Conditions: Breast Cancer
Keywords: Early stage Resectable Ipilimumab Nivolumab Cryoablation 16-495
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 5
Healthy volunteers: no

Brief summary:
This study is being done to evaluate the safety of combining two strategies called "cryoablation" and "immune therapy" in women with curable early stage breast cancer.

Detailed description:

Arms:
arm 1: Patients will undergo breast biopsy with cryoablation 7-10 days prior to the planned surgery, and ipilimumab with nivolumab administration 1-5 days before cryoablation. All patients will undergo surgery at the pre-determined day defined at the initial surgical consultation. Women will receive ipilimumab and nivolumab 1-5 days prior to US or MRI-guided core biopsy and cryoablation date. Intravenous ipilimumab will be administered as a single 1 mg/kg dose to be infused intravenously over 90 minutes. Intravenous nivolumab will be administered as a single 3 mg/kg dose to be infused intravenously over 60 minutes.

Interventions: intervention 1: Cryoablation intervention 2: Ipilimumab intervention 3: Nivolumab
Intervention descriptions: intervention 1: None intervention 2: None intervention 3: None

Protocol document:
Prot_SAP_000.pdf: 
Memorial Sloan Kettering Cancer Center 
IRB Protocol #: 16-495 A(5) 
Approved: 3-MAY-2017 
Page 1 of 55 
 
 
 
A Pilot Study of Pre-Operative, Single-Dose Ipilimumab, Nivolumab and 
Cryoablation in Early Stage/Resectable Breast Cancer 
PROTOCOL FACE PAGE FOR 
MSK THERAPEUTIC/DIAGNOSTIC PROTOCOL 
Principal Investigator/Department: 
Elizabeth Comen, MD 
Medicine- Breast 
Co-Principal 
Investigator(s)/Department: 
Larry Norton, MD 
Steve Solomon, MD 
Elizabeth Morris, MD 
Medicine- Breast 
Radiology 
Radiology 
Investigator(s)/Department: 
José Baselga, MD, PhD 
Ting Bao, MD, DABMA, MS 
Victoria Blinder, MD 
Jacqueline Bromberg, MD, PhD 
Sarat Chandarlapaty, MD, PhD 
Gabriella D’Andrea, MD 
Chau Dang, MD 
Maura Dickler, MD 
Monica Fornier, MD 
Devika Gajria, MD, MPH 
Teresa Gilewski, MD 
Shari Goldfarb, MD 
Ayca Gucalp, MD 
Neil Iyengar, MD 
Komal Jhaveri, MD 
Christopher Klebanoff, MD 
Diana Lake, MD 
Shanu Modi, MD 
Mary Ellen Moynahan, MD 
Pedram Razavi, MD, PhD 
Mark Robson, MD 
Rachel Sanford, MD 
Andrew Seidman, MD 
Nancy Sklarin, MD 
Lillian Smyth, MD 
Tiffany A. Traina, MD 
Monica Morrow, MD 
Hiram Cody, MD 
Mahmoud El-Tamer, MD 
Mary Gemignani, MD, MPH 
Alexandra Heerdt, MD, MPH 
Melissa Pilewskie, MD 
George Plitas, MD 
Virgilio Sacchini, MD 
Kimberly Van Zee, MD 
Laurie Kirstein, MD 
Andrea Barrio, MD 
Lisa Sclafani, MD 
Medicine- Breast 
Medicine- Breast 
Medicine- Breast 
Medicine- Breast 
Medicine- Breast 
Medicine- Breast 
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Surgery 
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Memorial Sloan Kettering Cancer Center 
IRB Protocol #: 16-495 A(5) 
Approved: 3-MAY-2017 
Page 2 of 55 
 
 
 
Deborah Capko, MD 
Surgery 
Edi Brogi, MD, PhD 
Pathology 
Sujata Patil, PhD 
Epidemiology and 
Biostatistics 
Christopher Comstock, MD 
Majid Maybody, MD 
Jeremy Durack, MD 
Joseph Erinjeri, MD 
Amy Deipolyi, MD 
Yolanda Bryce, MD 
Andrea F. Abramson, MD 
Donna D’Alessio, MD 
D. David Dershaw, MD 
Kimberly Feigin, MD 
Mary C. Hughes, MD 
Maxine Jochelson, MD 
Jennifer Kaplan, MD 
Delia Keating, MD 
Carol Lee, MD 
Janice Sung, MD 
Elizabeth Sutton, MD 
Alan Kotin, MD 
Alisa Thorne, MD 
Eric Kelhoffer, MD 
Anoushka M. Afonso, MD 
Cosmin Gauran, MD 
Sarah L. Bowman, MD 
Radiology 
Radiology 
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Radiology 
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Radiology 
Anesthesiology 
Anesthesiology 
Anesthesiology 
Anesthesiology 
Anesthesiology 
Anesthesiology 
Phillip Wong, MD 
SKI 
Karen Drucker, NP 
Maureen Cannon, NP 
Carrie Mancini, NP 
Valentina Sterlin, NP 
Nursing 
Nursing 
Nursing 
Nursing 
Lauren Wood, RN 
Tina Alano, RN 
Emer Heaney, RN 
Odessa Williams, NP 
Danielle Alleva, NP 
Nicole DeVine, NP 
Catherine Keane, NP 
Renee Wisniewski, NP 
Nursing 
Nursing 
Nursing 
Nursing 
Nursing 
Nursing 
Nursing 
Nursing 
Consenting 
Professional(s)/Department: 
Elizabeth Comen, MD 
Larry Norton, MD 
Medicine- Breast 
Medicine- Breast 


Memorial Sloan Kettering Cancer Center 
IRB Protocol #: 16-495 A(5) 
Approved: 3-MAY-2017 
Page 3 of 55 
 
 
 
José Baselga, MD, PhD 
Ting Bao, MD, DABMA, MS 
Victoria Blinder, MD 
Jacqueline Bromberg, MD, PhD 
Sarat Chandarlapaty, MD, PhD 
Gabriella D’Andrea, MD 
Chau Dang, MD 
Maura Dickler, MD 
Monica Fornier, MD 
Devika Gajria, MD, MPH 
Teresa Gilewski, MD 
Shari Goldfarb, MD 
Ayca Gucalp, MD 
Neil Iyengar, MD 
Komal Jhaveri, MD 
Christopher Klebanoff, MD 
Diana Lake, MD 
Shanu Modi, MD 
Mary Ellen Moynahan, MD 
Pedram Razavi, MD, PhD 
Mark Robson, MD 
Rachel Sanford, MD 
Andrew Seidman, MD 
Nancy Sklarin, MD 
Lillian Smyth, MD 
Tiffany A. Traina, MD 
Stephen Solomon, MD 
Jeremy Durack, MD 
Majid Maybody, MD 
Joseph Erinjeri, MD 
Amy Deipolyi, MD 
Yolanda Bryce, MD 
Christopher Comstock, MD 
Andrea F. Abramson, MD 
Donna D’Alessio, MD 
D. David Dershaw, MD 
Kimberly Feigin, MD 
Mary C. Hughes, MD 
Maxine Jochelson, MD 
Jennifer Kaplan, MD 
Delia Keating, MD 
Carol Lee, MD 
Janice Sung, MD 
Elizabeth Sutton, MD 
Monica Morrow, MD 
Hiram Cody, MD 
Medicine- Breast 
Medicine- Breast 
Medicine- Breast 
Medicine- Breast 
Medicine- Breast 
Medicine- Breast 
Medicine- Breast 
Medicine- Breast 
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Memorial Sloan Kettering Cancer Center 
IRB Protocol #: 16-495 A(5) 
Approved: 3-MAY-2017 
Page 4 of 55 
 
 
 
Mahmoud El-Tamer, MD 
Mary Gemignani, MD, MPH 
Alexandra Heerdt, MD, MPH 
Melissa Pilewskie, MD 
George Plitas, MD 
Virgilio Sacchini, MD 
Kimberly Van Zee, MD 
Laurie Kirstein, MD 
Andrea Barrio, MD 
Lisa Sclafani, MD 
Deborah Capko, MD 
Surgery 
Surgery 
Surgery 
Surgery 
Surgery 
Surgery 
Surgery 
Surgery 
Surgery 
Surgery 
Surgery 
 
 
 
 
Principal Investigator/Department: 
Co-Principal 
Investigator(s)/Department: 
Investigator(s)/Department: 
Consenting 
Professional(s)/Department: 
 
Please Note: A Consenting Professional must have completed the mandatory Human 
Subjects Education and Certification Program. 
Memorial Sloan Kettering Cancer Center 
1275 York Avenue 
New York, New York 10065 


Memorial Sloan Kettering Cancer Center 
IRB Protocol #: 16-495 A(5) 
Approved: 3-MAY-2017 
Page 5 of 55 
 
 
 
Table of Contents 
1.0 
PROTOCOL SUMMARY AND/OR SCHEMA ..................................................................... 6 
2.0 
OBJECTIVES AND SCIENTIFIC AIMS .............................................................................. 8 
3.0 
BACKGROUND AND RATIONALE .................................................................................... 8 
4.0 
OVERVIEW OF STUDY DESIGN/INTERVENTION........................................................... 20 
4.1 
Design ........................................................................................................................... 20 
4.2 
Intervention .................................................................................................................... 21 
5.0 
THERAPEUTIC/DIAGNOSTIC AGENTS .......................................................................... 22 
6.0 
CRITERIA FOR SUBJECT ELIGIBILITY .......................................................................... 23 
6.1 
Subject Inclusion Criteria ............................................................................................... 23 
6.2 
Subject Exclusion Criteria .............................................................................................. 24 
7.0 
RECRUITMENT PLAN ..................................................................................................... 25 
8.0 
PRETREATMENT EVALUATION ..................................................................................... 25 
9.0 
TREATMENT/INTERVENTION PLAN .............................................................................. 26 
10.0 EVALUATION DURING TREATMENT/INTERVENTION .................................................. 29 
11.0 TOXICITIES/SIDE EFFECTS ............................................................................................ 29 
12.0 CRITERIA FOR THERAPEUTIC RESPONSE/OUTCOME ASSESSMENT ...................... 31 
13.0 CRITERIA FOR REMOVAL FROM STUDY ...................................................................... 33 
14.0 BIOSTATISTICS ............................................................................................................... 33 
15.0 
RESEARCH PARTICIPANT REGISTRATION AND RANDOMIZATION PROCEDURES34 
15.1 Research Participant Registration .................................................................................. 34 
15.2 Randomization............................................................................................................... 34 
16.0 DATA MANAGEMENT ISSUES ....................................................................................... 34 
16.1 Quality Assurance ......................................................................................................... 34 
16.2 Data and Safety Monitoring ........................................................................................... 35 
17.0 PROTECTION OF HUMAN SUBJECTS ........................................................................... 35 
17.1 Privacy .......................................................................................................................... 35 
17.2 Serious Adverse Event (SAE) Reporting ........................................................................ 36 
17.2.1 
 37 
18.0 INFORMED CONSENT PROCEDURES ........................................................................... 42 
19.0 REFERENCES ................................................................................................................. 43 
20.0 APPENDICES ................................................................................................................... 48 


Memorial Sloan Kettering Cancer Center 
IRB Protocol #: 16-495 A(5) 
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1.0 
PROTOCOL SUMMARY AND/OR SCHEMA 
This is a pilot study evaluating the safety and tolerability of pre-operative, single-dose 
ipilimumab, nivolumab and cryoablation in patients with early stage/resectable breast cancer. 
The central idea for this project is to make a major advance for women with early stage breast 
cancer by augmenting an effected individual’s immune response to their recently diagnosed, 
resectable breast cancer prior to definitive surgical management, thereby conferring long-term 
immunity to the primary breast tumor, and ultimately, cure. 
A pilot study evaluating pre-operative, single-dose ipilimumab and/or cryoablation in patients 
with early stage/resectable breast cancer was recently completed at MSKCC and met the 
primary endpoint of safety/tolerability. In this study we are building on the success of this 
pilot study and the improvements observed in other tumor types with combined checkpoint 
blockade, to further evaluate the safety of a therapeutic strategy whereby an individual’s 
immune system is stimulated to recognize and respond to the specific features of their own 
tumor. Our strategy combines two interventions: first we induce activation and maturation of 
dendritic cells and tumor-specific T cells by cross-presentation of tumor antigens via local 
destruction of tumor tissue by cryoablation. Second, we administer ipilimumab, a CTLA4 
blocking antibody, and nivolumab, an antibody against programmed death receptor-1 (PD-1), 
that together enhance the magnitude and potency of the tumor specific T cell response. 
One study arm of 6 patients will be enrolled. At the time of the initial surgical consultation, 
potentially eligible candidates will be identified by their surgeon and referred for an 
appointment with the Principal Investigator (Dr. Elizabeth Comen) or a consenting medical 
oncologist and an appointment with consenting professionals from radiology, if feasible, to 
evaluate eligibility for cryoablation. Prior to protocol intervention, consent will take place at 
the first available appointment with a consenting professional. Consenting, eligible women 
will then be registered to receive single-dose ipilimumab with nivolumab and cryoablation. 
All study interventions (including the cryoablation, drug administration, and imaging dates) 
will be defined by the pre-determined, non-study surgical date. 
The purpose of the study is to determine the safety of pre-operative ipilimumab, nivolumab 
and cryoablation in patients with biopsy proven resectable breast cancer. If at least 5 of the 6 
patients are without an adverse event (AE) necessitating delay in surgery, the regimen will be 
considered safe/tolerable for further study. Secondary aims of this study are to explore and 
characterize pre- and post-intervention peripheral and tumor responses to ipilimumab, 
nivolumab and cryoablation. 


