent treatment is at the discretion of the attending physician. 
13.7 
Major violation 
A patient is deemed a major violation, if protocol requirements regarding treatment in 
cycle 1 of the initial therapy are severely violated that evaluability for primary end point 
is questionable. All data up until the point of confirmation of a major violation must be 
submitted. The patient will go directly to the event-monitoring phase of the study. The 
patient may continue treatment off-protocol at the discretion of the physician as long as 
there are no safety concerns, and the patient was properly registered. Event monitoring 
will be required per Section 4.0 of the protocol. 
13.8 
Cancel 
A patient is deemed a cancel if he/she is removed from the study for any reason before 
any study treatment is given. On-study material and the End of Active Treatment/Cancel 
Notification Form must be submitted. No further data submission is necessary. 
 

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14.0 
Body Fluid Biospecimens 
14.1 
Summary Table of Research Blood/Blood Products to Be Collected for This Protocol 
Collect and process all blood/blood products according to instructions and table below. For immunological monitoring both 
Panel 1 and Panel 2 will be collected. 
14.11 Collection During Treatment 
Correlative 
Study (Section 
for more 
information) 
Mandatory 
or Optional 
Blood or 
Body Fluid 
being 
Collected 
Component 
being 
harvested 
Type of 
Collection 
Tube (color 
of tube top) 
Volume to 
collect per tube 
(# of tubes to be 
collected) 
Day 1 of 
designated 
cycles1 
Days 2-5 
of Cycle 2 
Process at 
collecting 
site? (Yes 
or No) 
Temperature 
Conditions for 
Storage/ 
Shipping2 
Immune 
Monitoring 
Mandatory Whole Blood 
Plasma, 
leukocytes 
Heparin 
(green) 
10 mL (7) 
X 
 
No 
Room 
temperature 
EDTA 
(purple) 
6 mL (1) 
4 mL (1) 
Immune 
Monitoring 
Mandatory Whole Blood 
Plasma, 
leukocytes 
EDTA 
(purple) 
6 mL (1) 
 
X3 
No 
Room 
temperature 
1. Research blood should be collected per Section 4.0. 
2. All specimens should be sent to Dr. Lin’s laboratory c/o Anping Chen(pager 4-8864), Stabile 3-25 
3. Specimens may be collected at any laboratory. If not collected at Mayo Clinic, specimens should be shipped overnight at 
room temperature to Dr. Lin’s laboratory c/o Anping Chen(pager 4-8864), Stabile 3-25, Mayo Clinic, 200 First St SW, 
Rochester MN 55905 
 

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14.2 
Background/Methodology 
14.21 T cell functional assays 
If available, we will keep samples of mDC from each patient and use these cells 
to stimulate peripheral blood collected as per the test schedule and assay for 
anti-tumor response via proliferation, ELIspot, or intracellular cytokine 
expression. We will also stimulate T cells with a cocktail of multiple peptides 
from common melanoma antigens. 
14.22 Immune phenotyping 
We will immune phenotype blood and tumor for immune cells including but not 
limited to Tregs, central memory and effector memory T cells, quantitative T, B, 
and NK cell panel, dendritic cells and immune suppressor cells (CD14+DRneg) 
and other immune modulating cells per our prior experience. PD-1, PD-L1 and 
PD- L2 expression on leukocyte subset will be analyzed.  
14.23 Tetramer assays 
We will compare pre- and post-treatment frequencies of melanoma-specific CD8 
T cells via tetramer staining in HLA-A2-positive patients. PBMCs will be labeled 
with HLA-A2 tetramers complexed with melanoma-associated antigen peptides 
(MART-127-35, GP100209-217, survivin-- ELTLGEFLKL, and tyrosinase368-376), and 
control peptides. PBMCs will be counterstained with anti-CD8 (BD Biosciences, 
San Jose, CA). The stained samples will be analyzed by flow cytometry 
(FACScan and CellQuest software, Becton-Dickinson). 
14.24 Plasma cytokine assay 
We will profile the plasma cytokine changes as a result of vaccine administration 
using commercially available cytokine multiplex assays. We will also assess 
plasma levels of soluble PD-L1 via ELISA. 
 

