Bronchoscopic Cryo-Immunotherapy of Lung Cancer
Phase: EARLY_PHASE1
Conditions: Advanced Non-small Cell Lung Cancer
Sponsor: NYU Langone Health

Official Title:
Bronchoscopic Cryo-Immunotherapy of Lung Cancer  
NCT Number: 
NCT04049474 
Study Number:
17-00812
Document Type:  
Study Protocol and Statistical Analysis Plan 
Date of the 
Document: 
January 5, 2023

Page i
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writing by the study sponsor. 
Bronchoscopic Cryo-Immunotherapy of Lung Cancer   
 
Principal Investigator:
Daniel Sterman, MD
Director, Division of Pulmonary, Critical Care, and Sleep Medicine 
New York University School of Medicine  
462 First Avenue, OBV 605A 
New York, NY 10016 
Daniel.Sterman@nyulangone.org 
646-501-4182
Additional Investigators:
(see key roles for full details)
Andrew DeMaio, MD1
Vivek Murthy, MD1
Samaan Rafeq, MD1 
Jamie Bessich, MD1
Harvey Pass, MD2  
Chandra Goparaju, PhD2 
Jun-Chieh Tsay, MD1
Isaac Laniado, MD1
Andrea B. Troxel, ScD3 
Kwok-Kin Wong, MD, PhD5
Rosemary Schluger, RN, BSN1
 
1 Division of Pulmonary, Critical Care, and Sleep Medicine 
2 Department of Cardiothoracic Surgery 
3 Department of Population Health
5 Division of Hematology and Medical Oncology 
New York University School of Medicine 
NYULMC Study Number:
S17-00812
Funding Sponsor:
NIH Exploratory/Developmental Research Grant (R21CA229714-01A1)
Additional funding may be provided by: 
 
Division of Pulmonary, Critical Care, and Sleep Medicine 
NYU School of Medicine 
462 First Avenue, OBV 605A 
New York, NY 10016
IDE Number:  
G190098 
Study Product: 
ERBOKRYO® CA – Cryosurgical Unit with Flexible ERBECRYOTM Probe 
of 1.9 mm outer diameter
Study Product Provider:  
ERBE, Inc., Tubingen, Germany
ClinicalTrials.gov Number            NCT04049474
Initial version: October 23, 2017 
Amended: 
November 10, 2017 
Amended: 
April 12, 2019 
Amended: 
May 3, 2019 
Amended: 
May 16, 2019 
Amended: 
August 22, 2019 
Amended:          September 23, 2019 
Amended: 
October 7, 2019 
Amended:  
April 7, 2021 
Amended:  
October 4, 2022 
 
 
 

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writing by the study sponsor. 
Statement of Compliance 
This study will be conducted in accordance with the Code of Federal Regulations on the Protection of Human 
Subjects (45 CFR Part 46), 21 CFR Parts 50, 56, 312, and 812 as applicable, any other applicable US 
government research regulations, and institutional research policies and procedures. The International 
Conference on Harmonisation (“ICH”) Guideline for Good Clinical Practice (“GCP”) (sometimes referred to as 
“ICH-GCP” or “E6”) will be applied only to the extent that it is compatible with FDA and DHHS regulations. The  
 
Principal Investigator will assure that no deviation from, or changes to the protocol will take place without prior 
agreement from the sponsor and documented approval from the Institutional Review Board (IRB), except where 
necessary to eliminate an immediate hazard(s) to the trial participants. All personnel involved in the conduct of 
this study have completed Human Subjects Protection Training.

Study number: S17-00812
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writing by the study sponsor. 
Table of Contents
STATEMENT OF COMPLIANCE .............................................................................................................................. II
LIST OF ABBREVIATIONS......................................................................................................................................VI
PROTOCOL SUMMARY ........................................................................................................................................... 1
SCHEMATIC OF STUDY DESIGN ............................................................................................................................ 3
1
KEY ROLES......................................................................................................................................................4
2
INTRODUCTION, BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE .................................... 6
2.1 
BACKGROUND INFORMATION AND RELEVANT LITERATURE............................................................................. 6
2.2
BRONCHOSCOPIC CRYO-IMMUNOTHERAPY (BCI)........................................................................................10
2.2.1 
Preclinical Data ................................................................................................................................... 11
2.2.2 
Clinical Data to Date ........................................................................................................................... 12
2.2.3
BCI Dose Rationale............................................................................................................................13
2.2.4
Rationale for Performing BCI .............................................................................................................14
2.3 
POTENTIAL RISKS & BENEFITS ................................................................................................................... 15
2.3.1 
Known Potential Risks ........................................................................................................................ 15
2.3.2 
Known Potential Benefits ................................................................................................................... 16
3
OBJECTIVES AND PURPOSE ...................................................................................................................... 16
3.1 
PRIMARY OBJECTIVE: DEMONSTRATE FEASIBILITY AND DETERMINE THE MTD ............................................. 16
3.2 
SECONDARY OBJECTIVES: ASSESS BCI INDUCED ANTI-TUMOR IMMUNE RESPONSES................................... 16
4
STUDY DESIGN AND ENDPOINTS ............................................................................................................... 17
4.1 
DESCRIPTION OF STUDY DESIGN ............................................................................................................... 17
4.2 
STUDY ENDPOINTS.................................................................................................................................... 17
4.2.1 
Primary Study Endpoints: Feasibility and Safety ............................................................................... 17
4.2.2 
Secondary Study Endpoints: Immune Analysis ................................................................................. 17
4.2.3 
Sub-Set Analysis and Exploratory Endpoints ..................................................................................... 19
5
STUDY ENROLLMENT AND WITHDRAWAL ............................................................................................... 19
5.1 
INCLUSION CRITERIA ................................................................................................................................. 19
5.2
EXCLUSION CRITERIA................................................................................................................................19
5.3 
VULNERABLE SUBJECTS............................................................................................................................ 20
5.4 
INCLUSION OF MEN AND WOMEN ................................................................................................................ 20
5.5
STRATEGIES FOR RECRUITMENT AND RETENTION.......................................................................................20
5.5.1 
Use of DataCore/Epic Information for Recruitment Purposes ........................................................... 20
5.6 
DURATION OF STUDY PARTICIPATION......................................................................................................... 20
5.7 
PARTICIPANT WITHDRAWAL OR TERMINATION............................................................................................. 21
5.7.1 
Reasons for Withdrawal or Termination ............................................................................................. 21
5.7.2 
Handling of Participant Withdrawals or Termination .......................................................................... 21
5.8 
PREMATURE TERMINATION OR SUSPENSION OF STUDY ............................................................................... 21
6
PROCEDURAL INTERVENTION ................................................................................................................... 22
6.1 
DESCRIPTION OF BCI ................................................................................................................................ 22
6.1.1 
Administration of BCI .......................................................................................................................... 22
6.1.2 
Dose Escalation Protocol ................................................................................................................... 22
6.1.3 
Procedures for Training of Clinicians on Procedural Intervention ...................................................... 23
6.1.4 
Assessment of Compliance with Study Procedural Intervention ........................................................ 23
7
STUDY PROCEDURES AND SCHEDULE .................................................................................................... 23
7.1 
STUDY PROCEDURES/EVALUATIONS .......................................................................................................... 23
7.1.1 
Study Specific Procedures ................................................................................................................. 23