Memorial Sloan Kettering Cancer Center 
IRB Protocol #: 16-495 A(5) 
Approved: 3-MAY-2017 
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At First Visit For Potential Study Candidates in Surgery Clinic 
• 
Non-study surgery date is scheduled 
• 
Non-study pre-surgical testing date is scheduled 
• 
Informed consent is provided 
• 
Initial Med Onc and Cryoablation Evaluation appointments, if feasible, are scheduled to occur as soon 
as possible 
Med Onc and Cryoablation Evaluation Appointments 
• 
Potential study candidates evaluated for eligibility 
• 
If eligible then: 
o Informed consent signed 
o Core biopsy (+ cryoablation) date set for 7-10 days before surgery 
o Ipilimumab + nivolumab date set for 1-5 days before core biopsy/cryoablation date 
o Post-op follow-up appointment with Med Onc set 
Safety assessment 2-3 weeks post-surgery (coordinated with off-study surgery follow-up appt if 
feasible) and then every 2-3 weeks thereafter until at least 12 weeks post ipilimumab and nivolumab 
date. # 
All Arms: Research bloods 30 (+/-10) days after surgery.* 
 
Med Onc and Cryoablation Evaluation Appointments 
 
 
 
 
 
 
 
* Indicates time points for obtaining Research blood samples. Baseline study bloods may be drawn with routine pre- 
surgical testing or on the day of ipilimumab injection (if applicable), prior to drug administration. 
# Indicates time points for obtaining Safety blood Samples 
Eligibility/Baseline study bloods (coordinated with routine Pre-Surgical Testing whenever feasible) * 
Core Biopsy + Cryo Date* (7-10 days before surgery) 
Core biopsy x ≥3 
Cryoablation 
Ipilimumab + Nivolumab Date* (1-5days before biopsy/cryoablation) 
Appt. with Med Onc 
Ipilimumab + nivolumab administered 
Surgery Date* 


Memorial Sloan Kettering Cancer Center 
IRB Protocol #: 16-495 A(5) 
Approved: 3-MAY-2017 
Page 8 of 55 
 
 
2.0 
OBJECTIVES AND SCIENTIFIC AIMS 
Primary Objective 
• 
To assess the safety and tolerability of pre-operative ipilimumab, nivolumab and intratumoral 
cryoablation in patients with resectable breast cancer. 
 
Exploratory Objectives: 
To evaluate the effect of cryoablation and ipilimumab/nivolumab administration on intratumoral and 
serum lymphocyte phenotypes, intratumoral and serum dendritic cells (DCs), serum cytokines, 
disease related biomarkers, antibody responses to selected antigens, and humoral and cellular 
responses to tumor antigens and recall non-tumor antigens. 
• To assess the effect of treatment on ratios of specific intratumoral and serum T cell populations 
including CD4+FOXP3+ T-regulatory cells (Tregs) and CD8+Effector cells. 
• To explore T cell diversity and clonality changes by deep sequencing 
• To assess the pharmacodynamic effects of ipilimumab, nivolumab and cryoablation on Absolute 
Lymphocyte Count (ALC) and absolute T cell count (see below). 
 
3.0 
BACKGROUND AND RATIONALE 
Our goal is to make a major advance for persons with early stage breast cancer by augmenting the 
effected individual’s immune response to their recently diagnosed, resectable breast cancer prior to 
definitive surgical management, thereby conferring long-term immunity to the primary breast tumor, 
and ultimately, cure. 
 
Breast cancer is a global public health burden with more than 1 million new cases diagnosed 
worldwide and approximately 200,000 new cases diagnosed in the United States each year.1, 2 For 
most patients in the developed world, breast cancer is diagnosed at an early stage disease. In fact, the 
proportion of early stage diagnoses is increasing, largely as a result of significant improvements in 
screening and public education programs.3 However, despite ongoing therapeutic innovations, 
approximately one-third of women with early stage disease still develop distant metastases and 
ultimately die of metastatic breast cancer.1 
 
Some of the most notable recent therapeutic innovations in breast cancer reflect improvements in the 
ability to recognize biologically distinct breast cancer subsets and ultimately, to tailor treatment 
recommendations to these features. Currently, biologic subsets are primarily defined by hormone 
receptor and HER2 status outside of clinical trials. However, the subtyping of breast cancer 
represents both an advance (better targeted therapies and improved patient selection) and a challenge 
(ever smaller subsets make drug development difficult). Therefore, a therapeutic strategy that 
simultaneously utilizes the individual (and possibly unique) aspects of each patient’s tumor combined 
with greater generalizability (to the entire patient population) could represent a significant advance. 
Our goal is, therefore, to combine the biological promise of personalized therapy (via stimulation of 
the effected individual’s immune system) with a standard therapeutic intervention (cryoablation with 
ipilimumab/nivolumab administration) thereby overcoming the issue of heterogeneity and potentially 
increasing the probability of cure. 


Memorial Sloan Kettering Cancer Center 
IRB Protocol #: 16-495 A(5) 
Approved: 3-MAY-2017 
Page 9 of 55 
 
 
To induce the desired immune response, we selected local thermal ablation of the tumor using 
cryoablation specifically. This approach has emerged as alternative to surgical resection to treat many 
types of inoperable tumors including kidney, liver, adrenal, lung, and others and is therefore 
clinically feasible.5 Cryoablation involves the insertion of a probe into a tumor nodule in order to 
administer tissue ablative freezing temperatures.6 This will lead to immediate disruption of the tissue 
structure and cellular damage. The necrosis observed in the ablated lesion is mediated by mechanical 
disruption from crystallization, osmotic changes, and vascular stasis. The necrotic tumor lesion 
remains within the body, and the release of the tumor antigens by the dying cells could activate a 
specific immune response through antigen presentation by DCs to T-cells. Several feasibility studies 
have indicated that thermal ablation can be performed safely in small breast cancers and effectively 
eradicate breast cancer cells.7 
 
To further amplify the immune response, we plan to use two fully human monoclonal antibodies that 
blocks the normal host’s attenuation of immunity. Ipilimumab specifically binds to human cytotoxic 
T lymphocyte antigen 4 (CTLA4) resulting in immuno-stimulation. Treatment with ipilimumab has 
resulted in clinically important and durable tumor responses in several solid malignancies, including 
prostate, bladder, and renal cell carcinoma. However, it has been most extensively studied in 
malignant melanoma with overall response rates of 10-20% demonstrated in multiple studies and 
significant survival benefits when compared with a gp100 vaccine.11,12 The agent is generally well 
tolerated with few grade 3/4 adverse events.8,9 Nivolumab, an anti-PD1 antibody, has demonstrated 
activity not only in melanoma but also in multiple solid tumors.58-60 Furthermore, improved responses 
have been observed in untreated metastatic melanoma with the combination of ipilimumab and 
nivolumab when compared with either drug alone.61 
 
The combination of tumor cryoablation and immunomodulation has been shown in animal models, 
by our colleagues, to generate such a systemic anti-tumor response.4 Specifically, local tumor 
destruction with cryoablation can lead to exposure of DCs with sufficient quantities of tumor antigens 
to ultimately lead to DC maturation and activation. Recently, our collaborators (Dr. James Allison’s 
group at MSKCC) showed that combination therapy with cryoablation and CTLA4 blockade 
successfully mediated rejection of metastatic prostate cancer lesions and prevented the growth of 
secondary tumors in preclinical murine models.4 Furthermore, in a pilot study of single-dose pre- 
operative ipilimumab and/or cryoablation in women with early stage breast cancer completed at 
MSKCC, the combination of CTLA4 blockade with cryoablation was safe and tolerable 
Cryoablation and single dose ipilimumab at 10mg/kg were safe/well tolerated alone and in 
combination. The primary endpoint of this trial was reached, with all 18 patients receiving standard- 
of-care surgical mastectomy without delay. No treatment-associated grade III/IV adverse events, and 
one unrelated grade III/IV adverse event, were recorded on study. One subject from group C (cryo+ 
ipi) developed a grade 3maculopapular rash originating at the site of the mastectomy drain, which 
erupted hours after mastectomy and spread to involve the face, neck and chest. Based upon the 
timing, appearance, and distribution of the rash, the treating dermatologist concluded that the rash 
was related to peri-operative anti-septic wash and/or cephalexin administration, rather than the study 
intervention. Skin biopsy revealed mixed-pattern dermatitis with eosinophilic infiltrate, consistent 
with a hypersensitivity reaction. Combination therapy was also associated with sustained peripheral 
elevations in Th1-type cytokines, proliferating CD4+ and CD8+ T cells, and ICOS expression as well 
as post-treatment proliferation of T-effector cells relative to T-regulatory cells within the tumor.56 


Memorial Sloan Kettering Cancer Center 
IRB Protocol #: 16-495 A(5) 
Approved: 3-MAY-2017 
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Given the safety of ablation techniques in the early stage breast cancer setting, the efficacy and 
tolerability of ipilimumab with nivolumab administration in other solid tumors, and the success of a 
pilot study evaluating the safety/tolerability of pre-operative ipilimumab at 10mg/kg with 
cryoablation in early stage breast cancer, we aim to confirm the safety/tolerability of cryoablation, 
ipilimumab and nivolumab using the model of the successfully completed pilot study. 
 
This project directly translates the pre-clinical data developed at our institution demonstrating that 
cryoablation combined with CTLA4 blockade synergize to mediate tumor rejection into clinical 
practice, and the clinical data generated at our institution demonstrating the safety of single dose 
ipilimumab with cryoablation in the pre-operative setting. We hypothesize that by exploiting the 
biology of the tumor and the immune system of the effected individual, that this strategy will confer 
long-term immunity, and ultimately cure. Our immediate goal is to study the safety and tolerability of 
pre-operative cryoablation in combination with single dose ipilimumab and nivolumab. Because of 
toxicity observed with ipilimumab and nivolumab administered in combination in other settings, each 
drug will be administered at a conservatively low dose of 1 mg/kg in women with early stage breast 
cancer (notably, ipilimumab was previously administered at 10mg/kg in the pilot study).61 Our 
longer-term goal is to develop this strategy as a curative treatment for women with early stage breast 
cancer 
Principles of the Proposed Therapies 
Thermal Ablation in Cancer: 
Thermal ablation treatments such as cryoablation emerged as an alternative to surgical resection and 
as treatment for many inoperable tumors including breast, prostate, kidney, liver, adrenal and lung.13 
Cryoablation, or tissue destruction by freezing, involves percutaneous placement of a needle-like 
probe(s) through the skin and into the tumor under image guidance whereby the lesion is ablated in 
situ. The treatment success is dependent on achieving adequate freezing margin of the ice ball 
containing the tumor. The temperature necessary for reliable cryoablation induced cell death is - 
20oC.14 
In a series of in vivo animal experiments we compared the thermal map around a commercially 
available cryoprobe to the ice ball edge using thermocouple temperature probes. This revealed that 
reliable -20oC temperatures for porcine tissue were achieved at approximately 10mm inside the ice 
ball edge. We also observed that the treatment effect using thermal mapping varies with different 
organs. Cryoablation zones were larger in kidney than lung and liver and felt to represent variations 
in vascular perfusion relative to the tissue density of these different organs producing the 
phenomenon commonly referred to as the “thermal sink” effect, described by the Pennes bioheat 
transfer equation.15 
 
The procedure induced cell death is due to the immediate and direct damage to the cell membranes 
and cellular organelles through the formation of the ice crystals. Thereafter, thrombosis of small 
vessels is observed in the ablation bed within hours to days following the procedure. Cryoablation, 
unlike other ablative tools such as radiofrequency ablation (RFA), allows the operator to visualize the 
complete ice ball on intra-procedural images. This permits greater confidence that the tumor is 
confined within the treatment zone. Additionally, when comparing our institutional experience with 
cryotherapy versus RFA, it was noted that the procedural pain was significantly less with 


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cryotherapy.19 This experience primarily reflects renal ablation models; however, it is reasonable to 
expect that these advantages also apply in the ablation of the breast. 
 
The ice ball from cryoablation is well visualized on MR images as an area of low signal. This can 
allow intra-procedural monitoring of the treatment to ensure adequate coverage of the tumor and 
ensure avoidance of injury to nearby structures such as skin. Breast cancer lesions are also well seen 
on MR images allowing an ideal target for cryoablation needle placement. The same devices that are 
used in the MR environment to allow breast biopsy and localization will be available to guide 
cryoablation probe placement. 
 