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15.0 
Drug Information 
15.1 
Pembrolizumab (MK-3475, Keytruda®) (commercial supply) 
15.11 Background 
Highly selective anti-PD-1 humanized monoclonal antibody which inhibits 
programmed cell death-1 (PD-1) activity by binding to the PD-1 receptor on T-
cells to block PD-1 ligands (PD-L1 and PD-L2) from binding. Blocking the PD-1 
pathway inhibits the negative immune regulation caused by PD-1 receptor 
signaling. Anti-PD-1 antibodies reverse T-cell suppression and induce antitumor 
responses. 
15.12 Formulation 
Commercially available for injection 25mg/ml (4ml) [contains polysorbate 80] in 
a one-vial formulation 
15.13 Preparation and storage 
Appropriate dose required volume should be added to an IV bag of either 0.9% 
sodium chloride or dextrose 5% in water, and the final concentration should be 
between 1 mg/ml to 10 mg/ml. Mix diluted solution by gentle inversion. Discard 
any unused portion left in the vial. Store bag at room temperature for no more 
than 6 hours (includes duration of infusion) and refrigerate [2°C to 8°C or 36°F 
to 46°F] for no more than 24 hours. Allow solution to come to room temperature 
prior to administration. 
15.14 Administration: 
Infuse over 30 minutes through a 0.2 to 5 micron sterile, nonpyrogenic, low-
protein binding inline or add-on filter. Do not infuse other medications through 
the same infusion line. Compatible in either 0.9% sodium chloride or dextrose 
5% in water. 
15.15 Pharmacokinetic information: 
Note: Clearance is ~20% lower at steady state than with the first dose. With 
weight-based dosing (2 mg/kg), pembrolizumab concentrations in pediatric 
patients are comparable to those of adults (at the same dose). 
Distribution: Vdss: 6.1 L 
Excretion – mean estimated t1/2 values from the 28-day single dose profiles 
ranged from 14.1 to 21.6 days. Clearance is nonlinear and saturable. 
15.16 Potential Drug Interactions 
There are no known significant drug interactions. 
15.17 Known potential toxicities: 
Consult the package inset for the most current and complete information. 
Common known potential toxicities, >10%:  
Cardiovascular: Facial edema 
Central nervous system: Fatigue 
Dermatologic: Pruritus, skin rash 
Endocrine & metabolic: Hyperglycemia, hyponatremia, hypoalbuminemia, 
hypertriglyceridemia, hypocalcemia, decreased sodium bicarbonate 
Gastrointestinal: Nausea, decreased appetite, constipation, diarrhea, vomiting, 