Study number: S17-00812
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7.1.2
Standard of Care Study Procedures ..................................................................................................23
7.2
LABORATORY PROCEDURES/EVALUATIONS ................................................................................................24
7.2.1 
Clinical Laboratory Evaluations .......................................................................................................... 24
7.2.2
Other Assays or Procedures ..............................................................................................................24
7.2.3
Specimen Preparation, Handling, and Storage..................................................................................25
7.2.4 
Specimen Shipment ........................................................................................................................... 25
7.3 
STUDY SCHEDULE..................................................................................................................................... 25
7.3.1
Screening ...........................................................................................................................................25
7.3.2 
Enrollment .......................................................................................................................................... 25
7.3.3 
Study Visits ......................................................................................................................................... 26
7.3.4
Data Collection ...................................................................................................................................26
7.3.5 
Withdrawal/Early Termination Visit ..................................................................................................... 26
7.3.6
Unscheduled Visit...............................................................................................................................26
7.4
CONCOMITANT MEDICATIONS, TREATMENTS, AND PROCEDURES.................................................................26
7.5 
JUSTIFICATION FOR SENSITIVE PROCEDURES............................................................................................. 26
7.5.1 
Precautionary Medications, Treatments, and Procedures ................................................................. 26
7.6
PROHIBITED MEDICATIONS, TREATMENTS, AND PROCEDURES.....................................................................27
7.7 
PROPHYLACTIC MEDICATIONS, TREATMENTS, AND PROCEDURES................................................................ 27
7.8 
RESCUE MEDICATIONS, TREATMENTS, AND PROCEDURES .......................................................................... 27
7.9 
PARTICIPANT ACCESS TO STUDY AGENT AT STUDY CLOSURE ..................................................................... 27
8
ASSESSMENT OF SAFETY .......................................................................................................................... 27
8.1 
SPECIFICATION OF SAFETY PARAMETERS................................................................................................... 27
8.1.1 
Definition of Adverse Events (AE) ...................................................................................................... 28
8.1.2 
Definition of Serious Adverse Events (SAE) ...................................................................................... 28
8.1.3 
Definition of Unanticipated Problems (UP) ......................................................................................... 28
8.2 
CLASSIFICATION OF AN ADVERSE EVENT.................................................................................................... 29
8.2.1 
Severity of Event ................................................................................................................................ 29
8.2.2 
Relationship to BCI ............................................................................................................................. 29
8.2.3 
Expectedness ..................................................................................................................................... 30
8.2.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT ............................................................................... 30
8.3 
REPORTING PROCEDURES......................................................................................................................... 31
8.3.1 
Adverse Event Reporting ................................................................................................................... 31
8.3.2 
Unanticipated Problem Reporting ...................................................................................................... 31
8.3.3 
Reporting of Pregnancy ...................................................................................................................... 31
8.4 STUDY HALTING RULES .................................................................................................................................... 31
8.5 
SAFETY OVERSIGHT: ................................................................................................................................. 32
8.5.1 DATA SAFETY MONITORING BOARD (DSMB)..................................................................................................32
9
CLINICAL MONITORING ............................................................................................................................... 32
10 
STATISTICAL CONSIDERATIONS ............................................................................................................... 33
10.1
STATISTICAL AND ANALYTICAL PLANS......................................................................................................... 33
10.2
STATISTICAL HYPOTHESES ........................................................................................................................ 33
10.3
ANALYSIS DATASETS ................................................................................................................................. 33
10.4
DESCRIPTION OF STATISTICAL METHODS ................................................................................................... 33
10.4.1 
General Approach .......................................................................................................................... 33
10.4.2 
Analysis of the Primary Endpoint: Feasibility ................................................................................. 33
10.4.3 
Analysis of the Primary Endpoint: Safety ....................................................................................... 33
10.4.4 
Analysis of the Secondary Endpoint(s) .......................................................................................... 34
10.4.5 
Baseline Descriptive Statistics ....................................................................................................... 34
10.4.6 
Planned Interim Analysis................................................................................................................ 34
10.4.7 
Additional Sub-Group Analyses ..................................................................................................... 34
10.4.8 
Tabulation of Individual Response Data ........................................................................................ 34
10.5
SAMPLE SIZE ............................................................................................................................................ 35
10.6
MEASURES TO MINIMIZE BIAS .................................................................................................................... 35
10.6.1 
Enrollment/Randomization/Masking Procedures ........................................................................... 35

Study number: S17-00812
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10.6.2
Evaluation of Success of Blinding..................................................................................................35
10.6.3
Breaking the Study Blind/Participant Code....................................................................................35
11 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA ...................................................................... 35
12
QUALITY ASSURANCE AND QUALITY CONTROL ....................................................................................36
13
ETHICS/PROTECTION OF HUMAN SUBJECTS..........................................................................................36
13.1
ETHICAL STANDARD .................................................................................................................................. 36
13.2
INSTITUTIONAL REVIEW BOARD.................................................................................................................. 36
13.3
INFORMED CONSENT PROCESS .................................................................................................................37
13.3.1 
Consent Form Provided to Participants ......................................................................................... 37
13.3.2 
Consent Procedures and Documentation ...................................................................................... 37
13.4
PARTICIPANT AND DATA CONFIDENTIALITY .................................................................................................37
13.4.1 
Research Use of Stored Human Samples, Specimens, or Data ................................................... 38
13.5
OPTIONAL FUTURE USE OF STORED SPECIMENS ........................................................................................ 38
14
DATA HANDLING AND RECORD KEEPING................................................................................................39
14.1
DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES ........................................................................... 39
14.2
STUDY RECORDS RETENTION .................................................................................................................... 39
14.3
PROTOCOL DEVIATIONS ............................................................................................................................ 40
14.4
PUBLICATION AND DATA SHARING POLICY.................................................................................................. 40
15 
STUDY FINANCES ......................................................................................................................................... 41
15.1
FUNDING SOURCE ..................................................................................................................................... 41
15.2
COSTS TO THE PARTICIPANT ...................................................................................................................... 41
15.3
PARTICIPANT REIMBURSEMENTS OR PAYMENTS ......................................................................................... 41
16 
CONFLICT OF INTEREST POLICY ............................................................................................................... 41
17 
REFERENCES ................................................................................................................................................ 42
18 
ATTACHMENTS ............................................................................................................................................. 43
19 
SCHEDULE OF EVENTS ............................................................................................................................... 44
 

Study number: S17-00812
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List of Abbreviations 
AE
Adverse Event 
BAL
Bronchoalveolar Lavage 
BCI
Bronchoscopic Cryo-Immunotherapy
CTCAE 
Common Terminology Criteria for Adverse Events
DSMB
Data Safety Monitoring Board 
DLT
Dose-Limiting Toxicity
ECOG
Eastern Cooperative Oncology Group
EBUS
Endobronchial Ultrasound
EGFR 
Epidermal Growth Factor Receptor 
FACS
Fluorescence-Activated Cell Sorting 
ICI 
Immune Checkpoint Inhibitor
IRB
Institutional Review Board
MTD
Maximum Tolerated Dose
NYULMC 
New York University Langone Medical Center 
NSCLC 
Non-Small Cell Lung Cancer 
NSAID
Non-Steroidal Anti-Inflammatory Drug
ROSE
Rapid On-Site Cytology Evaluation
SAE 
Serious Adverse Event 
UP 
Unanticipated Problem 

Study number: S17-00812
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Updated 10/7/19 
 
 
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Protocol Summary 
Title 
Bronchoscopic Cryo-Immunotherapy of Lung Cancer  
Short Title 
BCI Study 
Brief Summary
This is a first-in-human, safety and feasibility study of bronchoscopic cryo-
immunotherapy (BCI) of peripheral lung tumors in advanced non-small cell 
lung cancer (NSCLC) and suspected NSCLC. BCI has the potential to 
stimulate anti-tumor immune responses. Pre- and post-BCI peripheral blood 
samples will be analyzed to assess for anti-tumor immune responses. Post-
BCI peripheral blood will be collected 7 and 14 days after the procedure. 
Additionally, pre-BCI bronchoalveolar lavage (BAL) specimens will be collected 
to correlate PD-1 phenotype on pre-BCI BAL lymphocytes by single cell RNA 
sequencing and BCI-induced anti-tumor immune responses.
Phase 
Phase I  
Objectives 
Primary:  
 
Establish safety and feasibility of BCI and determine the maximum 
tolerated dose (MTD) (i.e., freeze duration) 
 
Secondary:  
Assess the extent of BCI-induced anti-tumor immune responses
Methodology 
Dose-escalation trial using a 3+3 design 
Endpoints 
Primary: Feasibility and Safety  
 
Feasibility: successful performance of BCI in at least 80% of 
participants in whom BCI is attempted 
 
Safety endpoints: Dose-limiting toxicity (DLT), defined as incidence of 
bleeding complications and pneumothorax, length of time to perform 
BCI, length of fluoroscopy exposure during BCI, and other adverse 
events 
 
Secondary: Immune Analysis and Tumor Response 
 
Changes in peripheral blood CD8+ T cells expressing a combination of 
Ki67+,PD-1+, HLA-DR+, CD38+, and Bcl-2low after BCI 
 
Changes in peripheral blood lymphocyte gene expression after BCI, 
which will be assessed by the PanCancer OncoImmuneTM profile 
panel, a 770-plex gene expression panel covering innate and adaptive 
immune responses (NanoString® Technology, Seattle, WA) 
 
Correlation of PD-1 phenotype of pre-BCI BAL lymphocytes by 
single-cell RNA sequencing and BCI-induced anti-tumor immune 
responses. 
Study Duration 
12 months 
Participant Duration 
14 days 
Duration of IP 
administration 
BCI will be performed once at the time of a standard-of-care bronchoscopy for 
another indication. BCI is anticipated to take approximately 15-25 minutes. 