In addition to its effect on the localized tumor, it has been hypothesized that cryoablation can induce 
systemic anti tumor response that results in regression of distant metastatic lesions. Following 
ablation the necrotic tumor remains within the body, the vigorous inflammatory response to the 
necrotic tissue with the sustained release of tumor antigens from the dying cells, which are captured 
by the antigen presenting cells (APCs), subsequently this will lead to maturation activation and 
migration of the APCs to the draining lymph nodes (LNs) where eventually the activation of tumor 
specific T cells occurs.16,17 The immunological effect of cryoablation has been documented in many 
preclinical and clinical models. It was first demonstrated by Shulman and colleagues who described 
the production of anti-epididymal spermatozoal antibodies in rabbits after freezing.18 Also Levy et al 
showed that combining cryoablation with cyclophosphamide induces a potent anti tumor response in 
murine model of metastatic colon cancer.19 Clinically, Ablin et al, described remission of metastatic 
lesions in patients with prostate cancer whose primary tumors were treated with cryotherapy.20 More 
recently, Dr. James Allison’s laboratory has demonstrated that the combination of cryoablation and 
immune modulation (such as with CTLA4 blockade) can lead to rejection of tumor in murine models 
of prostate cancer (see below section on the rationale for the combination).4 
 
Cryoablation in Breast cancer 
With recent improvements in breast cancer screening and diagnosis, most newly diagnosed breast 
cancers in the developed world are small and non-palpable. Consequently, a number of lumpectomy 
alternatives, including mini-invasive ablation techniques, have been explored. For example, methods 
such as RFA and cryosurgery for the in situ ablation of breast cancer, indicate that these procedures 
are safe and well tolerated procedures.21 
Preclinical data from mouse models of mammary carcinoma showed that adoptive transfer of T cells 
from cryo-ablated tumor draining lymph nodes generated a significant increase in the fraction of 
tumor specific T-cells and anti-tumor activity. This increased activity ultimately translated into 
significant reduction of pulmonary metastases when compared to adoptive transfer of T-cells from 
mouse lymph nodes after surgical tumor excision or no treatment. These data demonstrate the 
effectiveness of the “cryo-immunotherapy” approach in the adjuvant treatment in breast cancer17 
Utilizing more modern techniques, researchers in Japan demonstrated the ability to manage locally 
advanced breast cancer with cryosurgery. In 49 patients with advanced or recurrent breast cancer 
treated with cryosurgery, alleviation of pain, control of hemorrhage, and reduction of tumor bulk 
were reported along with impressive survival benefits.22 Cryoablation is also used safely in treating 
benign breast tumors such as fibroadenomas.23 


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More recently, Sabel et al conducted a phase I multi institutional study to determine the safety and 
feasibility of cryoablation in patients with early stage breast cancer.24 Twenty-nine patients with 
tumors ≤ 2 cm treated with cryoablation followed by 1 to 4 weeks later by surgical resection. 
Cryoablation was successfully performed in an office-based setting with only local anesthesia. There 
were no complications to the procedure or post-procedural pain requiring narcotic pain medications. 
Cryoablation successfully destroyed 100% of cancers <1.0 cm. For tumors between 1.0 and 1.5 cm, 
this success rate was achieved only in patients with invasive ductal carcinoma without a significant 
ductal carcinoma-in-situ (DCIS) component. Morin et al explored the use of MRI-guided 
cryoablation in 25 patients.25 Complete pathologic ablation was achieved in only 13 of 25 patients; 
however, this study included much larger tumors, ranging from 1.2cm to 6cm (mean = 2.8cm). The 
authors were able to accurately predict success using a combination of MR images and post- 
procedure scinti-mammography and no delays in scheduled mastectomies where reported. Both of the 
aforementioned studies concluded that cryoablation is safe and well-tolerated in early stage breast 
cancer. 
 
Rationale for Immune Therapy with Ipilimumab and Nivolumab 
Full activation of naive T cells requires not only stimulation of the antigen receptor by peptide/major 
histocompatibility complexes, but also by co-stimulatory signals.27 These signals are provided by the 
engagement of CD28, which is constitutively expressed on T-cell surfaces, with CD80 and CD86 
molecules, which are present on APC. CD28-B7 co-stimulatory signals are critical for the induction 
of T-cell proliferation, cytokine secretion, and effector functions which ultimately translate into 
clinical effects (Kearney 1995). CTLA4 is an activation-induced T-cell surface molecule that binds 
CD80 and CD86 with greater avidity than CD28. CTLA4 ligation down-regulates T-cell responses, 
which results in an abrogation of the clinical effects provided by T-cell activation.29 Blockade of 
CTLA4/B7 interactions results in an increase in T-cell activation. 
Perhaps the most convincing demonstration of the down-regulatory role of CTLA4 came from 
examination of mice with a null mutation.30 These CTLA4 knockout mice appear to have 
spontaneously activated T cells evident at approximately 1 week after birth, followed by rampant 
lymphoproliferation and lymphadenopathy.31 These mice die at approximately 3 weeks of age, either 
as a result of polyclonal T cell expansion and tissue destruction or as a result of toxic shock resulting 
from lymphokine production by the T cells. Since thymocyte differentiation and selection proceed 
normally in CTLA4-deficient mice, the rampant T cell expansion that occurs in the mice indicates 
that CTLA4 plays a critical role in down-regulating T cell responses in the periphery.32 Many 
preclinical studies demonstrated that treatment with anti-murine CTLA4 monoclonal antibody 
resulted in tumor rejections, for example CTLA4 blockade led to rejection of immunogenic colon 
carcinoma and fibrosarcoma murine models.33 However, rejection of less immunogenic tumors, such 
as murine melanoma B16, was achieved only when CTLA4 blockade was combined with vaccines or 
radiation therapy. 
 
Ipilimumab is human monoclonal antibody specific to human cytotoxic T lymphocyte antigen 4 
(CTLA4) with immuno-stimulating properties. Treatment with ipilimumab resulted in clinically 
important and durable tumor responses in several malignancies, including melanoma, prostate cancer, 
and renal cell carcinoma but most extensively studied tumor type has been malignant melanoma and 
result of many clinical trials demonstrating an overall response rate of 10-20%.10 More recently, a 


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phase 3 study showed that ipilimumab, either alone or with gp100 vaccine, improved overall survival 
compared with gp100 alone in previously treated metastatic melanoma.11 Specifically, a 3.8 month 
increase in overall survival and a doubling of the 1 year survival rate was demonstrated in favor of 
the ipilimumab-treated group. 
PD-1 (or CD279) is a member of the CD28 family of T-cell costimulatory receptors that include 
immunoglobulin super family members CD28, CTLA-4, inducible co-stimulator (ICOS), and B and 
T-lymphocyte attenuator (BTLA). PD-1 is highly expressed on activated T cells and B cells. PD-1 
expression can also be detected on memory T-cell subsets with variable levels of expression. Two 
ligands specific for PD-1 have been identified: PD-L1 (also known as B7-H1 or CD274) and PD-L2 
(also known as B7-DC or CD273). PD-L1 and PD-L2 have been shown to down-regulate T-cell 
activation upon binding to PD-1 in both murine and human systems.69 The interaction of PD-1 with 
its ligands, PD-L1 and PD-L2, that are expressed on antigen-presenting cells (APC) and dendritic 
cells (DC), transmits negative regulatory stimuli to down-modulate the activated T-cell immune 
response. The absence or inhibition of PD-1 in murine models has resulted in the development of 
various autoimmune phenotypes and autoimmune diseases. Taken together, these results suggests that 
inhibition of PD-1 binding to its ligands has the potential to activate T-cell responses. Since these 
responses are variable and dependent upon various host genetic factors, PD-1 deficiency or inhibition 
is not accompanied by a universal loss of tolerance to self antigens. 
Tumors can express tumor-specific antigens as a result of mutational burden and ongoing immune 
surveillance is believed to control the development of many tumors. Tumor progression may depend 
upon acquisition of mechanisms which permit them to evade an effective immune response. One such 
mechanism of evasion may be the expression of ligands which engage inhibitory receptor(s) on anti- 
tumor T-cells of many tumors. PD-L1 expression has been found on a number of tumors including 
breast cancer, and may be a mechanism by which tumors can directly engage PD-1 to evade an 
effective anti-tumor immune response.70 Expression of INF-γ by T-cells is known to induce PD-L1 
expression in tumors. PD-L1 expression has been associated with poor prognoses multiple cancers, 
and binding of PD-L1 to PD-1 on T-cells may limit effective immune responses. Co-localization of 
lymphoid cell infiltrates and PD-L1 staining has been observed in human melanoma.71 
Studies in multiple tumor models using a chimeric murine anti-mouse PD-1 antibody showed that 
PD-1 blockade has anti-tumor activity. Blocking PD-1 in PD-L1+ tumors may reverse the 
inactivation of tumor-specific effector T-cells at the tumor site as well activate anti-tumor responses 
that are limited by PD-L1 expression on “host” DC or APC. The anti-tumor effects of anti-PD-1 
observed in several murine models suggest that both PD-L1+ and PD-L1- tumors may be targeted 
using this approach. In addition, in several tumor models in which anti-PD-1 has proved ineffective, 
PD-1 blockade can be combined with vaccines, tumor-destructive modalities, or other 
immunomodulatory antibodies for improved therapeutic efficacy.72,73 


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Nivolumab, an anti-programmed death-1 (PD1) antibody has demonstrated activity not only in 
melanoma but also in multiple solid tumors.58-60 Furthermore, improved responses have been 
observed in untreated metastatic melanoma with the combination of ipilimumab and nivolumab when 
compared with either drug alone.61 
 
 
Possible toxicities with ipilimumab may include: 
- 
Infusion reactions: Fever, chills, sweating, shakes, itching, rash, hyper- or hypotension, 
difficulty breathing, flushing, nausea, vomiting. It is likely that most infusion-related adverse 
events will occur within the first 24 hours after beginning the infusion, and may be treated by 
slowing or interruption of the infusion, or with supportive treatment as indicated in Section 
11.3. 
 
- 
Immune-related Adverse Events (irAE): Many of the adverse events considered related to 
ipilimumab are immune in nature and presumably a consequence of the intrinsic biological 
activity of ipilimumab. In prior ipilimumab clinical studies, an irAE was defined as any 
adverse event associated with drug exposure and consistent with an immune-mediated event. 
Disease progression, infections and other etiologic causes were ruled out or deemed unlikely 
as contributing to the event. Supportive data, such as autoimmune serology tests or biopsies, 
were considered to be helpful but not necessary to categorize an event as an irAE. Events of 
unclear etiology which were plausibly immune mediated were conservatively categorized as 
irAEs even if serologic or histopathology data were absent. These irAEs likely reflect a loss 
of tolerance to some self-antigens or an unchecked immune response to gut or skin flora. 
Some breakthrough of immunity may be inseparably linked to the clinical antitumor activity 
of ipilimumab. Immune-related adverse events predominately involve the GI tract, endocrine 
glands, liver or skin. Experience with ipilimumab indicates that irAEs are typically low grade 
and self-limited, with few exceptions; irAEs were clinically manageable and reversible with 
supportive care or corticosteroids (see treatment algorithm Appendix 2-6). Notably, IrAE 
typically occur after multiple doses of ipilimumab. In this study a single 1mg/kg dose of 
ipilimumab is being administered, which make irAE unlikely. This is supported by a recently 
reported study wherein presurgical ipilimumab administration in patients with bladder cancer 
was tolerable and safe. 12 
 
- 
Gastrointestinal irAEs: The most common grade 3 or greater irAE involved the lower GI 
tract and clinically manifested as diarrhea or hematochezia. Diarrhea resulting from treatment 
with ipilimumab ranged from mild to severe and was life-threatening in some cases. Some 
cases of diarrhea began as mild and became very severe (Beck 2006). Serious GI irAEs 
mostly involved diarrhea or colitis (e.g. hemorrhagic colitis that was unresponsive to medical 
management and necessitated colectomy, bowel wall perforations associated with and without 
ipilimumab-induced colitis). Diarrhea/colitis is one of the most serious AEs and must be 
evaluated in a timely fashion (see diarrhea management algorithm in Appendix 4). All 
patients with diarrhea will be seen by physician and evaluated with a CT Scan/colonoscopy if 
necessary. 


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- 
The first onset of GI irAEs generally occurred within the first 12 weeks of treatment and was 
generally reversible. Bowel wall biopsies demonstrated a pleomorphic infiltrate, including 
many lymphocytes, consistent with colitis due to an autoimmune process. In addition, upper 
GI tract involvement including ileitis, duodenitis, and esophagitis have been reported. 
 
- 
Gastrointestinal perforation in association with narcotic use: Isolated cases of intestinal 
perforation in association with narcotic use have been reported. The use of narcotics may 
potentially mask GI symptoms or delay detection of their relevant complications, such as 
bowel perforation. 
 
- 
Inflammatory Hepatotoxicity: Immune-related hepatic dysfunction, including hepatitis or 
abnormal liver function tests (LFTs) attributed to ipilimumab therapy, has been reported. 
Subjects may develop elevations in LFTs in the absence of clinical symptoms. Inflammatory 
hepatotoxicity includes non-infectious hepatitis (e.g., autoimmune hepatitis). Hepatic irAEs 
generally occur in the first 12 weeks of treatment and are primarily reversible (see 
hepatotoxicity management algorithm in Appendix 5). 
 
- 
Hypophysitis/Hypopituitarism and Other Endocrine Conditions: 
Hypophysitis/hypopituitarism, clinically manifested by fatigue, has been reported. Most 
subjects with hypopituitarism presented with nonspecific complaints such as fatigue, 
confusion, visual disturbance, or impotence. Some had headache as the predominant 
presentation. The majority of subjects with hypopituitarism demonstrated enlarged pituitary 
glands based on brain magnetic resonance imaging. Low adrenocorticotropic hormone and 
cortisol were the most common biochemical abnormality reported; low thyroid stimulating 
hormone, testosterone, or prolactin was also reported in some subjects.50 
The first onset of endocrine irAEs typically occurred between weeks 6 and 12 of treatment. 
Hypophysitis/hypopituitarism was controlled with appropriate hormone replacement therapy 
and may be dose related. Other endocrine glands may be targets of ipilimumab-related 
toxicity, and rare cases of primary adrenal insufficiency, hyperthyroidism and hypothyroidism 
were reported (see endocrinopathy management algorithm in Appendix 6). 
- 
Rash and Other Skin Conditions: Rash was one of the most common irAEs, and most cases 
were Grade 1 or 2 in intensity; pruritus has also been reported.51 
When biopsied, pleomorphic infiltrates were noted in the skin. Skin irAEs were generally 
reversible. Some subjects reported vitiligo associated with ipilimumab administration. 
 
- 
Ocular Inflammation: Ocular inflammation, manifested as Grade 2 or Grade 3 episcleritis or 
uveitis, was associated with concomitant diarrhea in a few subjects and occasionally occurred 
in the absence of clinically apparent GI symptoms.52 Most of the subjects with ocular 
inflammation and known outcome recovered or improved with or without corticosteroid 
therapy with a median duration of approximately 6 days (range: 5 to 23 days). 