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abdominal pain 
Hematologic & oncologic: Anemia, lymphocytopenia 
Hepatic: Increased serum AST, increased serum ALT, increased serum alkaline 
phosphatase 
Neuromuscular & skeletal: Arthralgia  
Respiratory: Cough, dyspnea  
Miscellaneous: Fever 
Less common known potential toxicities, 1% - 10%:  
Central nervous system: Confusion, peripheral neuropathy 
Endocrine & metabolic: Hypothyroidism, hyperthyroidism  
Gastrointestinal: Colitis 
Immunologic: Antibody development 
Neuromuscular and skeletal: Weakness, arthritis 
Respiratory: Pneumonitis, pleural effusion, pneumonia, respiratory failure 
Rare known potential toxicities, <1% (Limited to important or life- 
threatening): 
Adrenocortical insufficiency (immune-mediated), Bullous pemphigoid (immune- 
mediated), chronic inflammatory demyelinating polyradiculoneuropathy, diabetic 
ketoacidosis, exfoliative dermatitis (immune-mediated), Guillain-Barre syndrome 
(immune-mediated), hemolytic anemia (immune-mediated), hepatitis (including 
autoimmune hepatitis), hypophysitis, infusion-related reaction, interstitial 
nephritis (with renal failure), myasthenia gravis (immune-mediated), myositis 
(immune-mediated), nephritis (autoimmune), pancreatitis (immune-mediated), 
partial epilepsy (immune-mediated; in a patient with inflammatory foci in brain 
parenchyma), thyroiditis, type I diabetes mellitus, uveitis (immune-mediated), 
vasculitis (immune-mediated) 
15.18 Drug procurement:  
Commercial supplies. Pharmacist or clinics shall obtain supplies from normal 
commercial supply or wholesaler. 
15.19 Nursing guidelines 
15.191 
Pembrolizumab side effects vary greatly from those of traditional 
chemotherapy and can vary in severity from mild to life threatening. 
Instruct patients to report any side effects to the study team 
immediately. Side effects may be immediate or delayed up to months 
after discontinuation of therapy. Most side effects are reversible with 
prompt intervention of corticosteroids. 
15.192 
Diarrhea can be seen however is less common than that seen with anti-
CTLA-4 agents. However it can be severe, leading to colonic 
perforation. Instruct patients to report ANY increase in the number of 
stools and/or change in baseline, blood in the stool, abdominal pain to 
the study team immediately. 
15.193 
Rash/pruirits/dermatitis is seen. Patients should report any rash to 
the study team. Treat per section 9.0 and monitor for effectiveness. 
15.194 
Monitor LFTs closely as elevations in these levels could indicate 
early onset autoimmune hepatitis. Patients should also be instructed 
to report any jaundice, or right upper quadrant pain to the study 
team immediately. 

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15.195 
Pneumonitis can be seen and may be mild (only seen on imaging) to 
severe. Patients should be instructed to report any SOB, dyspnea, 
cough, chest pain, etc. to the study team immediately. Patients 
reporting these symptoms should have a pulse ox checked and 
consider immediate imaging per the treating MD. 
15.196 
Endocrinopathies (including hypopituitarism, hypothyroidism, 
hypophysitis, and adrenal insufficiency) are seen with this agent. 
Patients may present only with the vague sense of fatigue and “not 
feeling well.” Additional symptoms may be that of nausea, sweating 
and decreased activity tolerance. Instruct patients to report these signs 
or symptoms immediately and obtain appropriate labs as ordered by 
MD. 
15.197 
Patients who are started on steroid therapy for any side effects of 
pembrolizumab toxicity should be instructed to take the steroids as 
ordered, and not to discontinue abruptly as symptoms may return 
and be severe. Patients may be on steroid therapy for weeks. Instruct 
patients to report any increase or change in side effects with any 
dosage decrease as patients may need a slower taper. 
15.198 
Fatigue is common and may or may not be associated with immune 
related side effects. Assess patient’s fatigue level prior to each 
cycle of therapy and report any changes to the study team. 
15.199a Patients should avoid receiving live vaccines within 30 days of study 
drug administration or per other study guidelines. 
15.199b Patients who have undergone an allogeneic bone marrow transplant 
have an increased risk of severe complications including early GVHD 
and venoocclusive disease, if they have previously been treated with 
pembrolizumab. 
15.199c Myocarditis has been reported and associated with pembrolizumab. 
Instruct patients to report chest pain, SOB, or dyspnea to study team 
immediately and/or seek emergency medical attention. 
15.199d  Autoimmune hematologic disorders including ITP and hemolytic 
anemia  have been reported. Monitor blood counts closely and report 
any abnormalities to the study team. 
15.199e Rare neurologic disorders including Guillain-Barre syndrome and 
myasthenia gravis have been reported. Instruct patients to report any 
neurologic symptoms including weakness, parasthesias or numbness, 
tingling to the study team immedicately. 
 