Study number: S17-00812
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Population 
Patients with known or suspected advanced non-small cell lung cancer 
(stages IIIA/B/C and IVA/B based on the 8th edition TNM staging), with 
an accessible peripheral lung tumor, and who have been referred to 
NYU Interventional Pulmonary for bronchoscopy for diagnostic and/or 
palliative purposes unrelated to this study 
Patients with high risk clinical features (significant smoking history, 
history of COPD, presence of spiculated lesion with bronchus sign on 
chest CT, enlargement of mediastinal/hilar lymph nodes and/or 
evidence of distant metastatic disease) may be candidates for BCI if 
they meet all other enrollment criteria. 
 
Age: > 22 years of age  
 
Gender: All genders 
 
ECOG performance status 
 
Location: New York, New York
Study Sites
New York University Langone Medical Center
Number of participants
Up to 24 participants
Description of 
Intervention 
BCI is performed by advancing a flexible cryoprobe through a bronchoscope to 
reach a target peripheral tumor. Freeze-thaw cycles will be applied to the 
target peripheral tumor with the intention of inducing anti-tumor immune 
responses. The number of freeze-thaw cycles will depend on the size of the 
target tumor. One freeze-thaw cycle will be applied per one centimeter of 
largest tumor diameter. Providers will be instructed to remain greater than 2 
cm away from critical structures. Freeze times will start at 1 second, and will 
be increased by dose-escalation protocol based on tolerability. The thaw 
phase will last until the cryoprobe separates from the target tumor. 
Reference Therapy 
None 
Key Procedures 
Bronchoscopic Cryo-Immunotherapy 
 
Bronchoalveolar lavage in the lobe regions of the primary tumor and of 
the distal portion of the same lung 
Pre-BCI (day 0) and Post-BCI (day 7 and day 14 ± 3) blood draws 
Statistical Analysis 
A standard 3+3 dose escalation design will be implemented. 
 
Safety endpoints, including the incidence of bleeding and 
pneumothorax, will be descriptive. 
 
Wilcoxon signed-rank tests will be used to assess paired pre- and 
post-BCI biomarker levels.

Study number: S17-00812
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Schematic of Study Design
Screen potential subjects. Review history, laboratory studies (CBC, BMP, PT/PTT, INR when 
available) and CT chest. Obtain informed consent.
(n = 15-24)
Pre-BCI peripheral blood collection for immune analysis
During standard-of-care bronchoscopy:
BAL in regions of the primary tumor and distal portion of the same lung
BCI
Post-BCI peripheral blood collection #1 for immune analysis
Post-BCI peripheral blood collection #2 for immune analysis
Monitor for adverse effects
Day 0
Enrollment
Day 0 
Day 7 
± 3
Day 14 
± 3
Up to 1 
year

Study number: S17-00812
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1 Key Roles 
Principal Investigator: 
Daniel Sterman, MD  
Director, Division of Pulmonary, Critical Care, and Sleep Medicine 
New York University School of Medicine  
462 First Avenue, OBV 605A 
New York, NY 10016 
Daniel.Sterman@nyumc.org 
646-501-4182 
Additional Investigators: 
 
Andrew DeMaio, MD 
Assistant Professor (Clinical) of Medicine 
Division of Pulmonary, Critical Care, and Sleep Medicine 
New York University School of Medicine  
160 East 34th St, 8th Floor  
New York, NY 10016 
Andrew.Demaio@nyulangone.org 
212-731-5637 
 
Samaan Rafeq, MD 
Senior Associate Director of Interventional Pulmonology  
Division of Pulmonary, Critical Care, and Sleep Medicine 
New York University School of Medicine  
160 East 34th St, 8th Floor  
New York, NY 10016 
Samaan.Rafeq@nyulangone.org 
212-731-5452  
 
Jamie Bessich, MD 
Associate Director of Interventional Pulmonology and Director of Bronchoscopy, Tisch Hospital 
Division of Pulmonary, Critical Care, and Sleep Medicine 
New York University School of Medicine  
160 East 34th St, 8th Floor  
New York, NY 10016 
Jamie.Bessich@nyulangone.org 
212-731-5637 
 
Harvey Pass, MD 
Chief, Division of Thoracic Surgery 
New York University School of Medicine 
160 East 34th St, 8th Floor  
New York, NY 10016 
Harvey.Pass@nyulangone.org 
212-731-5414 
 
Chandra Goparaju, PhD 
Senior Scientist, Thoracic Oncology Research Lab 

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Department of Cardiothoracic Surgery
New York University Langone Medical Center 
550 1st Ave 
New York, NY 10016 
Chandra.Goparaju@nyulangone.org 
212-263-6479
 
Jun-Chieh Tsay, MD 
Assistant Professor 
Division of Pulmonary, Critical Care, and Sleep Medicine 
New York University School of Medicine 
423 E 23rd St, 13th Floor 
New York, NY 10010 
Jun-Chieh.Tsay@nyulangone.org 
212-263-0255
 
Isaac Laniado, MD 
Pulmonary and Critical Care Fellow 
Division of Pulmonary, Critical Care, and Sleep Medicine 
New York University School of Medicine 
462 First Avenue, 7N24 
New York, NY 10016 
Isaac.laniado@nyulangone.org
619-253-1547 
 
Andrea B. Troxel, ScD 
Professor and Director, Division of Biostatistics 
Department of Population Health 
New York University School of Medicine 
650 First Ave, 521 
New York, NY 10016 
Andrea.Troxel@nyulangone.org 
212-263-6527 
 
Kwok-Kin Wong, MD, PhD 
Professor of Medicine 
Director, Division of Hematology and Medical Oncology 
New York University School of Medicine 
160 East 34th Street, 8th Floor 
New York, NY 10016 
Kwok-Kin.Wong@nyulangone.org 
212-731-5662 
 
Rosemary Schluger, RN, BSN 
Senior Research Nurse 
Division of Pulmonary, Critical Care and Sleep Medicine
New York University School of Medicine 
530 First Ave, Suite 5D, HCC-Schwartz East 
New York, NY 10016
Rosemary.Schluger@nyulangone.org
646-501-9807 
 

Study number: S17-00812
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2 Introduction, Background Information and Scientific Rationale 
2.1
Background Information and Relevant Literature
I) 
Cryoablation and Bronchoscopic Cryo-Immunotherapy (BCI) 
 
Cryotherapy is the local application of extremely cold temperatures for tissue destruction, removal, 
and/or biopsy. Nitrous oxide is the most commonly utilized cryogen, and provides temperatures below -
400C at the tip of the cryoprobe(1). In cryoablation, repetitive freeze-thaw cycles cause tissue injury and 
necrosis. Cryoablation causes cell injury leading to necrosis and apoptosis by multiple mechanisms 
including formation of intracellular ice crystals (damage cell membranes and organelles), cell 
dehydration (extra-cellular fluid freezes and the osmolarity of the remaining non-frozen extracellular 
fluid increases leading to water shifts), and vascular compromise (tissue ischemia from micro-vascular 
thrombosis)(2-4). Additionally, cryoablation may induce an anti-tumor immune response by causing the 
release of tumor antigens from dying lung cancer cells, and ultimately stimulating anti-tumor specific T 
cell activation and proliferation. In contrast, in cryobiopsy and cryoadhesion, target tissue is frozen 
without a follow-up thaw phase. Frozen target tissue becomes adherent to the cryoprobe to facilitate 
removal from the airway. 
 
Bronchoscopic cryoablation has been utilized for over twenty years for safe and effective palliative 
treatment and debulking of endobronchial tumors in the central airways (trachea and proximal 
bronchi)(5, 6). Percutaneous cryoablation has been utilized for more than a decade to 
treat peripheral primary and secondary lung tumors that are not amenable to surgical resection(7).  
However, bronchoscopic cryoablation has not yet been applied to the treatment of peripheral lung 
tumors. Peripheral lung tumors comprise lung nodules and masses, which are completely surrounded 
by lung parenchyma. These tumors are located in airways distal to sub-segmental bronchi. 
 
Bronchoscopic cryoablation of peripheral lung tumors is anticipated to induce cytotoxicity to tumor cells.  
Anti-tumor immune responses are hypothesized to be active against any remaining tumor cells locally, 
as well as at microscopic and macroscopic distant sites of malignancy. In order to achieve anti-tumor 
responses, bronchoscopic cryoablation may only need to be performed on a portion of the target tumor. 
The target tumor will be measured in its largest diameter and one freeze-thaw cycle will be performed 
per centimeter from the distal portion advancing proximally. This will be referred to as bronchoscopic 
cryo-immunotherapy or BCI. In the context of treating peripheral lung tumors, the safety profile of BCI is 
currently unknown, but is anticipated to be safe and well tolerated. 
 