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Possible toxicities with Nivolumab may include: 
CA209003 is a Phase 1 open label, multiple dose escalation study in 306 subjects with select 
previously treated advanced solid tumors, including melanoma, RCC, NSCLC, colorectal cancer, and 
hormone-refractory prostate cancer. Subjects received nivolumab at doses of 0.1, 0.3, 1, 3 or 10 
mg/kg intravenously every 2 weeks, up to a maximum of 2 years of total therapy. As of 18-Mar- 
2013, a total of 306 melanoma subjects were treated with nivolumab in the dose range of 0.1 - 10 
mg/kg. 
 
No maximal tolerated dose was identified in CA209003. The incidence, severity and relationship of 
AEs were generally similar across dose levels and tumor types. Nivolumab related AEs of any grade 
occurred in 75.2% of subjects. Of the 306 treated subjects, 303 (99.0%) subjects have at least 1 
reported AE regardless of causality. The most frequently reported AEs were fatigue (54.9%), 
decreased appetite (35.0%), diarrhea (34.3%), nausea (30.1%), and cough (29.4%). 
 
Treatment-related AEs were reported in 230 (75.2%) of the 306 subjects. The most frequently 
reported treatment-related AEs were fatigue (28.1%), rash (14.7%), diarrhea (13.4%), and pruritus 
(10.5%). Most treatment-related AEs were low grade. Treatment-related high grade (Grade 3-4) AEs 
were reported in 52 (17.0%) of subjects. The most common treatment-related high grade AEs were 
fatigue (2.3%) and diarrhea (1%). 
Drug-related SAEs occurred in 11.5% of subjects. Grade 3-4 drug-related SAEs reported in at least 2 
subjects included: diarrhea (3 subjects, 1.0%), pneumonitis (3 subjects, 1.0%), pneumonia (2 
subjects, 0.7%) and lipase increased (2 subjects, 0.7%). Select AE categories (events with a potential 
inflammatory mechanism requiring more frequent monitoring and/or unique intervention such as 
immunosuppressants and/or endocrine replacement therapy) include: GI AEs, pulmonary AEs, renal 
AEs, hepatic AEs, skin AEs, and endocrinopathies. In addition, Select AEs include a category for 
infusion reactions. 
 
Each category is composed of a discrete set of preferred terms, including those of greatest clinical 
relevance. These Select AEs are considered events of interest based on the mechanism of action and 
were previously referred to as immune-related AEs or immune-mediated AEs. The 10 mg/kg cohort 
had numerically greater frequency of high-grade select AEs including the subcategories of 
endocrinopathies, GI, pulmonary, and infusion reactions (Table 11.1.3-1) Most high grade events 
resolved following the treatment guidelines for the treatment of pulmonary events, GI events, hepatic 
events, renal events, and endocrine events, respectively. 
 
Treatment-related AEs leading to discontinuation were reported in 32 (10.5%) of the 306 treated 
subjects on CA209003. The most frequent of these were pneumonitis (8 subjects; 2.6%) and colitis (3 
subjects; 1.0%).There were 3 (1%) drug related deaths; each occurred after development of 
pneumonitis. 
 
The phase 3 dose for nivolumab monotherapy is 3 mg/kg. Additional details on the safety profile of 
nivolumab, including results from other clinical studies, are also available in the BMS-936558 
(nivolumab) IB. 


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Possible toxicities for Ipilimumab and nivolumab in combination may include: 
As of 15-Feb-2013, in the Phase 1 study CA209004 of 86 subjects with unresectable or metastatic 
melanoma, ascending doses of nivolumab have been studied as monotherapy in sequence with 
ipilimumab (N = 33) or in combination with ascending doses of ipilimumab (N = 53). 
 
In the sequential cohorts, 1 mg/kg and 3 mg/kg nivolumab cohorts have been studied. Subjects were 
required to have prior ipilimumab and their last treatment must have occurred within 4 – 12 weeks. 
As such, based on ipilimumab pharmacokinetics, pharmacodynamically active ipilimumab was 
present at the outset of treatment in all subjects. Based on this, the monotherapy safety profile in the 
sequential cohorts was expected to differ from the monotherapy safety profile reported in CA209003. 
Therefore, the most relevant safety data for nivolumab monotherapy is from CA209003 (described 
above). The pooled safety data from CA209004 is only provided below for reference. 
 
In each of the combination cohorts in this multi-arm study, ipilimumab was administered once every 
3 weeks for 4 doses with nivolumab administered once every 3 weeks for 8 doses. Starting at week 
24, ipilimumab and nivolumab were administered once every 12 weeks for 8 doses. The three initial 
dose escalation cohorts consisted of Cohort 1 (nivolumab 0.3 mg/kg plus ipilimumab 3 mg/kg; n = 
14), Cohort 2 (nivolumab 1 mg/kg plus ipilimumab 3 mg/kg; n = 17) and Cohort 3 (nivolumab 3 
mg/kg plus ipilimumab 3 mg/kg; n = 6). The study was subsequently amended to include Cohort 2a 
which evaluated nivolumab 3 mg/kg plus ipilimumab 1 mg/kg (n = 16). Safety data were pooled to 
summarize the overall findings. 
 
At least one AE, regardless of whether they were attributed to the therapy, has been reported in 81 
(94.2%) of the 86 subjects (Table 11.2.1-1). A numerically higher number of subjects in the 
combination therapy treatment groups experienced severe AEs, regardless of causality and treatment 
related, than those in the monotherapy treatment group. 
 
In the combination treatment groups, the following DLTs were observed during the dose 
escalation in the combination cohorts: 
• Cohort 1: Grade 3 elevated AST/ALT (1 subject); 
• Cohort 2: Grade 3 uveitis (1 subject) and Grade 3 elevated AST/ALT (1 subject); 
• Cohort 3: Grade 4 elevated lipase (2 subjects) and Grade 3 elevated lipase (1 subject). 
Based on these data, Cohort 2 was identified as the maximum tolerated dose (MTD) and 
Cohort 3 exceeded the MTD. The most frequent treatment related AEs were rash (54.7%), pruritus 
(47.2%) and fatigue (37.7%). The most frequently reported treatment related severe AEs in the 
combination group were lipase and AST elevation (13.2% each), ALT elevation (11.3%), followed 
by diarrhea (5.7%) and rash (3.8%). 
 
Treatment related serious adverse events (SAEs) were reported in 49% of patients in the 
combination treated subjects (N = 53) and most frequently severe SAEs were: AST 
elevation (13.2%), ALT elevation (11.3%), lipase elevation (5.7%) and diarrhea, colitis and renal 
failure (3.8% each). Ten of the 53 (18.9%) treated subjects in the combination cohort 


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discontinued therapy due treatment-related adverse events, the most frequent of which were ALT, 
AST elevation, lipase elevation, renal failure or pneumonitis. No drug-related deaths were reported. 
Median time to event onset was approximately 61 days (range: 4 to 114 days). 
 
Immunotherapy and Breast Cancer: 
The interrelationship between the tumor, the tumor microenvironment and the function of infiltrating 
immune cells at the tumor site, is important for generating efficacious anti-tumor immune response 
(Naito 1998). It is also well established that the magnitude of the tumor burden leads to local 
suppression and apoptosis of the infiltrating T-Cells.37 In breast cancer, the influence of tumor burden 
on the generation of tumor antigen-specific cytotoxic T-lymphocytes (CTLs) was investigated in a 
phase I/II clinical trial in the metastatic setting. The study evaluated the cytotoxic function in an 
adoptive immunotherapy model in 2 patient study arms, one with macroscopic disease and other with 
a complete remission (CR). The results revealed that the cytotoxic function was sustained in only one 
CR patient, suggesting that tumor burden had an inverse effect on the function of the generated 
CTLs.38 
The tumor bearing host ability to mount an immune response to tumor antigens is dampened by the 
effect of Tregs, a subset of CD+ T cells. These cells have a potent ability to suppress immunity by 
inhibiting cytotoxic lymphocytes, and are thought to play role in tolerance to tumor and self antigens. 
39 In experimental models, when the number or function of Tregs was reduced, a surge in the anti- 
tumor response was shown.40 Furthermore, recent studies have demonstrated that immunophenotypic 
features of tumor infiltrating lymphocytes in breast cancer patients are associated with prognostic 
factors and survival rates. For example, investigators have shown increased numbers of tumor 
infiltrating Tregs and peripheral blood Tregs in patients with breast cancer and other tumors, compared 
to healthy donors.41 In addition, significantly higher numbers of Tregs have been demonstrated in 
patients with invasive breast cancer versus ductal carcinoma in situ (DCIS). Particularly high 
numbers of Tregs were present in patients with higher risk tumors including high-grade tumors, tumors 
with lymph node involvement, and estrogen receptor (ER) negative tumors. Furthermore, Tregs 
numbers correlated with relapse rates and even overall survival. 42 Thus, tumor infiltration by immune 
cells appears to play a critical role in the tumorigenesis of breast cancer and therapies that can 
effectively modulate this immune response likely represent a promising opportunity for therapeutic 
innovation. 
 
The presence of tumor infiltrating lymphocytes (TILs) has recently demonstrated prognostic (and 
predictive impact in breast cancer specifically.62-65 Furthermore, PD-1/PD-L1 blockade alone has 
demonstrated approximately 20% response rates in the palliative breast cancer setting. 66, 67 
 
Rationale for Combined Therapy with Cryoablation and Ipilimumab in Cancer 
Novel strategies that combine a cytoreductive modality (i.e. intra-tumoral cryoablation) with 
immune-modulation are promising strategies for the management of tumors. For example, 
combination of CTLA4 blockade with thermal ablation demonstrated synergistic anti tumor response 
in a B16 murine melanoma model.16 Furthermore, our collaborators at MSKCC (Dr. James Allison 
and colleagues) have shown that combination therapy with cryoablation and CTLA4 blockade 
successfully mediated rejection of metastatic prostate cancer lesions and prevented the growth of 
secondary tumors in mice (Figure 1). 


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Fig 1. Cryoablation and anti-CTLA-4 
combination therapy synergize to mediate 
rejection of a second prostate tumor challenge. 
Fig 2. CD4+FoxP3- effector T cells and 
CD8+ T cells infiltrate the secondary 
tumors of combination treated mice. 
Fig 3.Combination treated tumors have an 
increased Teff to Treg ratio compared to controls 
 
 
 
 
 
Dr. Allison and colleagues have also shown that secondary tumors from combination treated mice 
were infiltrated with tumor specific CD8+ T cells (Figure 2) and demonstrated an increase in the 
effector T cell (Teff) to regulatory T cell (T reg) ratio (Figure 3). Clinical and pre-clinical studies have 
suggested that an elevated CD8 to Treg ratio at the tumor site is associated with better clinical 
outcomes (Quezada 2006). 
 
 
 
The safety of cryoablation combined with CTLA4 blockade has been demonstrated in the pre- 
operative breast cancer setting by our group.56 


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Rationale for Biomarker Research 
As part of the completed pilot study of pre-operative ipilimumab and/or cryoablation, immunologic 
correlatives were conducted on both PBMCs and TILs. Tumors treated with combined ipilimumab 
plus cryoablation exhibited a higher ratio of CD8+Ki67+ proliferating Teff cells to 
CD4+CD25+FOXP3+ Treg cells suggesting that combined therapy may favorably shift the 
effector/regulatory T-cell ratio. 56 This effect was not seen with Ki67-negative Teff cells. One possible 
explanation is that cryoablation depletes both Teff and Treg cells, and that concurrent ipilimumab 
induces proliferation of residual or immigrating Teff cells. We also demonstrated that cryoablation 
and ipilimumab enhances systemic immune activation (as measured by IFNγ and ICOShi T-cells by 
flow cytometry) and intratumoral T-cell proliferation (as measured by TcR sequencing and 
Ki67+CD8+ T-cells by flow cytometry.57 
In addition, T-cell repertoire analysis was conducted on both core biopsy and mastectomy TILs (Page 
2014) In cryo-ablated specimens, T-cell counts were reduced, however the infiltrate was more 
polyclonal with low clonal overlap, indicating influx of new clones. Combination cryoablation plus 
ipilimumab resulted in an expansion of a greater number of T-cell clones intratumorally, versus 
cryoablation or ipilimumab alone (Table 3.1, unpublished data). 
 
 
 
Table 3.1: T-cell Clonal expansion following ipilimumab plus cryoablation 
Median # TIL clones expanding by 
Median # PBMC clones expanding by 
≥102 
amplicons 
≥103 
amplicons 
≥104 
amplicons 
≥102 
amplicons 
≥103 
amplicons 
≥104 
amplicons 
Cryo 
199 
2 
0 
697 
1 
0 
Ipi 
750 
22 
1 
545 
5 
0 
Cryo+Ipi 2010 
85 
1 
830 
1 
0 
 
These data suggest that cryoablation may induce new clones secondary to antigen presentation, and 
that the addition of ipilimumab may induce proliferation of a subset of tumor-reactive infiltrating 
clones. 
4.0 
OVERVIEW OF STUDY DESIGN/INTERVENTION 
4.1 
Design 
This is a pilot, single-institution study, wherein one group of 6 women ≥18 years of age with 
operable, ≥1.5 cm, histologically-confirmed, invasive carcinoma of the breast and no evidence of 
distant metastases, for whom surgery is planned, will be enrolled. The 6 participating women will be 
treated with ipilimumab, nivolumab and cryoablation of the dominant primary tumor prior to the 
planned surgery. 


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At the time of the initial MSKCC surgical consultation, potentially eligible candidates will be 
identified, informed consent forms provided, and surgery-related dates requested/set per standard-of- 
care. As soon as possible, potentially eligible patients will be evaluated by 1) the principal 
investigator (Dr. Elizabeth Comen), a designated consenting medical oncologist for a medical 
oncology assessment or the referring surgeon and 2) consenting professionals from radiology for 
assessment of amenability to cryoablation, if feasible. All available (per the standard of care) imaging 
(MMG +/- breast US +/- breast MRI) will be reviewed to determine eligibility for cryoablation and 
thus, study participation. Whenever feasible, the radiology evaluation will be arranged prior to the 
presurgical testing date so that baseline research bloods may be collected after the patient is 
confirmed eligible and together with the non-study presurgical bloods. Prior to protocol intervention, 
consent will take place at the first available appointment with a consenting professional. 
 