 
 

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15.2 
Mature Dendritic Cells (IND#17522) 
15.21 Background 
The DC used in this study is patient’s autologous dendritic cells (mDC). 
Dendritic cells are cells manufactured in lab from monocytes removed from 
patients’ blood by leukapheresis. The dendritic cells need to acquire tumor 
antigens in order to stimulate anti-tumor immunity. Patients will be treated with 
DC placed into a tumor killed using cryoablation where tumor antigen exposure 
will occur in vivo. 
15.22 Formulation 
DCs are supplied as recently thawed cells prepared from patient material. Cells 
are manufactured and released by the Human Cellular Therapy Lab, Mayo Clinic 
Rochester. Before released for use, cells will undergo testing for sterility and 
potency. 
15.23 Preparation and storage 
mDCs will be prepared and stored at the Human Cellular Therapy Lab, Mayo 
Clinic Rochester according to approved SOPs included in the IND. The drug will 
be supplied to the appropriate administration area by personnel from the Human 
Cellular Therapy Lab/Immune, Progenitor, and Cellular Therapy (IMPACT) 
Unit. 
15.24 Administration:  
See Section 7.5. 
15.25 Pharmacokinetic information 
NOTE: this drug is a biologic made up of cells from the patient. Classical drug 
pharmacokinetics do not apply. 
15.26 Potential Drug Interactions: Unknown. 
15.27 Known potential toxicities: Unknown. 
15.28 Drug procurement:  
Drug is manufactured on site in the Immune, Progenitor, and Cell Therapeutics 
(IMPACT) Laboratory (507) 284-4010 
 
15.3 
Pneumococcal 13-valent Conjugate Vaccine (Prevnar13®) 
15.31 Background:  
Pneumococcal 13-valent Conjugate Vaccine (Diphtheria CRM197 Protein), is a 
sterile solution of saccharides of the capsular antigens of Streptococcus 
pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F 
individually linked to non-toxic diphtheria CRM197 protein. Each serotype is 
grown in soy peptone broth. 
Potency of the formulated vaccine is determined by quantification of each of the 
saccharide antigens, and by the saccharide to protein ratios in the individual 
glycoconjugates. 
15.32 Formulation 

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Supplied as ready-to-use prefilled syringes (10 x 0.5-mL prefilled syringes per 
package). Pneumococcal 13-valent Conjugate Vaccine is manufactured as a 
liquid preparation for intramuscular injection. Each 0.5 mL dose of the vaccine is 
formulated to contain approximately 2.2 μg of each of Streptococcus pneumoniae 
serotypes 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F saccharides, 4.4 μg of 
6B saccharides, 34 μg CRM197 carrier protein, 100 μg polysorbate 80, 295 μg 
succinate buffer and 125 μg aluminum as aluminum phosphate adjuvant. 
15.33 Preparation and storage:  
Pneumococcal 13-valent Conjugate Vaccine is stored at refrigerated temperatures 
of 2ºC to 8ºC (36ºF to 46ºF) away from freezer compartment. DO NOT 
FREEZE. Discard if frozen. 
15.34 Administration 
Shake vigorously immediately prior to use to obtain a uniform suspension in the 
vaccine container. The vaccine should not be used if it cannot be resuspended. 
After shaking, the vaccine is a homogeneous, white suspension. Do not mix the 
vaccine with other products in the same syringe. 
15.35 Potential Drug Interactions 
Patients receiving therapy with immunosuppressive agents (large amounts of 
corticosteroids, antimetabolites, alkylating agents, cytotoxic agents) may not 
respond optimally to active immunization. 
As with other intramuscular injections, Pneumococcal 13-valent Conjugate 
Vaccine should be given with caution to patients on anticoagulant therapy. 
See complete prescribing information for information regarding co- 
administration of Pneumococcal 13-valent Conjugate Vaccine with other 
vaccines. 
15.36 Known potential toxicities 
There is no safety data available in the adult patient population. Please see 
Prevnar13® prescribing information for comprehensive toxicity data. 
The following systemic events were noted within 2-3 days of the Pneumococcal 
13-valent Conjugate Vaccine injection in pediatric patients: fever, irritability, 
drowsiness, restless sleep, decreased appetite, vomiting, diarrhea, and urticaria- 
like rash. The following local reactions occurred within 3 days of immunization 
pediatric patients: erythema, induration, tenderness and interference with limb 
movement. 
15.37 Drug procurement 
Prevnar13 will be purchased with study funds and provided free of charge to 
patients. 
 