II) 
Safety Profiles of Current Applications of Cryotherapy in Pulmonary Diseases and 
Anticipated Safety Profile of BCI 
 
Bronchoscopic cryoablation is an accepted, standard-of-care technique for safe and effective treatment 
of endobronchial tumors in the central airways. The American Thoracic Society and the American 
College of Chest Physicians have both endorsed the use of cryotherapy for palliation of central airway 
obstruction due to inoperable malignant tumors(8, 9). In a review of over five hundred consecutive 
patients with obstructive tracheobronchial malignant tumors, target tissue was freeze-thawed with a 
cryoprobe and accessible necrotic tumor material was then removed with biopsy forceps.  Cryoprobes 
of 2.2 mm or 5 mm, depending on target tumor size, were able to safely freeze endobronchial tumors 
using freeze times of 3 minutes. Complication rates were low, including no reported pneumothoraces 
and only a four percent rate of post-procedure hemoptysis (6). Overall, hemoptysis improved in 76% of 
symptomatic patients post-endoluminal cryotherapy.(6) Additionally, early case series using smaller 1.1 
mm probes in conjuction with robotic bronchoscopy to biopsy peripheral nodules have not reported 
significant adverse events (37, 38). 

Study number: S17-00812
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Percutaneous cryoablation is also an established approach for the treatment of peripheral primary and 
secondary lung tumors that are not amenable to surgical resection. In percutaneous cryoablation, rigid 
cryotherapy probes are inserted via transthoracic CT guidance and the target tumor becomes 
completely engulfed in an ice ball during freezing. In contrast, in our proposed novel technique of BCI, 
only a portion of the target tumor will be frozen. Percutaneous cryoablation has been shown to be 
effective for local tumor control.  In percutaneous cryoablation, the target necrotic tumor remains in-situ, 
and does not undergo any biopsy or tissue removal, which is similar to BCI.  In an observational study 
of over two hundred percutaneous cryoablation procedures of thoracic masses, 86% of masses were 
reduced or remained stable in size over a six-month period(7). In percutaneous cryoablation of 
peripheral lung tumors, the cryoprobe must pass through lung parenchyma in order to reach the target 
peripheral tumor, which carries a significant risk of causing a pneumothorax.  In a systematic review of 
observational studies on percutaneous cryoablation of lung tumors, overall rate of pneumothoraces 
ranged from 12-62% with up to 17.6% of induced pneumothoraces requiring insertion of thoracostomy 
tubes(10, 11). In BCI, however, the cryoprobe will reach the target peripheral lung tumor via the airway 
directly without traversing through the lung parenchyma. Therefore, BCI is anticipated to have a 
significantly lower risk of pneumothoraces compared to percutaneous cryoablation.   
 
Reports of hemoptysis after percutaneous cryoablation have been small volume and self-limited in the 
majority of cases (10, 11). For example, in a review of 117 consecutive patients treated with 193 
cryoablation sessions of lung tumors, there were no episodes of life-threatening pulmonary hemorrhage 
(11).  However, there have been rare case reports of life-threatening pulmonary hemorrhage 
previously(12).  Post- percutaneous cryoablation hemoptysis may be due to tumor necrosis from 
freezing or due to lung injury from the cryoprobe traversing through lung parenchyma.  In percutaneous 
cryoablation, cryoprobes with diameters up to 3mm have been used to safely administer freeze times 
lasting up to 20 minutes 7, 10). In BCI, a smaller cryoprobes of 1.1mm or 1.9mm will administer 
significantly shorter freeze times based on a dose escalation protocol (see figure). Based on the 
tolerability of percutaneous cryoablation with non-significant bleeding (if bleeding occurs), significant 
hemoptysis is not anticipated to occur in BCI. Additionally, the shorter freeze phases, smaller diameter 
(1.9mm) of the cryoprobe, and avoiding the need for the cryoprobe to traverse through lung 
parenchyma will further decrease the risk of bleeding in BCI compared to percutaneous cryoablation. 
 
Over the past several years, bronchoscopic cryobiopsy has been studied for the diagnosis of interstitial 
lung diseases, lung transplant rejection, and peripheral lung lesions, including lung cancer.  In contrast 
to cryoablation, cryobiopsy involves the freezing of target tissue for typically 3-5 seconds without a 
follow-up thaw phase (13-15).  Frozen target tissue becomes adherent to the cryoprobe to facilitate 
removal via the airway and transport to Pathology for analysis. In observational studies, the rates of 
pneumothoraces and significant bleeding after trans-bronchial cryobiopsies for the diagnosis of 
interstitial lung disease and lung transplant rejection are highly variable. For example, in a retrospective 
review of three hundred consecutive trans-bronchial cryobiopsies for evaluation of interstitial lung 
disease at a single center, the rates of pneumothoraces and bleeding were 5.0% and 5.2%, 
respectively(13).  In another retrospective review of twenty-five consecutive trans-bronchial 
cryobiopsies at a single center, the rates of pneumothoraces and significant bleeding (quantified as at 
least 100cc of hemoptysis post procedure) were 8% and 12%, respectively(14). There are significant 
differences in technique between our proposed novel procedure of BCI and trans-bronchial cryobiopsy. 
Most notably, trans-bronchial cryobiopsy does not have a thaw phase and involves the removal of 
frozen tissue which becomes adherent to the cryoprobe. In BCI, as long as passive thaw phases are of 
sufficient time to allow for target tissue and cryoprobe to separate, no target tissue will be removed, and 
significant bleeding and pneumothoraces are not anticipated to occur. For these reasons, BCI of 
peripheral lung tumors is anticipated to be safe and well-tolerated.   
   
III) 
Anticipated Feasibility of BCI 

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The feasibility of performing cryobiopsy of peripheral pulmonary lesions with the use of radial 
endobronchial ultrasound (rEBUS) guidance has been previously demonstrated (16). In a prospective 
study of thirty-eight consecutive subjects with peripheral lung lesions of less than four centimeters 
(excluding one patient with a visible endobronchial tumor), thirty-one peripheral lesions were identified 
by rEBUS.  The average time duration for tumor detection by rEBUS was 7.6 minutes. All thirty-one 
peripheral lesions underwent transbronchial cryobiopsies successfully. Based upon these experiences, 
the utilization of rEBUS to guide BCI of peripheral lung tumors is anticipated to be technically feasible 
(16). 
 
IV) 
 Proposed Mechanism for BCI Inducing Anti-Tumor Immune Responses 
 
BCI is anticipated to induce anti-tumor immune responses. Cryo-immunotherapy involves the killing of 
tumor cells by freezing, generating an “in-situ vaccination” associated with CD8+ T cell activation and 
proliferation, which has been confirmed in murine models as well as in early-phase human clinical 
trials(17). A proposed mechanism is that cryoablation may, in part, induce the release of tumor antigens 
from dying lung cancer cells. These released tumor antigens will be taken up by antigen presenting 
cells, which may interact with T cells and lead to anti-tumor specific T cell activation and proliferation.   
 
BCI induced anti-tumor immune responses may be further enhanced in combination with other 
treatments. Cryoablation combined with other therapies have demonstrated enhanced anti-tumor 
immune responses, slower tumor growth, and have been associated with improved outcomes 
compared to treatments without cryoablation in pre-clinical and experimental clinical trials of non-small 
cell lung cancer (18-21). We are particularly interested in the possibility of combining BCI with anti-PD-1 
monoclonal antibodies and other checkpoint inhibitors for the treatment of advanced non-small cell lung 
cancer.  Synergistic effects of BCI and immune checkpoint inhibitors (ICI) may enable tumor responses 
in patients refractory to treatment with ICI alone. 
 
V) 
BCI Combined with Immune Checkpoint Inhibitors 
 
Immune checkpoint inhibitors (ICI) have emerged as first-line treatment in select patients with 
advanced non-small cell lung cancer who have tumor PDL-1 expression of greater than 50% (22).  
However, only approximately twenty percent of patients with non-small cell lung cancer respond to ICI 
in the majority of clinical trials (23). Individual response to ICI therapy in advanced NSCLC is affected 
by PD-1/PD-L1 expression and the presence of T cell infiltration within the tumor microenvironment 
(24).  One strategy to increase ICI response in advanced NSCLC would be to combine ICI with other 
modalities capable of stimulating tumor-specific T cell responses in the tumor microenvironment such 
as BCI.  BCI may increase the presence of activated, tumor-reactive CD8+ T cells within the tumor 
microenvironment and peripheral blood, and may ultimately improve clinical responses to ICIs.   
The combination cryoablation and ICI has demonstrated enhanced immune responses in the 
treatment of a murine prostate cancer model and in a phase I clinical trial in breast cancer (25, 26).  In 
a murine prostate cancer model, prostate tumor cryoablation combined with an anti-CTLA-4 
monoclonal antibody (Mab) resulted in growth inhibition, greater CD4+ and CD8+ T cell infiltration, 
and higher T effector to T regulatory cell ratios in distant prostate tumors compared to cryoablation 
or anti-CTLA-4 MAb alone (25). A phase I clinical trial of neoadjuvant cryo-immunotherapy in breast 
cancer demonstrated that the combination of percutaneous cryoablation and ipilimumab was safe 
and associated with increased expression of CD4+ and CD8+ T cell activation markers (e.g. ICOS 
and Ki67) in peripheral blood mononuclear cells (26). The combination of cryoablation and immune 
checkpoint inhibitors is currently being investigated for the treatment of non-small cell lung cancer in a 
clinical trial based at Brown University (ClinicalTrials.gov identifier: NCT02469701). 