Consenting, eligible women, with tumors amenable to MRI or US-guided cryoablation will be 
enrolled. All study interventions (including the cryoablation, ipilimumab/nivolumab administration, 
and imaging dates) will be defined by the pre-determined, non-study surgical date. Research study 
appointments/interventions will not interfere with the planned, standard-of-care, surgery 
arrangements. 
 
4.2 
Intervention 
During study intervention planning, the standard of care for patients with early stage primary breast 
cancer diagnosis and treatment of as well as the timing of the definitive surgery schedule will not be 
altered in any way for patients participating in this study. If a patient experiences side effects from 
intervention resulting in a delay of their planned surgery date, this will be a failure of the primary 
endpoint. The planned intervention of this study is to obtain image guided biopsies, percutaneous 
MRI or US guided breast tumor cryoablation, and ipilimumab/nivolumab administration. 
Most patients will have an outside biopsy confirming the diagnosis of breast cancer at the time of the 
initial surgical consultation. However, because the cryoablation procedure will cause near complete 
tumor destruction (that in turn will cause immunologic effect and antitumor response), pre- 
cryoablation breast biopsies will be performed. At least three 18G MRI or US-guided core biopsies of 
the primary breast tumor will be obtained (together with core biopsies of any suspicious lymph nodes 
as clinically or radiographically indicated) if feasible. Two core breast specimens will be provided to 
the MSKCC Breast and Imaging Center (BAIC) pathology department for routine (standard of care) 
testing. Additional core biopsy specimens will be sent to the immune monitoring facility (IMF) for 
research purposes as detailed in section 12. If not all three core biopsies can be performed, this will 
not make the patient ineligible. 
The MRI or US-guided biopsies will be performed by investigators from the Breast Radiology 
Service with the assistance of investigators from Interventional Radiology and Anesthesiology as 
needed. Image guided percutaneous cryoablation can be performed through a small nick making it a 
minimally invasive procedure that has been performed safely in cancer patients for many years. The 
cryoablation will be performed 7-10 days prior to the surgery date. 
Ipilimumab and nivolumab are fully human, monoclonal anti-CTLA4 antibodies that have shown 
anti-tumor activity and are now US FDA approved for the treatment of patients with unresectable 
advanced melanoma (unresectable stage III or stage IV). 


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Recent studies have shown that administration of ipilimumab in the pre-operative setting is safe and 
does not cause delay of surgery in bladder cancer.12 In this study, ipilimumab and Nivolumab will be 
administered 1-5 days prior to core biopsy + cryoablation and 8-15 days prior to the planned surgery. 
 
 
5.0 
THERAPEUTIC/DIAGNOSTIC AGENTS 
5.1 Cryoablation 
Cryoablation has been successfully adopted for the treatment of solid tumors for many years. Initially 
it was an intraoperative procedure, but with advanced engineering devices for percutaneous 
cryoablation has been made possible. In addition the use of imaging to guide the ablation provides 
increased confidence of appropriate targeting of the tumor. The cryoablation will be performed 
percutaneously under MRI or US image guidance. Once the probes are properly placed in the tumor 
freeze-thaw cycles will be performed. The probes will then be removed. The patients will be 
observed for a short time prior to expected same day discharge. 
 
 
5.2 Ipilimumab and Nivolumab: 
Ipilimumab (BMS-734016), a fully human monoclonal antibody that recognizes CTLA4, is 
considered an immunotherapeutic drug and is currently in development as an anti-cancer agent. The 
proposed mechanism of action for ipilimumab is interference of the interaction of CTLA4 with CD80 
(B7-1) and CD86 (B7-2) molecules expressed on antigen-presenting cells (APC), with subsequent 
blockade of the inhibitory modulation of T-cell activation promoted by the CTLA4:CD80/CD86 
interaction. 
 
Nivolumab (BMS-936558), a fully human monoclonal antibody that recognizes PD1, is considered 
an immunotherapeutic drug and is currently in development as an anti-cancer agent. The proposed 
mechanism of action for nivolumab is interference with the interaction between PD1 and PDL1. T 
cell influx into tumors results in the release of IFN-γ, which up-regulates PD-L1 expression by tumor 
cells. PD-L1 binds to PD-1, which is expressed by activated T cells, generating a negative signal that 
causes T cell exhaustion (inhibiting the ability of T cells to recognize and kill their targets). During 
antigen presentation by dendritic cells, PD1 can also act as a checkpoint inhibitor where a negative 
signal can be sent by its binding to either PD-L1 or the closely related (and dendritic cell–specific) 
negative regulatory ligand PD-L2.68 
 
Concomitant Medications 
Participants will be advised not to take steroids, immunosuppressants, or anti-inflammatory 
medications such as NSAIDS from the day of trial entry and until 30 days after surgery unless 
recommended by the study Principal Investigator (or co-investigators) for the treatment of immune 
related adverse events (irAEs) or adrenal insufficiency. 


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6.0 
CRITERIA FOR SUBJECT ELIGIBILITY 
Describe the characteristics of the patient/subject population. 
6.1 
Subject Inclusion Criteria 
Subjects must meet the following criteria at screening to be eligible to participate in the study. 
• Women age 18 years or older 
• Confirmed histologic diagnosis of invasive adenocarcinoma of the breast, including 
MSKCC pathology confirmation 
• ER, PR and HER2 testing in progress (i.e. on outside or MSKCC biopsy report) 
o HER2-positive pathology is permitted 
• Operable tumor measuring ≥1.5 cm in maximal diameter 
o Any nodal status 
o Multifocal and multicentric disease is permitted. 
o Synchronous bilateral invasive breast cancer is permitted 
o The tumor should be more than 5 mm from the skin. 
• No indication of distant metastases 
• Breast surgery planned 
• Tumor amenable to cryoablation as determined by radiologist 
• ECOG performance status score of 0 or 1. 
• Screening laboratory values must meet the following criteria: 
i. White blood cells (WBCs) ≥ 2000/μL 
ii. Absolute neutrophil count (ANC) ≥ 1500/μL 
iii. Platelets ≥ 100 x 103/μL 
iv. Hemoglobin ≥ 11.0 g/dL 
v. Serum creatinine ≤ 2 mg/dL (or glomerular filtration rate ≥ 40 ml/min) 
vi. AST ≤ 2.5 x upper limit of normal (ULN) 
vii. ALT ≤ 2.5 x ULN 
viii. Bilirubin within normal limits (except subjects with Gilbert’s syndrome, who 
must have total bilirubin < 3.0 mg/dL) 
ix. Negative HIV screening test 
x. Negative screening tests for Hepatitis B and Hepatitis C. 
Patients with positive results that do not indicate true active or chronic 
infection may enroll after discussion and consensus agreement by the 
treating physician and principal investigator. 
• Women of childbearing potential (WOCBP) must be using an acceptable method of 
contraception to avoid pregnancy throughout the study and for at least 3 months after 
the last dose of ipilimumab in such a manner that the risk of pregnancy is minimized. 
See below for the definition of WOCBP. 
• WOCBP must have a negative serum pregnancy test within 14 days prior to the first 
dose of ipilimumab/nivolumab. 
• Women must not be breastfeeding. 


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• Willing to adhere to the study visit schedule and the prohibitions and restrictions 
specified in this protocol. 
Definition of WOCBP 
Women of childbearing potential include any female who has experienced menarche and who has not 
undergone successful surgical sterilization (hysterectomy, bilateral tubal ligation, or bilateral 
oophorectomy) or is not postmenopausal. Post menopause is defined as: 
• Amenorrhea ≥ 12 consecutive months without another cause and a documented serum 
follicle stimulating hormone (FSH) level >35 mIU/mL 
• Women with irregular menstrual periods and a documented FSH level > 35 mIU/mL 
• Women on hormone replacement therapy (HRT) 
 
Women who are using oral contraceptives, other hormonal contraceptives (vaginal products, skin 
patches, or implanted or injectable products), or mechanical products such as an intrauterine device 
or barrier methods (diaphragm, condoms, spermicides) to prevent pregnancy, or are practicing 
abstinence or where their partner is sterile (e.g., vasectomy) should be considered to be of 
childbearing potential. 
 
6.2 
Subject Exclusion Criteria 
1) 
Inflammatory breast cancer 
2) 
Medical history and concurrent diseases 
a) Subjects with active, known or suspected autoimmune disease. Subjects with vitiligo, type I 
diabetes mellitus, residual hypothyroidism due to autoimmune condition only requiring 
hormone replacement, psoriasis not requiring systemic treatment, or conditions not expected 
to recur in the absence of an external trigger are permitted to enroll. 
b) Any serious or uncontrolled medical disorder that, in the opinion of the investigator, may 
increase the risk associated with study participation or study drug administration, impair the 
ability of the subject to receive protocol therapy, or interfere with the interpretation of study 
results. 
 
3) 
Prohibited Treatments and/or Therapies 
a) Chronic use of immunosuppressants and/or systemic corticosteroids (used in the 
management of cancer or non-cancer-related illnesses). However, use of 
corticosteroids is allowed for the treatment of immune related Adverse Events (irAEs), 
or adrenal insufficiency. 
b) Any non-oncology vaccine therapy used for prevention of infectious diseases within 4 
weeks prior to first dose of ipilimumab/Nivolumab. 
c) Prior treatment with a CD137 agonist, ipilimumab or other CTLA4 inhibitor; 
d) Prior 
investigational 
agents 
within 
2 
weeks 
prior 
to 
first 
dose 
of 
ipilimumab/Nivolumab; 
e) Prior therapy with any anti-cancer agents including chemotherapy, adjuvant 
chemotherapy, immunosuppressive agents, surgery or radiotherapy within 2 weeks 
prior to first dose of ipilimumab/Nivolumab. 


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7.0 
RECRUITMENT PLAN 
The Evelyn H. Lauder Breast Center, in the Breast and Imaging Center at MSKCC provides a large 
referral base, with approximately 2000 new breast cancer referrals each year, with the vast majority 
of these representing early stage diagnoses. In 2014, the last year for which we have complete data, 
719 (41%) of the 1774 women undergoing surgery at MSKCC for resectable invasive breast cancer 
underwent total mastectomy (with 59% undergoing breast conserving surgery). We anticipate an 
average accrual rate of 2-3 patients per month with complete accrual of 6 patients within 2-3 months. 
At the time of the initial MSKCC surgical consultation, potentially eligible candidates will be 
identified, informed consent forms provided, and surgery-related dates requested/set per standard-of- 
care. As described in detail in Section 4.1, at the time of initial presentation in the surgery clinic, 
potentially eligible study candidates will be identified by the surgeon and referred for an appointment 
with the Principal Investigator (Dr. Elizabeth Comen) or a consenting medical oncologist and an 
appointment with consenting professionals from radiology, if feasible, to evaluate eligibility for 
cryoablation. Prior to protocol intervention, consent will take place at the first available appointment 
with a consenting professional. 
8.0 
PRETREATMENT EVALUATION 
After the patient is identified as a potentially appropriate study candidate at the time of the initial 
breast surgery consultation, an appointment will be made with the Principal Investigator (Dr. 
Elizabeth Comen) or another consenting medical oncologist. An appointment will also be made with 
consenting professionals from radiology, if feasible, to evaluate eligibility for cryoablation and to 
review potential complications. Eligibility for safe cryoablation will include an assessment of how 
close the tumor is to various structures. The tumor should be more than 5 mm from the skin. The 
chest wall and associated structures represent less of a complication risk. The observation of the ice 
ball with imaging during the procedure should mitigate these potential risks. Prior to protocol 
intervention, consent will take place at the first available appointment with a consenting professional. 
If the patient’s tumor is amenable to cryoablation and informed consent has been signed, additional 
appointments will be arranged accordingly. Informed consent must be performed within 28 days prior 
to ipilimumab/nivolumab administration. 
Baseline blood tests to be drawn with non-study presurgical blood tests, within 14 days prior to 
ipilimumab/nivolumab administration: 
Comprehensive metabolic panel (COMP): aspartate aminotransferase (AST), alanine aminotransferase 
(ALT), albumin, alkaline phosphatase (ALP), total bilirubin, calcium, creatinine, glucose, total protein, 
BUN, sodium, potassium, chloride, bicarbonate 
Complete blood cell count with differential and platelet count (CBC) 
Thyroid stimulating hormone (TSH), free T4, T3, ACTH, FSH/LH, prolactin, testosterone levels 


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HIV, Hepatitis C, and Hepatitis B screening tests 
Research tests: Blood (4 CPT tubes) for immune response studies will be sent to the IMF (currently 
located on the 15th floor of the Zuckerman building) for exploratory studies. These baseline blood 
samples may be drawn on the day of the ipilimumab/Nivolumab injection (if applicable), but should 
be drawn prior to the injection. 
All other screening assessments should be performed within 14 days prior to ipilimumab/nivolumab 
administration: 
• Medical history, demographic data, adverse events, physical examination, ECOG PS, 12-Lead 
ECG. 
No delay in surgery will be made to accommodate participation in the protocol. 
9.0 
TREATMENT/INTERVENTION PLAN 
Patients will undergo breast biopsy with cryoablation 7-10 days prior to the planned surgery, and 
ipilimumab with nivolumab administration 1-5 days before cryoablation. All patients will undergo 
surgery at the pre-determined day defined at the initial surgical consultation as detailed in section 4.1. 
9.1 Ipilimumab and Nivolumab: 
Women will receive ipilimumab and nivolumab 1-5 days prior to US or MRI-guided core biopsy and 
cryoablation date. Intravenous ipilimumab will be administered as a single 1 mg/kg dose to be 
infused intravenously over 90 minutes in the chemotherapy suite of the BAIC. Intravenous 
nivolumab will be administered as a single 3 mg/kg dose to be infused intravenously over 60 minutes 
in the chemotherapy suite of the BAIC. 
In a study reported by Dr. Wolchok of various doses of ipilimumab and nivolumab in combination in 
metastatic melanoma, there was a 98% AE rate, a 93% treatment related AE rate, a 72% grade 3/4 
AE rate, a 53% treatment related grade 3/4 AE rate and a 49% serious AE rate.61 Toxicity with both 
drugs administered at 3mg/kg was considered unacceptable, and nivolumab at 1mg/kg with 
ipilimumab at 3mg/kg was considered the maximum dose with an “acceptable” level of AEs - a 
threshold which is unlikely to be “acceptable” in the curative setting. Thus, combination therapy with 
ipilimumab administered at 1mg/kg and nivolumab administered at 3mg/kg was recommended and 
will be the dose we will administer for this study. 
9.1.1 Dose Calculations 
Calculate Total Dose as follows for ipilumumab: 
Patient body weight in kg x [1 mg/kg] = total dose in mg 
Calculate Total Infusion Volume as follows: 
Total dose in mg ÷ 5 mg/mL = infusion volume in mL 


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Calculate Rate of Infusion as follows: 
Infusion volume in mL ÷ 90 minutes = rate of infusion in mL/min. 
For example, a patient weighing 114 kg (250 lb) would be administered 114 mg of ipilimumab (114 
kg x 1 mg/kg = 114 mg) with an infusion volume of 22.8 mL (114 mg ÷ 5 mg/mL = 22.8 mL) at a 
rate of approximately .25mL/min (22.8 mL ÷ 90 minutes) in 90 minutes. 
9.1.2 Storage, Preparation, and Administration 
Identification 
Ipilimumab is available in 5 mg/mL single-use vials (10 mL or 40 mL). The sterile solution in the 
vial is clear and colorless to pale yellow. Ipilimumab is administered via intravenous infusion only. 
 