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16.0 
Statistical Considerations and Methodology 
16.1 
Study Design 
Dose-limiting toxicity (DLT) is defined as one of the following events occurring 
during the first two cycles of protocol therapy and attributed as possibly, probably, 
or definitely related to protocol therapy. 
- Grade 3+ infusion reactions, acute kidney injury, chronic kidney disease, 
pneumonitis; or Grade 2 infusion reactions, acute kidney injury, chronic 
kidney disease or pneumonitis that does not resolve to Grade 0-1 within 21 
days. 
Primary Endpoint: The primary endpoint is tumor response rate (RR) defined as 
the number of patients whose disease meets the RECIST criteria for a partial (PR) 
or complete (CR) on two consecutive evaluations at least 4 cycles (approximately 84 
days) apart divided by the total number of patients who started protocol treatment 
at the continuation dose level. (That is, patients in the run-in phase assigned the 
dose level chosen enrolled onto the schedule carried forward into the continuation 
phase will be for the continuation phase will be included in the analysis of this 
endpoint). A 90% exact binomial confidence interval for the tumor response rate 
will be constructed.  
16.11 Run-in phase (Phase Ib) 
Six patients will be enrolled onto Schedule 1. If at most one of these 6 patients 
develops a DLT during the first 3 cycles of treatment then Schedule 1 will be 
carried forward to the continuation phase (Phase II). 
If two or more patients enrolled onto Schedule 1 develop a DLT during the first 3 
cycles of treatment, then an additional 6 patients will be treated on Schedule -1 
(See Section 7.2). 
16.12 Continuation phase (Phase II) 
Schedule of pembrolizumab, cryoablation, mDCs, and Prevnar13 will be based 
on the tolerability findings of the run-in phase 
16.13 Adverse Event Stopping Rules 
Enrollment to the trial will temporarily suspended for close examination of 
treatment safety and tolerability: 
- at each instance of a treatment related death; 
- if 2 or more of the first 6 patients develops one of the following toxicities 
that is considered to be possibly, probably, or definitely related to protocol 
treatment: a Grade 4 hematologic toxicity or Grade 3 or worse non-
hematologic toxicity; 
- or at any time after the first 6 patients have been enrolled, if 25% or more 
patients develops one of the following toxicities that is considered to be 
possibly, probably or definitely related to protocol treatment: a Grade 4 
hematologic toxicity or Grade 3 or worse non- hematologic toxicity. 
All toxicity data will be examined to assess whether changes should be made in 
the treatment or eligibility criteria to increase the tolerability of the treatment. All 
modifications to the protocol will be forwarded to the IRB for review. 

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16.2 
Sample Size and accrual rate 
If a cohort of 33 patients is enrolled onto the schedule carried forward into the 
continuation phase, a one-sided exact binomial test of proportions with significance level 
of 0.10 will have a 90% chance of rejecting the null hypothesis that the tumor response 
rate is at most 12% when the alternative hypothesis that the tumor response rate is at least 
30% is true. 
Thus, the minimum sample size for this trial will be 33 patients (if at most 1 DLT is seen 
on Schedule 1in the run-in phase) or a maximum of 39 patients (if 2 or more DLTs are 
seen on Schedule 1 and at most 1 DLT is seen on Schedule -1)  
It is anticipated that 2 patients per month will be enrolled onto this study with a 
temporarily break in enrollment after the first 6 patients are enrolled onto Schedule 
1 (and if necessary after the first 6 patients are enrolled onto Schedule -1) to 
establish a schedule to be carried forward into the continuation phase. 
 