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VI)
Biomarkers to Assess for BCI Induced Anti-Tumor Immune Responses
There is no well-established peripheral blood biomarker panel to assess for immune responses after 
cryo-immunotherapy. After percutaneous cryoablation, previous studies have not demonstrated 
meaningful changes in total lymphocyte counts in peripheral blood, suggesting that biomarkers of 
cryoablation-induced immune responses lie within finer subsets of lymphocytes. Consistent with this, Lin, 
et al. recently reported that IL-2 and IFN-gamma were elevated in serum after cryoablation (25), 
suggesting that changes in T-cell function may follow the procedure. Assays of secreted cytokines in 
serum, however, fail to identify the exact cell type producing the cytokines, and these cytokines can be 
released by non-T-cells.  Moreover, tumor-specific T-cells are capable of expressing many cytokines 
other than IL-2 and IFN-gamma, each of which provides different mechanistic information about the 
nature of the immune response. Therefore, cellular assays that combine global markers of T-cell 
activation with cell-by-cell analysis of many different cytokines will be required to determine if cryoablation 
induces T-cell responses. Additionally, given the rationale and potential for combining cryoablation and 
checkpoint inhibitor therapies, an analysis of checkpoint molecule expression will also be valuable in this 
setting.  
 
Recently, a co-investigator on this study (Dr. Pratip Chattopadhyay, Director of Precision Immunology 
Incubator at NYU Langone Health) developed 30+ parameter flow cytometry technology (which has now 
been commercialized by BD Biosciences as the Symphony A5).  This technology is ideally suited to the 
analyses described above, because of the bright fluorescent dyes used (Brilliant Dyes, BD Biosciences) 
and the low electronic noise of the system.  (The technology provides better 
resolution than alternate cytometry technologies, like mass cytometry (CyTOF), 
with higher throughput, less cell loss, and a more robust hardware platform.)  
Dr. Chattopadhyay developed a number of antibody staining panels for the 
system, including a panel that assesses many immune checkpoint molecules at 
once, and a pan-cytokine/global T-cell activation panel.  Table 1 lists the 
markers studied with each panel.  There are three notable advantages of highly 
multiplexed flow cytometry panels like these.  First, they allow broad screening 
of many markers in order to find the most sensitive biomarkers in a particular 
disease setting.  Second, by finely and precisely characterizing combined 
expression of every marker studied at the single cell level, a more detailed, 
mechanistic understanding of responses is possible than with serum, secretion, 
or bulk population assays.  Finally, the technology is sample sparing; it provides 
maximal information (about protein expression) from a single vial of cells; this 
will be important given the relatively low numbers of cells collected from BAL.  
Though a number of institutions now have high-parameter flow cytometry 
technology, these panels have not been successfully developed elsewhere, and 
those institutions do not have Dr. Chattopadhyay’s expertise in application of 
this technology. 
 
Although this approach is novel in immuno-oncology, there is precedent, 
particularly from studies of vaccine responses.  In healthy individuals, baseline 
peripheral blood CD8+ T cell HLA-DR+CD38+ and Ki67+Bcl-2low expression is 
0.5-2% and less than 0.5%, respectively (26).  After immunization of healthy 
volunteers with the smallpox vaccine, up to 40% of peripheral blood CD8+ T 
cells displayed HLA-DR+CD38+Ki67+Bcl-2low (26). Similarly, in 29 patients with advanced NSCLC started 
on PD-1/PDL-1 axis inhibitory antibodies, approximately 70% of patients had a 1.5-fold increase in 
peripheral blood Ki67+CD8+ T cells demonstrating high levels of expression of DR+CD38+ Bcl-2low and 
PD-1 post-treatment (27).  Approximately 25-80% of these responding Ki67+CD8+PD-1+T cells expressed 
intra-cellular granzyme B, consistent with cytotoxic potential (27). Epstein-Barr Virus (EBV) -specific 
CD8+ T cells had minimal activation and proliferation after PD-1 targeted therapy suggesting these CD8+
Panel 1
Panel 2
CD45
CD45
Live/Dead
Live/Dead 
CD3
CD3
CD4
CD4
CD8
CD8
CD45RO
CD45RO
CCR7
CD154
CD27
CD107A
CD28
IFNG
CD127 
IL2
CD57
TNF
CD25
IL3
CD95
IL4
CD69
IL5
CD103 
IL8
CXCR6
IL9
CXCR3
IL10
BTLA
IL13
TIGIT
IL17
CTLA4 
IL21
4-1BB
GMCSF
2B4 
RANTES
GITR 
LTA
TIM3 
BCL2 
ICOS 
KI67 
PD1 
PD1
TIGIT
CD38
HLA-DR
HLA-DR
OX40
Granzyme B
CD69
CD69

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T cell responses may be tumor specific (27). Importantly, 80% of patients who developed these CD8+ T 
cell responses within 4 weeks of starting anti-PD-1 therapy had a clinical benefit.  
In addition to assessing for pan-cytokine/global T cell activation and expression of immune checkpoint 
molecules in peripheral blood mononuclear cells utilizing flow cytometry technology, the NanoString™ 
can assess for a variety of other immune biomarkers from peripheral blood.  The NanoString™ platform 
offers the convenience of straightforward high-throughput mRNA quantitation using RNA barcodes to 
probe and count mRNA transcripts of interest (28). The pre-designed OncoImmune™ Profile 
panel, which measures 770 gene targets most relevant in tumor immunobiology. 
2.2 
Bronchoscopic Cryo-Immunotherapy (BCI) 
 
BCI is a novel method to cryoablate portions of peripheral lung tumors through a bronchoscope. BCI 
will be performed using the ERBOKRYO® CA – Cryosurgical Unit with Flexible ERBECRYOTM Probe of 
1.9 mm outer diameter or using the ERBECRYOTM2 single-use flexible 1.1mm outer diameter 
cryoprobe for use with robotic bronchoscopy (ERBE, Inc., Tubingen, Germany). This device is FDA 
approved for tissue destruction of target pulmonary tumors (see attached FDA 510K Approval Form). 
Current and accepted bronchoscopic procedures using this device are bronchoscopic cryoablation 
of endobronchial tumors in the central airways and bronchoscopic transbronchial cryobiopsies of lung 
parenchyma. This device has not yet been used to perform bronchoscopic cryoablation of peripheral 
lung tumors. However, BCI is only a slight modification of known and accepted current uses of this 
device. 
Pre-BCI Planning:  
The clinical team, as per standard protocol, will conduct pre-procedural evaluation and care with no 
input from the researchers. The bronchoscopy with BCI will be performed by the NYU Interventional 
Pulmonology team under general anesthesia in the bronchoscopy suite or in the operating room at 
NYU Langone Medical Center. The pre-procedural chest CT scan will be carefully evaluated to identify 
the airway path leading to the peripheral lung tumor prior to performing BCI. 
If the patient does not have biopsy-proven non-small cell lung cancer prior to the procedure, the clinical 
team will obtain diagnostic biopsies per standard protocol without input from researchers. Exact 
location and type of biopsies (i.e. transbronchial needle aspiration of mediastinal or hilar lymph nodes 
and/or transbronchial biopsies of peripheral tumor) will be determined by the clinical team. The trans-
bronchial biopsies will be prepared for frozen section and/or transbronchial needle aspirates for rapid 
on-site cytology evaluation (ROSE), as indicated. If ROSE is performed a certified cytotechnologist will 
perform a rapid microscopic analysis of the ROSE specimen to confirm presence or absence of 
malignant cells. In cases where non-small lung cancer cannot be confirmed, but it is highly suspected 
based on clinical criteria, ROSE or frozen section, select patients with high-risk features for NSCLC, 
including significant smoking history, history of COPD, spiculated lesion with bronchus sign on chest 
CT, enlarged of mediastinal/hilar lymph nodes and or distant metastatic disease on chest CT or PET 
CT scan, may be enrolled.  
Description of BCI: 
In BCI, a radial endobronchial ultrasound (EBUS) probe (Olympus Ultrasonic Probe UM-S20-17S or 
UM-S20-20R, Tokyo, Japan) with a flexible guide sheath will be inserted into the working channel of a 
flexible bronchoscope, or may be inserted through the working channel of a robotic bronchoscope, and 
advanced into the target bronchial sub-segment to identify the peripheral tumor.  If the peripheral tumor 
is unable to be identified by radial ultrasound within twenty minutes, then the patient will not be a 
candidate to undergo BCI. Once the peripheral tumor is identified by radial EBUS, the radial EBUS 