Nivolumab is available in 10 mg/mL single-use vials (10 mL). The sterile solution in the vial is clear 
and colorless to pale yellow. Nivolumab is administered via intravenous infusion only. 
 
Packaging and Labeling 
BMS will provide ipilimumab and nivolumab at no cost for this study. Ipilimumab and nivolumab 
will be provided in open-label containers. The labels will contain the protocol prefix, batch number, 
content, storage conditions, and dispensing instructions along with the Investigational New Drug 
(IND) caution statement. Ipilimumab will be supplied at a concentration of 5 mg/mL in vials 
containing 10 ml or 40 mL solution. Nivolumab will be supplied at a concentration of 10 mg/mL in 
vials containing 10 ml solution. 
Storage, Handling, and Dispensing 
Storage 
Ipilimumab and nivolumab must be stored in a secure area according to local regulations. The 
investigator must ensure that it is stored in accordance with the environmental conditions as 
determined by BMS and defined in the Investigator Brochure or SmPC/reference label. Ipilimumab 
and nivolumab must be stored at a temperature  2C and  8C. 
Handling and Disposal 
As with all injectable drugs, care should be taken when handling and preparing ipilimumab and 
nivolumab. Whenever possible, ipilimumab and nivolumab should be prepared in a laminar flow 
hood or safety cabinet using standard precautions for the safe handling of intravenous agents 
applying aseptic technique. Latex gloves are required. If ipilimumab or nivolumab concentrate or 
solution comes in contact with skin or mucosa, immediately and thoroughly wash with soap and 
water. After final drug reconciliation, unused ipilimumab solution should be disposed at the site 
following procedures for the disposal of anticancer drugs. 


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Dispensing 
It is the responsibility of the investigator to ensure that ipilimumab and nivolumab is only dispensed 
to study subjects. The ipilimumab and nivolumab must be dispensed only from official study sites by 
authorized personnel according to local regulations. 
 
Drug product administration 
Ipilimumab is to be administered as an IV infusion with a 0.2μm or 1.2μm in-line filter using a 
volumetric pump, at the 1 mg/kg dose, to complete the infusion in 90 minutes with at least 10 cc 
normal saline flush at the end. The total dose needed should be prepared and administered per 
institutional guidelines. The total dose must be calculated using the most recent subject weight 
(obtained on the same day of, and prior to, the infusion). 
Nivolumab is to be administered as an IV infusion with a 0.2μm or 1.2μm in-line filter using a 
volumetric pump, at the 3 mg/kg dose, to complete the infusion in 60 minutes with at least 10 cc 
normal saline flush at the end. The total dose needed should be prepared and administered per 
institutional guidelines. The total dose must be calculated using the most recent subject weight 
(obtained on the same day of, and prior to, the infusion). 
Separate infusion bags and filters must be used for each infusion. Nivolumab is to be administered 
first. The nivolumab infusion must be promptly followed by a saline flush to clear the line of 
nivolumab before starting the ipilimumab infusion. 
9.2 
Pre-cryoablation core biopsies: 
At least three 18G MRI or US-guided core breast biopsies will be obtained at the core biopsy 
appointment for all participants, if feasible. If not all three core biopsies can be performed, this will 
not make the patient ineligible. Core biopsies of clinically or radiographically suspicious lymph 
nodes will also be obtained as indicated. Two core breast specimens will be provided to the MSKCC 
BAIC pathology department for routine (standard of care) testing. Additional core biopsy specimens 
will be sent to the IMF for research purposes as detailed in section 12. The MRI or US-guided 
biopsies will be performed by investigators from the Breast Radiology Service with the assistance of 
investigators from Interventional Radiology and Anesthesiology as needed. 
9.3 
Cryoablation: 
Percutaneous, MRI or US-guided cryoablation will be performed with a multiple freeze-thaw cycle. 
Freeze-thaw cycle strategies will be individualized to the patient and their tumor. Generally two 
cycles will be performed with each cycle being approximately 10 minutes of freeze time. Intermittent 
imaging will guide the procedure to ensure the ice ball does not encroach on critical structures, 
including skin. The number of probes used will depend on the lesion size and location. 
Prior to the procedure the operator will rate the level of technical difficulty. Immediately after the 
procedure the operator will rate the expectation of completeness of therapy. This information will be 


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used for data collection purposes for this study. Cryoablation will be performed under the care of a 
radiologist with support from anesthesiology as needed. 
9.4 
Blood samples: 
All participants will undergo 4 research blood draws as indicated in the protocol schema in Section 1 
(at ipilimumab/nivolumab administration, cryoablation/biopsy, surgery, and 30 (+/-10) days after 
surgery). Detailed collection/processing instructions are included in Section 12. 
 
 
10.0 EVALUATION DURING TREATMENT/INTERVENTION 
The focus of the evaluation during the treatment will be directed to assess the safety of the procedures 
described above. 
 
All patients will be observed for 30 minutes after drug injection to monitor for acute allergic 
reactions. All participants will be observed in the PACU after cryoablation and/or image guided core 
biopsies for a couple of hours prior to expected same day discharge if sedation or anesthesia is 
required. 
No additional testing, treatment, or intervention will be performed on patient accrued to the protocol. 
Efforts should be made to synchronize post procedure pain management, and to try to avoid NSAIDs 
(specifically COX-2 inhibitors) given their potential for immune modulation. 
 
11.0 TOXICITIES/SIDE EFFECTS 
In routine care for breast cancer, multiple cores are taken to insure adequate material for diagnosis. 
The potential complication associated with these procedures, such as bleeding and infection are the 
same as those associated with these procedures when done as part of routine care. Refer to Appendix 
2 for management of immune related adverse events. 
11.1 
Cryoablation 
Patients will be monitored for adverse events related to ablation. These events may include bleeding, 
infection, injury to the breast, skin injury, and pain at the ablation site. Other potential complications 
include paresthesias. 
11.2 
Infusion Reactions 
Ipilimumab and/or nivolumab may induce infusion or hypersensitivity reactions. If such a 
reaction were to occur, it may manifest with fever, chills, rigors, headache, rash, pruritus, 
arthralgias, hypo or hypertension, bronchospasm, or other symptoms. Infusion reactions 
should be graded according to Common Terminology Criteria for Adverse Events (CTCAE, 
Version 4.0) guidelines. Treatment recommendations are provided below and may be 


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modified based on clinical judgment, local treatment standards and guidelines, and/or specific 
symptoms, as appropriate: 
 
For Grade 1 symptoms: Mild reaction [e.g., localized cutaneous reactions including mild 
pruritus, flushing, rash] requires infusion rate to be decreased; intervention may be indicated. 
 
• Decrease the rate of the study drug infusion until recovery from symptoms. 
• Remain at 
bedside 
and monitor subject 
until 
resolution 
of symptoms. 
Diphenhydramine 50 mg may be administered at the discretion of the treating 
physician. 
• When symptoms resolve, restart the infusion at the original infusion rate. 
For Grade 2 symptoms: Moderate reaction [i.e., any symptom not listed above (mild 
symptoms) or below (severe symptoms) such as generalized pruritus, flushing, rash, dyspnea, 
hypotension with systolic blood pressure > 80 mm Hg], requires infusion interruption but 
responds promptly to symptomatic treatment [e.g., antihistamines, nonsteroidal anti 
inflammatory drugs, narcotics, corticosteroids, i.v. fluids]; prophylactic pre-infusion 
medications indicated for ≤ 24 hours. 
 
• Discontinue the study drug infusion. 
• Begin an i.v. infusion of normal saline, and treat the subject with diphenhydramine 
50 g i.v. (or equivalent) and/or paracetamol 325 to 1000 mg (acetaminophen). 
• Remain at bedside and monitor subject until resolution of symptoms. Corticosteroid 
therapy may be administered at the discretion of the treating physician. 
• When symptoms resolve, restart the infusion at 50% of the original infusion rate; if no 
further complications ensue after 30 minutes, the rate may be increased to 100% of the 
original infusion rate. 
• Monitor subject closely. If symptoms recur, immediately discontinue the infusion; no 
further study drug will be administered at that visit. Administer diphenhydramine 50 
mg i.v., and remain at bedside and monitor the subject until resolution of symptoms. 
• The amount of ipilimumab infused must be recorded. 
For Grade 3 or Grade 4 symptoms: Severe reaction [e.g. bronchospasm, generalized 
urticaria, systolic blood pressure < 80 mm Hg, or angioedema], Grade 3: prolonged [i.e. 
requiring 6 or more hours to respond to symptomatic medication and/or discontinuation of 
infusion]; recurrence of symptoms following initial improvement; hospitalization indicated 
for other clinical sequelae [e.g., renal impairment, pulmonary infiltrates]. 
 
Grade 4: life-threatening; pressor or ventilatory support indicated. 
• Immediately discontinue the study drug infusion. No further study drug will be 
administered. The amount of study drug infused must be recorded on the case report 
form (CRF). 
• Begin an i.v. infusion of normal saline, and treat the subject as follows: Recommend 
bronchodilators, epinephrine 0.2 to 1.0 mg of a 1:1,000 solution for subcutaneous 
administration or 0.1 to 0.25 mg of a 1:10,000 solution injected slowly for i.v. 


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administration, and/or diphenhydramine 50 mg i.v. with methylprednisolone 100 mg 
i.v. (or equivalent), as needed. 
• Remain at bedside and monitor subject until recovery from symptoms. 
• Subject should be monitored until the treating physician is comfortable that the 
symptoms will not recur. 
• Treating physician should follow their institutional guidelines for the treatment of 
anaphylaxis 
In the case of late-occurring hypersensitivity symptoms (e.g., appearance of a localized or 
generalized pruritus within 1 week after treatment), symptomatic treatment may be given 
(e.g., oral antihistamine or corticosteroids). 
 
12.0 CRITERIA FOR THERAPEUTIC RESPONSE/OUTCOME ASSESSMENT 
12.1 Safety: 
Safety/tolerability is the primary end point for this project. 
 
Safety will be evaluated for all treated subjects using National Cancer Institute (NCI) Common 
Terminology Criteria for Adverse Events v4.0 (CTCAE). Safety assessments will be based on 
medical review of adverse event reports and the results of vital sign measurements, physical 
examinations, and clinical laboratory tests. The incidence of observed adverse events will be 
tabulated and reviewed for potential significance and clinical importance. The reporting period for 
safety data will be from the date of first on-study dose to 12 weeks after ipilimumab/nivolumab 
administration. 
 
If there are no clinical signs or symptoms of toxicity, safety bloods will be done every 2-3 weeks and 
will include the following: 
- 
Comprehensive metabolic panel (COMP): aspartate aminotransferase (AST), alanine 
aminotransferase (ALT), albumin, alkaline phosphatase (ALP), total bilirubin, calcium, 
creatinine, glucose, total protein, BUN, sodium, potassium, chloride, bicarbonate 
- 
Complete blood cell count with differential and platelet count (CBC) 
- 
Thyroid stimulating hormone (TSH) 
 
If 5 of the 6 study patients are without an AE necessitating delay in surgery, the regimen will be 
considered safe/tolerable for further study. 
 