 

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16.3 
Secondary endpoints 
16.31  Safety profile: 
All eligible patients that have initiated treatment will be considered 
evaluable for assessing the safety profile. Toxicities will be graded in terms 
of severity and relationship to study treatment using CTC-CAE criteria. For 
each patient who initiated treatment, the maximum grade of each toxicity 
noted during treatment will be recorded. Frequency tables will be 
constructed by treatment schedule. 
16.32  Feasibility 
A point estimate of the proportion of patients who received both intra-tumoral 
DC injections among those who initiated treatment will be determined. A 95% 
exact binomial confidence interval for this proportion will be constructed.  
16.33 Clinical Benefit Rate (CBR) 
Clinical Benefit Rate (CBR) is defined as the proportion of patients who have 
completed 6 cycles of treatment without disease progression (that is, their 
objective disease status is a CR, PR, or stable for 6 cycles or more) A 90% exact 
binomial confidence interval for this proportion will be constructed.  
16.34  Progression-free survival (PFS) 
Time from randomization to the first 2 consecutive evaluations approximately 6 
weeks apart documenting disease progression. The distribution of progression-
free survival times will be estimated using the Kaplan-Meier approach. 
16.35 Overall survival (OS) 
Time from registration to death due to any cause. The distribution of overall 
survival times will be estimated using the Kaplan-Meier approach 
 
16.4 
Correlative endpoints 
A time series plot of individual patient TILs will be constructed. Also, the change in the 
number of tumor infiltrating lymphocytes (TILs) in tumor biopsies taken prior to and 
following cryoablation and intra-tumoral mDCs on Cycle 2 and on Cycle 3 will be 
determined. For a given cycle, the percent change in the number of TILs following 
cryoablation and intra-tumoral mDCs from pre- cryoablation and intra-tumoral mDCs 
levels will be determined.  
A times series plot of individual patient PD-L1 levels found in tumor biopsies as well as 
blood biopsies will be constructed and visually assess for trends within and between 
biopsy types. Wilcoxon rank sum tests will be used to assess whether the amount of the 
change in PD-L1 levels after first cryoablation and intra-tumor or the PD-L1 levels at 
completion of second cryoablation and intra-tumor differ among those patients who met 
the criteria for clinical benefit (progression-free and on study for at least 6 months) and 
those who do not. 
 
16.5 
Data & Safety Monitoring: 
The study statistician will provide the Mayo Clinic Cancer Center (MCCC) Data Safety 
Monitoring Board (DSMB) a report encompassing accrual, adverse events, and feasibility 

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issues that might be developing at least twice a year. After every cohort of 10 patients is 
treated at the continuation phase dose level, a formal safety check will be carried out. If 3 
or more patients in a given cohort of 10 patients develop a DLT, enrollment will be 
temporarily suspended to assess whether changes should be made in the treatment or 
eligibility criteria to increase the tolerability of the treatment. All modifications to the 
protocol will be forwarded to the IRB for approval. 
 
16.6 
Subset Analyses for Minorities 
16.61 Study availability 
This study will be available to all eligible patients, regardless of gender, race or 
ethnic origin.  
16.62 Statistical analysis by subset 
There is no information currently available regarding differential effects of this 
regimen in subsets defined by race, gender, or ethnicity, and there is no reason to 
expect such differences to exist. Therefore, although the planned analyses will 
look for differences in treatment effect based on racial groupings, the sample size 
is not increased in order to provide additional power for subset analyses.  
16.63 Regional population 
The geographical region served by MCCC has a population which includes 
approximately 3% minorities. Based on prior MCCC studies involving similar 
disease sites, we expect about 3-5% of patients will be classified as minorities by 
race and about 33% of patients will be women. Expected sizes of racial by 
gender subsets are shown in the following table: 
Accrual Targets 
Ethnic Category 
Sex/Gender 
Females 
Males 
Total 
Hispanic or Latino 
0 
1 
1 
Not Hispanic or Latino 
15 
23 
38 
Ethnic Category: Total of all subjects* 
15 
24 
39 
Racial Category 
American Indian or Alaskan Native 
 