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probe will be removed, but the guide sheath will remain in position to serve as a position marker and as 
a conduit from the bronchoscope to the location of peripheral tumor, if robotic bronchoscopy is being 
performed, the bronchoscope itself serves as the guide sheath. This will be performed under 
fluoroscopic guidance to confirm stability of guide sheath or robotic bronchoscopy position. 
The Flexible ERBECRYOTM Probe of 1.9mm outer diameter (ERBE, Inc., Tubingen, Germany) will then 
be inserted through the guide sheath or the ERBECRYOTM2 single-use flexible 1.1mm outer diameter 
cryoprobe through the robotic bronchoscope and advanced to reach the peripheral tumor. The 
cryoprobe will be applied to the target tumor. The cryoprobe will be maintained and calibrated based on 
manufacturer standards. In this study, freeze time will be selected based on a dose escalation protocol 
(Please see Section 2.2.3 for further details.) The freeze phase will be followed by a passive thaw 
phase, which will last until the flexible cryoprobe has separated from the target tissue. Freese-thaw 
cycles will be performed (with exact number based on tumor size and dose escalation protocol) from 
distal to proximal within the target tumor. One freeze-thaw cycle will be performed per one cm diameter 
of the tumor. Only one airway will be used to access the target tumor. Providers will be instructed to 
remain greater than 2 cm away from critical structures. The objective will not be to completely engulf 
the peripheral tumor in an “ice ball”, but to cryoablate a portion of the peripheral tumor. 
 
We will evaluate for formation of an ice ball with fluoroscopy as we perform BCI. We will note the 
presence or absence of discernible ice ball formation for each patient at each dose level after 
completion of BCI. 
2.2.1 Preclinical Data 
Cryoablation Induced Anti-Tumor Immune Response in Lewis Cell Lung Cancer Murine Model 
The Lewis lung carcinoma model is a syngeneic and reproducible non-small cell cancer line in 
mice(29).  In a syngeneic mouse model, immunologically compatible cancer cells are injected into an 
immunocompetent mouse.  In this study, immunocompetent mice were implanted with Lewis lung 
cancer tumors in bilateral flanks and treated with cryoablation.  In the control group, mice received a 
subcutaneous administration of LPS alone without undergoing cryoablation.  Cryoablation of 
heterotopic lung tumors in the flanks of mice led to significantly slower growth of contralateral flank 
tumors consistent with an “abscopal-like” effect of cryoablation.  At day 14 post-cryoablation, 
contralateral tumor volume was approximately five times smaller in the group receiving two cycles of 
cryoablation compared to the control group (17).  Furthermore, the percentage of CD4+ T cells and 
CD8+ T cells infiltrating the contralateral tumors increased by over five and six times, respectively in the 
group receiving two cycles of cryoablation compared to the control group.  Additionally, the 
subcutaneous peritumoral space of the cryoablated tumors had higher levels of pro-inflammatory 
cytokines, including IL-
-2, IL-6, IL-
-
-
(17).  These findings were highly suggestive that the “abscopal-like” effect of cryoablation of lung 
cancer was immunological in nature, and mediated both by cellular and cytokine effector mechanisms. 
Enhancement of Anti-Tumor Immune Response with Cryoablation Combined with 
Immunotherapy in Lewis Lung Cancer Murine Model 
Immunocompetent mice (C57BL/6J strain) were implanted with Lewis Lung carcinoma tumors in their 
intra-foot pads.  In addition to treatment with cryoablation, bone marrow derived immature dendritic 
cells (DC) were isolated, cultured ex-vivo, and then co-administered with an immune adjuvant, CpG-
ODN, by intratumoral injection one hour after cryoablation and again seven days after cryoablation.  
Additional treatment arms for comparison included treatment with and without cryoablation followed by 
intratumoral PBS, intratumoral DC or CpG-ODN, or intratumoral DC cells and CpG-ODN.  The tumor 
draining lymph nodes were analyzed seven days after the second treatment (14 days after 

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cryoablation).  In these lymph nodes, there were significant increases in CD4+ T cells and CD8+ T cells, 
and a significant decrease in T regulatory cells in the combined cryoablation and treatment with 
intratumoral DC and CpG-ODN group compared to all other arms.  (T regulatory cells act to maintain 
tolerance to self-antigens and to downregulate immune responses.  A decrease in the number of T 
regulatory cells can increase tumor immunity.(30)) Additionally, there were significantly increased CD4+ 
T cells and CD8+ T cell expression of IFN-gamma and TNF-alpha levels (involved in TH1 mediated 
immune responses), but no increase in CD4+ T cells or CD8+ T cell expression of Il-4 (involved in TH2
mediated immune responses)(18).  Overall, combined cryoablation with intratumoral DC and CpG-ODN 
led to slower tumor growth, less lung cancer metastasis, and improved overall survival compared to 
other treatment groups (18).   
 
Cryotherapy Combined with Anti-CTLA-4 Therapy for Enhanced Anti-Tumor Immune Response 
in Prostate Carcinoma Murine Model 
In immunocompetent mice (C57BL/6J strain), cryoablation of a primary prostate tumor (Tramp C2 
prostate cancer model) combined with anti-CTLA-4 therapy led to slower tumor growth of a secondary 
prostate tumor or complete rejection of prostate cancer cells upon after inoculation at a distant site 
compared to treatment with cryoablation alone, anti-CTLA-4 therapy alone, and placebo(31).  After 50 
days, tumor-free survival was 44% when treated with combination therapy compared to 0% in all other 
groups.   
Additionally, secondary prostate tumors were infiltrated with higher levels of CD4+ and CD8+ cells and 
had a higher T effector cell to T regulatory cell ratio when treated with combined therapy compared to 
cryoablation alone, anti-CTLA-4 therapy alone, and placebo(31).  The total number of CD8+ T cells per 
mg of tumor increased 10-fold compared to cryoablation alone and placebo, and two-fold over anti-
CTLA-4 therapy alone.  The total number of CD4+Foxp3- T cells per mg of tumor increased 8-fold 
compared to cryoablation alone and placebo, and 4-fold over anti-CTLA-4 therapy alone (31).  In 
contrast to the significant anti-tumor immune response to cryoablation alone observed in the prior study 
using the Lewis Lung Cancer Murine Model, in this study, treatment of primary prostate tumor with 
cryoablation alone did not demonstrate an effect on secondary tumor growth or secondary tumor T cell 
infiltration.  Despite this finding, the above significant synergistic immune and survival effects of 
cryoablation with anti-CTLA-4 therapy were observed.   
2.2.2 Clinical Data to Date 
Improved Outcomes When Targeted Therapy is Combined with Cryoablation for the Treatment 
of Advanced Non-Small Cell Lung Cancer   
In a small, randomized controlled trial, 36 female patients who were non-smokers received either 
percutaneous cryoablation prior to gefitinib (epidermal growth factor receptor [EGFR] transcription 
kinase inhibitor) or gefitinib alone for treatment of stage IIIb and stage IV, EGFR positive, non-small cell 
lung cancer.  Treatment with cryoablation prior to initiation of gefitinib was associated with significantly 
more partial regression (55.6% versus 27.8%), increased progression free survival (8.4 months versus 
5.2 months), and increased 1-year survival rate (66.7% versus 33.3) (19).  These studies support the 
use of combination therapies with cryoablation to improve clinical outcomes for treatments of advanced 
non-small cell lung cancer. 
 