12.2 Efficacy: 
Although not a primary endpoint, exploratory correlative studies will be performed to evaluate anti- 
tumor and immunological response pre- and post-treatment in individuals. Comparisons with other 
study participants will also be conducted. Breast cancer tissue specimens, axillary lymph nodes 
specimens where available and peripheral blood samples will be studied. The planned exploratory 
correlative studies currently include: 


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Immune response: 
The phenotypic and functional impact of the treatment will be explored by examining specimens 
from tumor tissue; peripheral blood (post treatment only). These tests will be done at the IMF 
(currently located in the Zuckerman building). 
The exploratory objectives of this study are: 
1. To evaluate the effect of cryoablation and ipilimumab/nivolumab administration on: lymphocyte 
phenotype and serum cytokines, disease related biomarkers, antibody responses to selected 
antigens, and humoral and cellular responses to tumor antigens and recall non-tumor antigens. 
2. To examine tumor samples for pathologic correlates of clinical activity before and after treatment, 
including (but not limited to) the abundance and characteristics of inflammatory infiltrates (e.g., 
CD8 and CD4 cells and expression of some molecules on lymphocytes and tumors, respectively), 
and, 
3. To evaluate serological and cellular immune correlates of toxicity and/or clinical activity 
 
To this end, the specimens to be collected for IMF evaluation include: 
• At least 1 core biopsy sample (collected on the core biopsy date) 
• Surgery specimen 
• Four research blood samples: 
1. Baseline blood on the day of pre-surgical testing or on the day of ipilimumab/nivolumab 
injection prior to drug administration 
2. Blood on day of core biopsy/cryoablation 
3. Blood on the day prior to or day of surgery 
4. Blood 30 (+/-10) days after surgery. 
For the each blood draw, 4 CPT tubes will be collected. 
• Flow cytometric analysis: 
1) T-cells: activation and memory markers 
2) Dendritic 
cells: 
activation 
and 
maturation 
markers 
and 
markers 
of 
inhibitory/tolerogenic DCs 
• ELISA: to identify relevant peripheral blood cytokines 
• CBC: to quantify Absolute lymphocyte count (ALC). 
Pathology: Pre (core biopsies) and post (surgical tissue) treatment evaluation of the tumor will be 
performed for each study participant. The planned exploratory studies to evaluate treatment response 
include: 
 
• Evaluation of T cell proliferation as measured by Ki-67. 
• Expression of the B7-H1 inhibitory molecule (Ghebeh 2007) 
• Lymphocyte infiltration including perivascular infiltration. 
• Specific immunohistochemistry (IHC) staining to further define particular 
lymphocytic populations such as T-regulatory cells (Tregs) (CD4+CD25+FOXP3+), 
effector T cells (Teff) (CD4+/CD8+) and the ratio between Tregs and Teff. 
• Using immunohistochemistry (IHC) staining to identify subsets of dendritic cell (DC) 
populations (i.e. tolerogenic versus immunogenic)Sentinel or non-sentinel lymph 


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node tissue, where available, will also be evaluated by immune profiling as described 
above. 
 
13.0 
CRITERIA FOR REMOVAL FROM STUDY 
Subjects participating may withdraw from the study at any time and for any reason. 
Subjects may be removed from the study at any time due to severe unacceptable side effects, life- 
threatening events, (as described in Section 11), patient’s non-compliance with the defined treatment 
plan, the patient becomes pregnant or the patient’s request to withdraw consent. 
For all participants, failure to have the planned surgery at MSKCC will result in removal from the 
study. 
Patients will be replaced if they withdraw from the study after signing consent but prior to 
completing all study assessments. 
14.0 
BIOSTATISTICS 
Definitive surgery confers cure for many women with early stage breast cancer and it is therefore 
imperative that any experimental pre-operative therapeutic innovation not undermine the timing of 
curative-intent surgery. Consequently, the primary endpoint for this study is safety/tolerability, as 
defined by the number of patients who do not develop grade 3 or 4 adverse events that necessitates a 
delay in surgery. Therefore, if 5 or 6 of the 6 patients are without an AE necessitating delay in 
surgery, the regimen will be considered safe/tolerable for further study. The probability of observing 
5 or 6 patients (out of 6) who for whom therapy is safe/tolerable is 0.53 if the true safety/tolerability 
rate is 0.75, 0.65 if the true safety/tolerability rate is 0.80, and 0.89 if the true safety/tolerability rate 
is 0.90. This rule is based on the time period from start of the investigation regimen to the date of 
surgery, which is estimated to be approximately 15 days. Safety data up to 12 weeks following 
surgery will also be evaluated and reported. 
Study design and analysis of the data will be performed with the assistance of Sujata Patil, PhD, 
Division of Epidemiology and Biostatistics. 
This is a pilot study using innovative methods and if cryoablation and single-dose 
ipilimumab/nivolumab is deemed safe, will result in the design of a larger trial. As such, there is no 
formal statistical analysis or sample size calculation for this study. All analysis will be descriptive in 
nature and the sample size is dictated by drug availability and budget constraints. 
We have several exploratory secondary analyses. As noted above, all analyses will be evaluated 
descriptively and graphically. Serum and tumor biomarker levels (including lymphocytes and DC’s) 
and serum cytokines will be plotted over the study timepoints. The ratio of specific intra-tumoral and 
serum t cell populations (i.e. CD4+FOXP3+ T-regulatory cells (Tregs) to CD8+Effector cells) will 
also be plotted over study timepoints. Other endpoints that will be summarized descriptively include 
ALC, and absolute T cell count. 


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Estimated accrual will be approximately 2-3 patients per month, making targeted completion of 
accrual within approximately 2 to 3 months of the study initiation. 
15.0 
RESEARCH PARTICIPANT REGISTRATION AND RANDOMIZATION PROCEDURES 
15.1 
Research Participant Registration 
Confirm eligibility as defined in the section entitled Criteria for Patient/Subject Eligibility. 
Obtain informed consent, by following procedures defined in section entitled Informed 
Consent Procedures. 
During the registration process registering individuals will be required to complete a protocol 
specific Eligibility Checklist. 
All participants must be registered through the Protocol Participant Registration (PPR) Office 
at Memorial Sloan Kettering Cancer Center. PPR is available Monday through Friday from 
8:30am – 5:30pm at 646-735-8000. Registrations must be submitted via the PPR Electronic 
Registration System (http://ppr/). The completed signature page of the written consent/RA or 
verbal script/RA, a completed Eligibility Checklist and other relevant documents must be 
uploaded via the PPR Electronic Registration System. 
15.2 
Randomization 
The study will not include any randomization. 
16.0 
DATA MANAGEMENT ISSUES 
A Research Study Assistant (RSA) will be assigned to the study. The responsibilities of the RSA 
include project compliance, data collection, abstraction and entry, data reporting, regulatory 
monitoring, problem resolution and prioritization and coordination of activities of the protocol study 
team. 
The data collected for this study will be entered into a secure database at MKSCC. Data from this 
trial will be entered in the Clinical Research Data Base (CRDB). Source documentation will be 
available to support the computerized patient record. 
16.1 
Quality Assurance 
Weekly registration reports will be generated to monitor patient accruals and completeness of 
registration data. Routine data quality reports will be generated to assess missing data and 
inconsistencies. Accrual rates and extent and accuracy of evaluations and follow-up will be 
monitored periodically throughout the study period. 
 
Random-sample data quality and protocol compliance audits will be conducted by the study team, at 
a minimum of two times per year, more frequently if indicated. 


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16.2 
Data and Safety Monitoring 
The Data and Safety Monitoring (DSM) Plans at Memorial Sloan Kettering Cancer Center were 
approved by the National Cancer Institute in September 2001. The plans address the new policies set 
forth by the NCI in the document entitled “Policy of the National Cancer Institute for Data and Safety 
Monitoring of Clinical Trials” which can be found at: 
http://cancertrials.nci.nih.gov/researchers/dsm/index.html. The DSM Plans at MSKCC were 
established and are monitored by the Office of Clinical Research. The MSKCC Data and Safety 
Monitoring Plans can be found on the MSKCC Intranet at: http://mskweb2.mskcc.org/irb/index.htm 
There are several different mechanisms by which clinical trials are monitored for data, safety and 
quality. There are institutional processes in place for quality assurance (e.g., protocol monitoring, 
compliance and data verification audits, therapeutic response, and staff education on clinical research 
QA) and departmental procedures for quality control, plus there are two institutional committees that 
are responsible for monitoring the activities of our clinical trials programs. The committees: Data 
and Safety Monitoring Committee (DSMC) for Phase I and II clinical trials, and the Data and Safety 
Monitoring Board (DSMB) for Phase III clinical trials, report to the Center’s Research Council and 
Institutional Review Board. 
During the protocol development and review process, each protocol will be assessed for its level of 
risk and degree of monitoring required. Every type of protocol (e.g. NIH sponsored, in-house 
sponsored, industrial sponsored, NCI cooperative group, etc.) will be addressed and the monitoring 
procedures will be established at the time of protocol activation. 
 
17.0 
PROTECTION OF HUMAN SUBJECTS 
Prior to the enrollment of each patient, the risks, benefits and objectives of the study will be reviewed 
with the participant, including a discussion of the possible toxicities and side effects. Every effort will 
be made to keep study records private. Neither the patient's name nor anything else that could 
identify the patient will be used in any reports or publications that result from this study. Trained staff 
at Memorial Hospital, the Food and Drug Administration will be able to review the medical records if 
necessary. The patient may terminate her participation in the study at any time during the trial. 
Patients will not be charged for the following costs associated with this study: 
• The cost of ipilimumab and nivolumab (administration will be charged). 
• Cryoablation procedure 
• Research blood tests 
• Research biopsy for all patients 
 
17.1 
Privacy 
MSK’s Privacy Office may allow the use and disclosure of protected health information pursuant to a 
completed and signed Research Authorization form. The use and disclosure of protected health 
information will be limited to the individuals described in the Research Authorization form. 


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A Research Authorization form must be completed by the Principal Investigator and approved by the 
IRB and Privacy Board (IRB/PB). 
17.2 
Serious Adverse Event (SAE) Reporting 
 
 
An adverse event is considered serious if it results in ANY of the following outcomes: 
• 
Death 
• 
A life-threatening adverse event 
• 
An adverse event that results in inpatient hospitalization or prolongation of existing 
hospitalization 
• 
A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• 
A congenital anomaly/birth defect 
• 
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 
Note: Hospital admission for a planned procedure/disease treatment is not considered an 
SAE. 
 
SAE reporting is required as soon as the participant signs consent. SAE reporting is 
required for 30-days after the participant’s last investigational treatment or intervention. Any 
events that occur after the 30-day period and that are at least possibly related to protocol 
treatment must be reported. 
 
If an SAE requires submission to the IRB office per IRB SOP RR-408 ‘Reporting of Serious 
Adverse Events’, the SAE report must be sent to the IRB within 5 calendar days of the event. 
The IRB requires a Clinical Research Database (CRDB) SAE report be submitted 
electronically to the SAE Office as follows: 
For IND/IDE trials: Reports that include a Grade 5 SAE should be sent to 
saegrade5@mskcc.org. All other reports should be sent to saemskind@mskcc.org. 
 
For all other trials: Reports that include a Grade 5 SAE should be sent to 
saegrade5@mskcc.org. All other reports should be sent to sae@mskcc.org. 
The report should contain the following information: 
Fields populated from CRDB: 
• 
Subject’s initials 
• 
Medical record number 
• 
Disease/histology (if applicable) 


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• 
Protocol number and title 
Data needing to be entered: 
• 
The date the adverse event occurred 
• 
The adverse event 
• 
The grade of the event 
• 
Relationship of the adverse event to the treatment (drug, device, or intervention) 
• 
If the AE was expected 
• 
The severity of the AE 
• 
The intervention 
• 
Detailed text that includes the following 
o A explanation of how the AE was handled 
o A description of the subject’s condition 
o Indication if the subject remains on the study 
• 
If an amendment will need to be made to the protocol and/or consent form 
• 
If the SAE is an Unanticipated Problem 
The PI’s signature and the date it was signed are required on the completed report. 
For IND/IDE protocols: 
The CRDB SAE report should be completed as per above instructions. If appropriate, the 
report will be forwarded to the FDA by the SAE staff through the IND Office 
 
 
17.2.1 Additional Information on Serious Adverse Event (SAE) Reporting 
Collection of Safety Information 
An Adverse Event (AE) is defined as any new untoward medical occurrence or worsening of a pre- 
existing medical condition in a patient or clinical investigation subject administered an 
investigational (medicinal) product and that does not necessarily have a causal relationship with this 
treatment. An AE can therefore be any unfavorable and unintended sign (including an abnormal 
laboratory finding, for example), symptom, or disease temporally associated with the use of 
investigational product, whether or not considered related to the investigational product. 
Although overdose and cancer are not always serious by regulatory definition, these events should be 
reported on an SAE form and sent to BMS in an expedited manner. An overdose is defined as the 
accidental or intentional ingestion or infusion of any dose of a product that is considered both 
excessive and medically important. 
Note that all pregnancies, regardless of outcome, must be reported to the sponsor on a Pregnancy 
Surveillance Form, not an SAE form. All pregnancies must be reported and followed to outcome, 
including pregnancies that occur in the female partner of a male study subject. See Section 8.4 for 
instructions on reporting pregnancies. 