 
 
Asian 
 
 
 
Black or African American 
 
 
 
Native Hawaiian or other Pacific Islander 
 
 
 
White 
15 
24 
39 
Racial Category: Total of all subjects* 
15 
24 
39 
 
Ethnic 
Categories: 
Hispanic or Latino – a person of Cuban, Mexican, Puerto Rican, South or Central 
American, or other Spanish culture or origin, regardless of race. The term “Spanish 
origin” can also be used in addition to “Hispanic or Latino.” 
Not Hispanic or Latino 

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Racial 
Categories: 
American Indian or Alaskan Native – a person having origins in any of the original 
peoples of North, Central, or South America, and who maintains tribal affiliations or 
community attachment. 
Asian – a person having origins in any of the original peoples of the Far East, 
Southeast Asia, or the Indian subcontinent including, for example, Cambodia, China, 
India, Japan, Korea, Malaysia, Pakistan, the Philippine Islands, Thailand, and 
Vietnam. (Note: Individuals from the Philippine Islands have been recorded as Pacific 
Islanders in previous data collection strategies.)  
Black or African American – a person having origins in any of the black racial 
groups of Africa. Terms such as “Haitian” or “Negro” can be used in addition to 
“Black or African American.” 
Native Hawaiian or other Pacific Islander – a person having origins in any of the 
original peoples of Hawaii, Guam, Samoa, or other Pacific Islands. 
White – a person having origins in any of the original peoples of Europe, the Middle 
East, or North Africa. 
 

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17.0 
Pathology Considerations/Tissue Biospecimens: 
17.1 
Summary Table of Research Tissue Specimens to be collected for this Protocol 
Correlative Study 
(Section for more 
information) 
Mandatory 
or Optional 
Type of 
Tissue to 
Collect 
Block, Slides, 
Core, etc.  
(# of each to 
submit) 
Cycles 2 
and 3 
Prior 
to 
Cycle 5 
Process 
at site? 
(Yes or 
No) 
Temperature 
Conditions 
for Storage/ 
Shipping1 
Immunohistochemistry, 
immunofluorescence, 
gene sequencing for 
tumor antigen and T cell 
receptors (17.2) 
Mandatory 
tumor in 
normal 
saline 
18 gauge core 
needle biopsy 
(6 cores) 
X 
X 
No 
2 tissue 
samples for 
Cryostore. 
4 tissue 
samples for 
formalin-fixed 
paraffin-
embedded 
(FFPE) 
1. All specimens should be sent to PRC for processing (into FFPE) and then to Dr. Block’s 
laboratory c/o Courtney Erskine (pager 127-14057, Guggenheim 3-23 
 
17.2 
Background and Methodology 
17.21 Analysis of tumor biopsies 
We will examine tumors for tumor-infiltrating lymphocyte and dendritic 
cell phenotype and compare changes prospectively over time. Immuno-
histochemistry, flow, and gene sequencing analysis will be performed. 
Exploratory biomarkers to predict treatment response include but are not 
limited to tumor-infiltrating CD4 and CD8 T cells, NK cells, and PD-
1/PDL1/PDL2 expression on melanoma and stromal cells. Leftover samples 
may be used for genomic or proteomic analysis. 
 
18.0 
Records and Data Collection Procedures 
18.1 
Submission Timetable 
Data submission instructions for this study can be found in the Case Report Form packet. 
18.2 
Event monitoring 
See Section 4.0 and Data Submission Table in the Case Report Form packet for the 
event monitoring schedule. 
18.3 
CRF completion 
This study will use Medidata Rave® for remote data capture (rdc) of all study data. 
18.4 
Site responsibilities 
Each site will be responsible for insuring that all materials contain the patient’s 
initials, MCCC registration number, and MCCC protocol number. Patient’s name 
must be removed. 
18.5 
Supporting documentation 