Enhanced Anti-Tumor Response and Slower Tumor Growth with Cryoablation Combined with 
Immunotherapy for the Treatment of Advanced Non-Small Cell Lung Cancer  
In an experimental clinical trial, 60 patients with stage III and stage IV non-small cell lung cancer were 
randomized to receive percutaneous cryoablation alone or percutaneous cryoablation followed by 

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allogeneic natural killer (NK) cell immunotherapy.  Percutaneous cryoablation of lung, liver, and bone 
tumors due to non-small cell lung cancer were performed. In the 30 patients who were treated with 
combination therapy, allogeneic donor peripheral blood was collected for NK cell culture and isolation.   
NK cell immunotherapy was administered 3-5 days after percutaneous cryoablation.  Peripheral blood 
was collected 9 days after cryoablation. Compared to peripheral blood post-cryoablation alone, 
peripheral blood post-combined therapy was remarkable for significant increases in number of T cells, 
CD8+ T cells, and specific cytokines involved in TH1 mediated immune responses (Il-2 and IFN-gamma) 
(20).  Maximal tumor diameters at 1 month post-treatment were significantly smaller in both groups 
compared to pre-treatment diameters. At 3 months post-treatment, maximal tumor diameter was 
significantly smaller in the combined group compared to the cryoablation alone group.  Effective clinical 
response rates included complete responders (arterial enhancement imaging of tumor disappeared) 
and partial responders (total reduction in tumor diameter of more than 30%).  The response rate 3 
months post-treatment was 63.3% in the combined group compared to 43.3% in the cryoablation alone 
group (p< 0.01) (20). 
Improved Outcomes When Chemotherapy and/or Immunotherapy Is Combined with 
Cryoablation for the Treatment of Advanced Non-Small Cell Lung Cancer 
In a single center retrospective review of 161 patients treated for stage IV non-small cell lung cancer, 
there was an association between combination treatment regimens involving percutaneous cryoablation 
and improved survival compared to treatment regimens without percutaneous cryoablation(21). Eighty-
six patients’ treatment regimens included percutaneous cryoablation compared to 75 patients whose 
treatment regimens did not include percutaneous cryoablation. Treatment regimens also included 
platinum-based chemotherapy, dendritic cells co-cultured with cytokine-induced killer cells (DC-CIK), or 
both. Median overall survival was significantly longer at 20 months in patients whose treatment included 
cryoablation versus 10 months in patients treated without cryoablation. In 33 patients receiving all three 
treatments, median overall survival was 27 months. In comparison, median overall survival in 32 
patients treated with cryoablation and chemotherapy and 21 patients treated with cryoablation and 
immunotherapy were 18 months and 17 months, respectively. In 44 patients who received 
chemotherapy alone and 31 patients who received chemotherapy with immunotherapy, median overall 
survival was 8.5 months and 12 months, respectively(21). 
Pre- and Post- Percutaneous Cryoablation Peripheral Blood Biomarkers  
 
In a small clinical trial of five patients treated with percutaneous cryoablation for various malignancies, 
there were no significant changes in pre- cryoablation and post- cryoablation peripheral blood T cell 
subsets (CD4+, CD8+, and regulatory T cells)(32).
Similarly, in a small clinical trial of 30 patients with advanced non-small cell lung cancer treated with 
percutaneous cryoablation alone, post-cryoablation peripheral blood total T cells and CD8+ T cells both 
only marginally increased by 1.1-fold compared to pre-cryoablation peripheral blood (20).  However, 
there were significant increases in specific cytokines involved in TH1 mediated immune responses 
levels in post-cryoablation peripheral blood compared to pre-cryoablation peripheral blood:  Il-2 and 
IFN-gamma levels increased by 1.9-fold and 2.3-fold, respectively (20). 
2.2.3 BCI Dose Rationale 
 
BCI of peripheral lung tumors has not been performed previously. To maximize safety, the initial freeze 
time is shorter than standard freeze times for cryobiopsy of peripheral lesions (15). Our dose escalation 
will initiate patients at a safe freeze time for which there is extensive clinical experience with related 
procedures. If there is no complication from this, in the next cohort of patients we will gradually escalate 
dose to those higher than are routinely performed in bronchoscopic cryobiopsy. 

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Freeze times will be escalated up to 30 seconds based on tolerability. In comparison, freeze times of up 
to 20 minutes have been safely administered during percutaneous cryoablation of peripheral lung 
tumors (7, 10).  Although an optimal freeze time duration is unknown, longer freeze times may be 
needed in order to induce anti-tumor immune responses.  Based on tolerability, freeze times will be 
increased as described in the following table: 
 
 
 
 
Dose Level
Freeze Time (per site)
Thaw Time
Level 1
1 second
Will last until the probe separates from target 
tissue* 
Level 2
2 seconds
Will last until the probe separates from target 
tissue* 
Level 3
3 seconds
Will last until the probe separates from target 
tissue* 
Level 4
5 seconds
Will last until the probe separates from target 
tissue* 
Level 5
10 seconds
Will last until the probe separates from target 
tissue* 
Level 6
20 seconds
Will last until the probe separates from target 
tissue* 
Level 7
30 seconds
Will last until the probe separates from target 
tissue* 
 
* Thaw times at each dose level will be captured for each site. 
 
2.2.4 Rationale for Performing BCI 
 
Although anti-PD-1 monoclonal antibody therapy has emerged as first-line treatment in select 
patients with advanced non-small cell lung cancer who have tumor PDL-1 expression of over 50% 
(22), only around 20% of patients respond to immune checkpoint inhibitors in the majority of clinical 
trials (23).  Combination therapy of cryoablation and anti-PD-1 therapy has the potential to enhance 
anti-tumor immune responses and improve clinical efficacy compared to treatment with anti-PD-1 
therapy alone.  In theory, BCI could stimulate anti-tumor specific T cell activation and proliferation, 
and PD-1 inhibition would prevent these tumor specific T cells from developing T cell anergy or 
immune tolerance by blocking PD-1/PD-L1 interactions. 
 
Currently, percutaneous cryoablation is an accepted technique for establishing local control of 
peripheral lung tumors which are not amenable to surgical resection. There are, however, potential 
benefits to performing BCI instead of percutaneous cryoablation of peripheral lung tumors.  Major 
innovations of BCI over percutaneous cryoablation of NSCLC include a lower theoretical rate of 
pneumothorax; significantly shorter treatment time; the ability to perform BCI during the same 
procedure as diagnostic bronchoscopy, and to concomitantly obtain tissue samples from primary 
tumor, draining lymph nodes, and periphery of lung. Additionally, unlike percutaneous cryoablation, 
BCI may allow for a greater capacity for safe, repeated procedures for future combination trials with 
ICI. 
 
We hope that this trial will demonstrate that BCI is feasible and safe, and that BCI induces 
significant anti-tumor immune responses. These results would support future investigations of the 

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efficacy of BCI combined with systemic and/or local delivery of anti-PD-1 therapy for the treatment 
of select patients with advanced non-small cell lung cancer.  
2.3
Potential Risks & Benefits 
2.3.1
Known Potential Risks
Potential Risks of Bronchoscopic Cryo-Immunotherapy (BCI)
 
The radial endobronchial ultrasound probe, which will be used to identify lung tumors in BCI, could potentially 
cause airway trauma, bleeding, or pneumothorax in rare cases.  When lung tumors undergo cryoablation in 
BCI, potential risks include bleeding, hemoptysis, and pneumothorax.  Because BCI does not involve taking 
any biopsies or removing any lung tissue, the risk of bleeding, hemoptysis, and pneumothorax directly 
related to this intervention are anticipated to be low.  After applying freezing temperatures, rewarming 
will occur to ensure that the cryoprobe does not remain frozen to the target lung tumor. This will avoid 
accidentally removing or performing a biopsy on the target tumor, which could cause bleeding, 
hemoptysis, or pneumothorax.  However, if a large pneumothorax occurs and is not properly treated, or 
if severe bleeding occurs, which is not properly controlled, then these potential complications could 
theoretically cause severe hypoxemia and be life-threatening. If a pneumothorax occurs, treatment 
with oxygen and possibly tube thoracostomy, depending on the size of the pneumothorax, will be 
provided. If significant bleeding or hemoptysis occurs, treatment to stop the bleeding may include 
applying iced saline, topical epinephrine, or placement of an endobronchial blocker. There may be 
additional risks of BCI that are currently unforeseeable. We will take all due precautions to determine if 
a potential participant is at increased risk for any adverse events and to minimize any potential 
complications in advance of undergoing BCI.      
 
BCI will involve exposure to radiation from fluoroscopy to guide the placement of the probe. The risks of 
receiving very small doses of radiation are thought to be low. These risks are not actually 
known. Pregnant women cannot be exposed to radiation. Women of child-bearing age must have a 
negative pregnancy test before they can have an X-ray and undergo appropriate lead shielding to 
minimize exposure to reproductive organs. 
 