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Nonserious Adverse Events 
All adverse events that are not classified as serious. 
Assignment of Adverse Event Intensity and Relationship to Investigational Product 
All adverse events, including those that are serious, will be graded according to the National Cancer 
Institute Common Toxicity Criteria for Adverse Events (NCI CTCAE), Version 4.0. 
The following categories and definitions of causal relationship to investigational product as 
determined by a physician should be used for adverse events: 
• Definite: The AE is clearly related to the intervention. 
• Probable: The AE is likely related to the intervention. 
• Possible: The AE may be related to the intervention. 
• Unlikely: The AE is doubtfully related to the intervention. 
• Unrelated: The AE is clearly NOT related to the intervention. 
Collection and Reporting 
Adverse events can be spontaneously reported or elicited during open-ended questioning, 
examination, or evaluation of a subject. (In order to prevent reporting bias, subjects should not be 
questioned regarding the specific occurrence of one or more AEs.) 
If known, the diagnosis of the underlying illness or disorder should be recorded, rather than its 
individual symptoms. The following information should be captured for all AEs: onset, duration, 
intensity, seriousness, relationship to investigational product, action taken, and treatment required. If 
treatment for the AE was administered, it should be recorded in the medical record. 
The investigator shall supply the sponsor and Ethics Committee with any additional requested 
information, notably for reported deaths of subjects. 
Collection and Reporting for Serious Adverse Events 
The investigator should notify BMS of any SAE occurring after the previously specified time period 
that is believed to be certainly, probably, or possibly related to the investigational product or 
protocol-specified procedure. 
All SAEs whether related or unrelated to the ipilimumab, must be immediately reported to BMS 
Worldwide Safety (by the investigator or designee) within 24 hours of becoming aware of the event. 
If only limited information is initially available, follow-up reports are required. The original SAE 
form must be kept on file at the study site. 
All SAEs should be faxed or emailed to BMS at: 


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Global Pharmacovigilance & Epidemiology 
Bristol-Myers Squibb Company 
Fax Number: 609-818-3804 
Email: Worldwide.safety@bms.com 
For studies conducted under an Investigator IND, any event that is both serious and unexpected must 
be reported to the FDA as soon as possible and, in no event, later than 7 days (death or life- 
threatening event) or 15 days (all other SAEs) after the investigator’s or institution’s initial receipt of 
the information. BMS will be provided with a simultaneous copy of all adverse events filed with the 
FDA. SAEs should be reported on the MedWatch Form 3500A, which can be accessed at: 
http://www.accessdata.fda.gov/scripts/medwatch/. 
MedWatch SAE forms should be sent to the FDA at: 
MEDWATCH 
5600 Fishers Lane 
Rockville, MD 20852-9787 
Fax: 1-800-FDA-0178 (1-800-332-0178) 
http://www.accessdata.fda.gov/scripts/medwatch/ 
All SAEs should simultaneously be faxed or e-mailed to BMS at: 
Global Pharmacovigilance & Epidemiology 
Bristol-Myers Squibb Company 
Fax Number: 609-818-3804 
Email: Worldwide.safety@bms.com 
Serious adverse events, whether related or unrelated to investigational product, must be recorded on 
the SAE page and reported expeditiously to BMS (or designee) to comply with regulatory 
requirements. An SAE report should be completed for any event where doubt exists regarding its 
status of seriousness. 
All SAEs must be immediately reported by confirmed facsimile transmission (fax) and mailing of the 
completed SAE page. In some instances where a facsimile machine is not available, overnight 
express mail may be used. If only limited information is initially available, follow-up reports are 
required. (Note: Follow-up SAE reports should include the same investigator term(s) initially 
reported.) In selected circumstances, the protocol may specify conditions that require additional 
telephone reporting. 
If the investigator believes that an SAE is not related to the investigational product, but is potentially 
related to the conditions of the study (such as withdrawal of previous therapy, or a complication of a 
study procedure), the relationship should be specified in the narrative section of the SAE page. 


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If an ongoing SAE changes in its intensity or relationship to the investigational product, a follow-up 
SAE report should be sent immediately to the sponsor. As follow-up information becomes available it 
should be sent immediately using the same procedure used for transmitting the initial SAE report. All 
SAEs should be followed to resolution or stabilization. 
Handling of Expedited Safety Reports 
In accordance with local regulations, BMS will notify investigators of all SAEs that are suspected 
(certainly, probably, or possibly related to the investigational product) and unexpected (i.e., not 
previously described in the Investigator Brochure). In the European Union (EU), an event meeting 
these criteria is termed a Suspected, Unexpected Serious Adverse Reaction (SUSAR). investigator 
notification of these events will be in the form of an expedited safety report (ESR). 
Other important findings which may be reported by the sponsor as an ESR include: increased 
frequency of a clinically significant expected SAE, an SAE considered associated with study 
procedures that could modify the conduct of the study, lack of efficacy that poses significant hazard 
to study subjects, clinically significant safety finding from a nonclinical (eg, animal) study, important 
safety recommendations from a study data monitoring committee, or sponsor decision to end or 
temporarily halt a clinical study for safety reasons. 
Upon receiving an ESR from BMS, the investigator must review and retain the ESR with the 
Investigator Brochure. Where required by local regulations or when there is a central IRB/IEC for the 
study, the sponsor will submit the ESR to the appropriate IRB/IEC. The investigator and IRB/IEC 
will determine if the informed consent requires revision. The investigator should also comply with 
the IRB/IEC procedures for reporting any other safety information. 
In addition, suspected serious adverse reactions (whether expected or unexpected) shall be reported 
by BMS to the relevant competent health authorities in all concerned countries according to local 
regulations (either as expedited and/or in aggregate reports). 
Nonserious Adverse Events 
The collection of nonserious AE information should begin at initiation of investigational product. 
Nonserious AE information should also be collected from the start of a placebo lead-in period or 
other observational period intended to establish a baseline status for the subjects. 
If an ongoing nonserious AE worsens in its intensity, or if its relationship to the investigational 
product changes, a new nonserious AE entry for the event should be completed. Nonserious AEs 
should be followed to resolution or stabilization, or reported as SAEs if they become serious. Follow- 
up is also required for nonserious AEs that cause interruption or discontinuation of investigational 
product, or those that are present at the end of study participation. Subjects with nonserious AEs at 
study completion should receive post-treatment follow-up as appropriate. 
All identified nonserious AEs must be recorded and described in the medical record. 


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Pregnancy 
Sexually active WOCBP must use an effective method of birth control during the course of the study, 
in a manner such that risk of failure is minimized. Before enrolling WOCBP in this clinical study, the 
investigator must review the guideline about study participation for WOCBP which can be found in 
the GCP Manual for Investigators. The topics include the following: 
• General Information 
• Informed Consent Form 
• Pregnancy Prevention Information Sheet 
• Drug Interactions with Hormonal Contraceptives 
• Contraceptives in Current Use 
• Guidelines for the Follow-up of a Reported Pregnancy. 
Before study enrollment, WOCBP must be advised of the importance of avoiding pregnancy during 
study participation and the potential risk factors for an unintentional pregnancy. The subject must 
sign an informed consent form documenting this discussion. 
All WOCBP MUST have a negative pregnancy test within 72 hours before receiving ipilimumab. 
The minimum sensitivity of the pregnancy test must be 25 IU/L or equivalent units of HCG. If the 
pregnancy test is positive, the subject must not receive ipilimumab and must not be enrolled in the 
study. 
In addition, all WOCBP should be instructed to contact the investigator immediately if they suspect 
they might be pregnant (e.g., missed or late menstrual period) at any time during study participation. 
If, following initiation of the investigational product, it is subsequently discovered that a study 
subject is pregnant or may have been pregnant at the time of investigational product exposure, 
including during at least 6 half-lives after product administration, the investigational product will be 
permanently discontinued in an appropriate manner (eg, dose tapering if necessary for subject safety). 
The investigator must immediately notify BMS of this event and record the pregnancy on the 
Pregnancy Surveillance Form (not an SAE form). Initial information on a pregnancy must be reported 
immediately to BMS, and the outcome information provided once the outcome is known. Completed 
Pregnancy Surveillance Forms must be forwarded to BMS according to SAE reporting procedures. 
Any pregnancy that occurs in a female partner of a male study participant should be reported to the 
sponsor. Information on this pregnancy will be collected on the Pregnancy Surveillance Form. 
Protocol-required procedures for study discontinuation and follow-up must be performed on the 
subject unless contraindicated by pregnancy (e.g., x-ray studies). Other appropriate pregnancy 


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follow-up procedures should be considered if indicated. In addition, the investigator must report to 
BMS, and follow-up on information regarding the course of the pregnancy, including perinatal and 
neonatal outcome. Infants should be followed for a minimum of 8 weeks. 
Other Safety Considerations 
Any significant worsening noted during interim or final physical examinations, electrocardiograms, 
x-rays, and any other potential safety assessments, whether or not these procedures are required by 
the protocol, should also be recorded in the medical record. 
18.0 
INFORMED CONSENT PROCEDURES 
Before protocol-specified procedures are carried out, consenting professionals will explain full details 
of the protocol and study procedures as well as the risks involved to participants prior to their 
inclusion in the study. Participants will also be informed that they are free to withdraw from the study 
at any time. All participants must sign an IRB/PB-approved consent form indicating their consent to 
participate. This consent form meets the requirements of the Code of Federal Regulations and the 
Institutional Review Board/Privacy Board of this Center. The consent form will include the 
following: 
1. The nature and objectives, potential risks and benefits of the intended study. 
2. The length of study and the likely follow-up required. 
3. Alternatives to the proposed study. (This will include available standard and 
investigational therapies. In addition, patients will be offered an option of supportive 
care for therapeutic studies.) 
4. The name of the investigator(s) responsible for the protocol. 
5. The right of the participant to accept or refuse study interventions/interactions and to 
withdraw from participation at any time. 
Before any protocol-specific procedures can be carried out, the consenting professional will 
fully explain the aspects of patient privacy concerning research specific information. In 
addition to signing the IRB Informed Consent, all patients must agree to the Research 
Authorization component of the informed consent form. 
Each participant and consenting professional will sign the consent form. The participant must 
receive a copy of the signed informed consent form. 


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20.0 
APPENDICES 
Appendix 1: Study Timetable 
 
 
Visit 
Visit #1: 
1st Surgical 
Appointment 
Visits #2 and #3: 
MD and IR 
evaluation 
appointments (to 
be coordinated 
whenever 
feasible) 
Visit #4: 
Ipilimumab/ 
nivolumab 
date 
Visit #5: 
Core biopsy/ 
cryoablation 
date 
Visit #6: 
Safety 
assessment (1 
day prior or 
day of 
surgery) 
Visit #7: 
Surgery 
date 
Visit #8 +: 
Safety 
assessment 2- 
3 weeks post- 
surgery and 
every 2-3 
weeks 
thereafter until 
12 weeks post 
ipi/nivo 
Potential candidates 
identified 
X 
Surgery date set 
X 
Presurgical testing date 
set 
X 
Cryoablation 
amenability determined 
by Radiology 
X 
Consent signed 
X 
Ipilimumab/nivolumab 
date set/appt with MD 
X 
Ipi/nivo infusion 
X 
Core biopsy x≥3 
X 
Research Blood 
samples* 
X 
X 
X 
X1 
Safety blood work 
(CBC, Comp, TSH) 
X 
Pathology review 
X 
History & Physical 
exam 
X 
X 
X 
X 
X 
* Baseline study bloods (CBC, COMP, TSH, Free T4, T3, ACTH, FSH, LH, Prolactin, Testosterone levels, 
HIV, Hep B, Hep C), EKG, vital signs, and research bloods may be tested with routine pre-surgical testing or 
on the day of ipilimumab/nivolumab administration. HIV, Hep B, and Hep C can be drawn within 60 days of 
study registration. 


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§this appointment will be coordinated with off-study surgery follow-up appt if feasible, visits 9 and 10 (if 
applicable) will be done every 2-3 for safety assessments up to 12 weeks after ipilimumab/nivolumab 
administration 
1. Post surgery research bloods will be collected 30 days (+/-10 days) after surgery completion. 
 
 
Appendix 2:  General Recommendation for Management of 
Suspected Immune Related Adverse Events (irAE) 
A general principle is that differential diagnoses should be diligently evaluated according to standard medical 
practice. Non-inflammatory etiologies should be considered and appropriately treated. 
 
Corticosteroids are a primary therapy for immuno-oncology drug-related adverse events. The oral equivalent 
of the recommended IV doses may be considered for ambulatory patients with low-grade toxicity. The lower 
bioavailability of oral corticosteroids should be taken into account when switching to the equivalent dose of 
oral corticosteroids. 
 
Consultation with a medical or surgical specialist, especially prior to an invasive diagnostic or therapeutic 
procedure, is recommended. 


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GI ADVERSE EVENT MANAGEMENT ALGORITHM 
Rule out non-inflammatory causes. If non-inflammatory cause is identified, treat accordingly and continue 
therapy. Opiates/narcotics may mask symptoms of perforation. Infliximab should not be used in cases of 
perforation or sepsis. 
 
 
 
 
 
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) at start 
of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of oral 
corticosteroids should be taken into account when switching to the equivalent dose of oral corticosteroids. 


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RENAL ADVERSE EVENT MANAGEMENT ALGORITHM 
Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue therapy 
 
 
 
 
 
 
 
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) at start 
of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of oral 
corticosteroids should be taken into account when switching to the equivalent dose of oral corticosteroids. 


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HEPATIC ADVERSE EVENT MANAGEMENT ALGORITHM 
Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue therapy. 
Consider imaging for obstruction. 
 
 
 
 
 
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) at start 
of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of oral 
corticosteroids should be taken into account when switching to the equivalent dose of oral corticosteroids. 
** The recommended starting dose for grade 4 hepatitis is 2mg/kg/day methylprednisolone IV. 


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ENDOCRINOPATHY MANAGEMENT ALGORITHM 
Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue therapy. 
Consider visual field testing, endocrinology consultation, and imaging. 
 
 
 
 
 
 
 
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) at start 
of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of oral 
corticosteroids should be taken into account when switching to the equivalent dose of oral corticosteroids. 


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SKIN ADVERSE EVENT MANAGEMENT ALGORITHM 
Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue therapy. 
 
 
 
 
 
 
 
 
 
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) at start 
of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of oral 
corticosteroids should be taken into account when switching to the equivalent dose of oral corticosteroids. 
*Refer to NCI CTCAE v4 for term specific grading criteria. 


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NEUROLOGICAL ADVERSE EVENT MANAGEMENT ALGORITHM 
Rule out non-inflammatory causes. If non-inflammatory cause, treat accordingly and continue therapy. 
 
 
 
 
 
 
Patients on IV steroids may be switched to an equivalent dose of oral corticosteroids (e.g. prednisone) at start 
of tapering or earlier, once sustained clinical improvement is observed. Lower bioavailability of oral 
corticosteroids should be taken into account when switching to the equivalent dose of oral corticosteroids.