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This study requires supporting documentation of histologic diagnosis and staging 
which includes the most recent tumor tissue biopsy pathology report, bone marrow 
biopsy report, and imaging reports. To submit these materials, they can be uploaded 
into the Supporting Documentation form in Medidata Rave®. These reports should be 
submitted within 14 days of registration. 
For response to treatment, supporting documentation includes imaging reports and, 
if applicable, a bone marrow biopsy report. 
For patients who progress after study therapy, supporting documentation including 
imaging reports is required. 
18.6 
Labelling of materials 
Each site will be responsible for insuring that all materials contain the patient’s initials, 
MCCC registration number, and MCCC protocol number. Patient’s name must be 
removed. 
18.7 
Incomplete materials 
Any data entered into a form will result in that form being marked as “received.” 
However, missing data will be flagged by edit checks in the database. 
18.8 
Overdue lists 
A list of overdue materials is automatically available to each site at any time. A list of 
overdue materials and forms for study patients will be generated monthly. The listings 
will be sorted by location and will include the patient study registration number. The 
appropriate co-sponsor/participant will be responsible to obtain the overdue material. 
18.9 
Corrections forms 
If a correction is necessary the QAS will query the site. The query will be sent to the 
appropriate site to make the correction in the database and respond back to the QAS. 
 
19.0  
Budget 
19.1 
Costs charged to patient: Routine clinical care, pembrolizumab (including administration) 
19.2 
Tests to be research funded:  
• 
Leukapheresis  
• 
Cryoablation/Anesthesia 
• 
DC and Prevnar13 injections 
• 
Correlative studies outlined in Section 14.0 and 17.0 
19.3 
Other budget concerns: None 
 

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20.0 
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272:903-910 
 
  
 

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Appendix I 
ECOG Performance Status 
 
ECOG PERFORMANCE STATUS* 
Grade ECOG 
0 
Fully active, able to carry on all pre-disease performance without restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out 
work of a light or sedentary nature, e.g., light house work, office work 
2 
Ambulatory and capable of all selfcare but unable to carry out any work 
activities. Up and about more than 50% of waking hours 
3 
Capable of only limited selfcare, confined to bed or chair more than 50% of 
waking hours. 
4 
Completely disabled. Cannot carry on any selfcare. Totally confined to bed or 
chair. 
5 
Dead 
*As published in Am. J. Clin. Oncol.: 
Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E., McFadden, E.T., Carbone, P.P.: 
Toxicity And Response Criteria Of The Eastern Cooperative Oncology Group. Am J Clin Oncol 
5:649- 655, 1982. 
 
The ECOG Performance Status is in the public domain therefore available for public use. To 
duplicate the scale, please cite the reference above and credit the Eastern Cooperative Oncology 
Group, Robert Comis M.D., Group Chair. 
 
From http://www.ecog.org/general/perf_stat.html 
 

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Appendix II 
New York Heart Association Classification of Congestive Heart Failure 
 
NYHA 
Class 
Symptoms 
I 
Cardiac disease, but no symptoms and no limitation in ordinary physical activity, e.g. no 
shortness of breath when walking, climbing stairs etc. 
II 
Mild symptoms (mild shortness of breath and/or angina) and slight limitation during ordinary 
activity. 
III 
Marked limitation in activity due to symptoms, even during less-than-ordinary activity, e.g. 
walking short distances (20–100 m). Comfortable only at rest. 
IV 
Severe limitations. Experiences symptoms even while at rest. Mostly bedbound patients. 
 
Adapted from Dolgin M, Association NYH, Fox AC, Gorlin R, Levin RI, New York Heart Association. 
Criteria Committee. Nomenclature and criteria for diagnosis of diseases of the heart and great vessels. 
9th ed. Boston, MA: Lippincott Williams and Wilkins; March 1, 1994. 
Original source: Criteria Committee, New York Heart Association , Inc. Diseases of the Heart and Blood 
Vessels. Nomenclature and Criteria for diagnosis, 6th edition Boston, Little, Brown and Co. 1964, p 114.