Participation in this study will lengthen the overall time of bronchoscopy and exposure to general 
anesthesia by approximately 15-25 minutes. In rare cases, risks associated with general anesthesia 
include arrhythmias, heart attack, stroke, and death. All due precautions will be taken to determine if 
you a potential participant is at increased risk for any of these complications. General anesthesia will be 
provided by an anesthesiologist throughout the procedure. 
Potential Risks of Bronchoalveolar Lavage (BAL) 
BAL is a low risk procedure, and sometimes is included as a standard of care component in routine 
bronchoscopy for lung cancer. BAL will be performed for research purposes in this study.  Potential 
rare risks of bronchoalveolar lavage (BAL) are bronchospasm, airway trauma, bleeding, fever, 
infection, pneumothorax, and hypoxemia. 
Potential Risks of Peripheral Blood Draw  
Potential risks of blood draws are localized bruising, redness or swelling at the venipuncture site. This 
is normally self - limited, requiring no treatment. 
Other Potential Risks 

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Participating in clinical research involving the access to, use, and sharing of health information involves 
a risk of loss of confidentiality. However, privacy procedures in place and good clinical practice 
guidelines are followed for the study to minimize risks associated with research procedures and 
participation. We will do our best to minimize this risk by labeling information and samples with a 
unique code and securely keeping identifiable information separates from data and samples.  
Enrollment in this study will have no impact on the standard of care portion of the bronchoscopy. 
Participation will also not delay you from receiving standard of care lung cancer treatment.  
2.3.2 Known Potential Benefits 
Although bronchoscopic cryo-immunotherapy may cause tissue necrosis of a portion of the target 
peripheral lung tumor and has the potential to induce anti-tumor immune responses, no potential 
benefits are anticipated to be conferred to participants in this study. Information derived from this study 
may benefit patients with lung cancer in the future. 
3
Objectives and Purpose
3.1
Primary Objective: Demonstrate Feasibility and Determine the MTD
1) Demonstrate feasibility of BCI 
 
We hypothesize that BCI will be feasible, defined as successful performance in at least 80% of patients 
in whom BCI is attempted. Successful performance of BCI will require identification of the target 
peripheral lung tumor by radial endobronchial ultrasound within 20 minutes followed by completed 
cryoablation of target tumor. 
 
2) Determine the safety and MTD of BCI 
 
We hypothesize that BCI will prove safe at each planned dose level. Using a standard 3+3 design, the 
trial will determine the maximally tolerated dose (MTD) by sequentially assessing increasing freeze 
times during the procedure. Dose-limiting toxicity (DLT) is defined as the occurrence of any of the 
following: grade 2 or 3 bleeding, pneumothorax necessitating tube thoracostomy, or any NCI CTCAE 
grade 4 or 5 adverse events, which may be possibly, probably, or definitely related to BCI (33). The 
monitoring period for dose limiting toxicity will be for seven days post-BCI. 
3.2
Secondary Objectives: Assess BCI Induced Anti-Tumor Immune Responses 
1) Pre- and Post-BCI Peripheral Blood Analysis
 
We hypothesize that BCI will induce activation and proliferation of peripheral blood T cells .  Based on 
previous work (26, 27) (see section 2.1.IV for full details), we hypothesize that some combination of 
the following markers will be displayed on peripheral blood CD8+ T cells after BCI: HLA-DR and CD38 
(surface markers of CD8+ T cell activation), Ki-67 (a cell cycle marker of proliferation), Bcl-2 (an anti-
apoptotic protein downregulated on activated CD8+ T cells), and PD-1. We also hypothesize that there 
will be significant changes in peripheral blood lymphocyte gene expression after BCI, which will be 
assessed by the PanCancer OncoImmuneTM profile panel, a 770-plex gene expression panel covering 
innate and adaptive immune responses (NanoString® Technology, Seattle, WA). 
2) Investigate BCI Association with PD-1 Phenotype of Effector T cells  in NSCLC 
 

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We hypothesize that BCI will induce PD-1 phenotype in circulating effector T cells on single-cell
RNA sequence analysis and that CD8+ T cell responses to BCI will be greater among subjects with high 
PD1 expressing T cells in BAL effluent. 
4 Study Design and Endpoints 
4.1
Description of Study Design
This is an open-label, phase I pilot study to primarily evaluate the feasibility and safety of 
bronchoscopic cryo-immunotherapy (BCI) of peripheral lung tumors in patients with non-small cell lung 
cancer or suspected non-small cel lung cancer who are undergoing a standard-of-care bronchoscopy 
for palliative and/or diagnostic purposes. Dose escalation will proceed using a standard 3+3 design. 
4.2
Study Endpoints
4.2.1 Primary Study Endpoints: Feasibility and Safety  
 
1) Each patient will be classified as having successfully undergone BCI or not. Feasibility of BCI is 
defined as successfully performing BCI in at least 80% of patients in whom BCI is attempted.    
Successful performance of BCI will require identification of the target peripheral lung tumor by 
radial endobronchial ultrasound within 20 minutes followed by completed cryoablation of target 
tumor. The target tumor will have freeze-thaw cycles applied every one centimeter of the largest 
diameter. Additional BCI features will be described as follows: 
a) Ability of radial endobronchial ultrasound to identify peripheral lung tumor 
b) Length of time to perform BCI 
c) Length of time of fluoroscopy exposure during BCI 
 
2) Dose-limiting toxicities (DLT) will be used to guide the dose-escalation procedure and determine 
the maximum tolerated dose. A DLT is defined as any of the following occurring up to 7 days 
post-BCI: 
 
 
Grade 2 or 3 bleeding, based on the following scale (34): 
a) Grade 0: absence of bleeding 
b) Grade 1: bleeding requiring suctioning to clear 
c) Grade 2: bleeding requiring an endoscopic procedure (iced saline, 1:20,000 
topical epinephrine, or bronchial occlusion) 
d) Grade 3:  severe bleeding (>100cc total volume).  Severe bleeding will also be 
defined as bleeding which cannot be controlled endoscopically, requires ICU 
admission or interventional radiology/ surgical intervention, or results in 
respiratory or hemodynamic instability (34) 
 
Pneumothorax necessitating tube thoracostomy; recorded features of pneumothorax to 
include: 
a) size 
b) associated symptomatology 
c) respiratory impairment 
d) need for chest tube placement 
 
Any NCI CTCAE (33) grade 3, 4, or 5 adverse events, which may be possibly, probably, 
or definitely related to BCI 
4.2.2 Secondary Study Endpoints: Immune Analysis  

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1)  Assess BCI Induced Anti-Tumor Immune Responses in Peripheral Blood
Research Samples: Peripheral blood will be collected by the investigatory team prior to BCI on the day 
of procedure, and at approximately 7 days and 14 days after BCI (± 3 days). Specimens may be 
processed in the William N. Rom Environmental Lung Disease Laboratory, the Segal Translational 
Lung Biology Laboratory, or in the Chattopadhyay Laboratory on the campus of the New York 
University Langone Medical Center/Bellevue Hospital Center. 
 
Flow cytometry:  Peripheral blood samples will undergo Ficoll-Paque separation. The buffy coat 
containing peripheral blood mononuclear cells (PBMCs) will be isolated, and then washed with 
phosphate buffered saline.  Isolated PBMC will undergo staining with antibodies against the markers 
described in Table 1 (Section 2.1: Background Information and Relevant Literature – Subsection VI 
Biomarkers to Assess for BCI Induced Anti-Tumor Immune Response) in Dr. Pratip Chattopadhyay’s 
laboratory in the Perlmutter Cancer Center at NYU School of Medicine.  
 
Data will be acquired on a BD Symphony A5 flow cytometer, and analyzed using FlowJo software and 
various R-based bioinformatics algorithms developed previously by Dr. Chattopadhyay and colleagues. 
The 30-parameter flow cytometry panels we plan to use (Table 1) will query multiple markers of 
activated cells (including a broad range of cytokines), and multiple markers associated with immune 
checkpoints (along with markers of T-cell differentiation). To ensure that our results do not reflect global 
T-cell activation or exhaustion, in follow-up experiments, we will substitute a cocktail of peptide MHC 
Class I multimers (“tetramers”) against influenza and EBV responses into panel 1 (the cocktail will 
cover various epitopes and HLA-types) and stimulate a parallel aliquot of cells from panel 2 with a CEF 
(CMV/EBV/influenza) cocktail of peptides or the same tetramer cocktail used for Panel 1. 
 
mRNA analysis: We will also be assessing a variety of other immune biomarkers from peripheral blood 
utilizing the NanoString™ platform, including cytokine gene expression, that may allow for the 
establishment of a variety of peripheral blood biomarkers to correlate with the effects of BCI in 
advanced NSCLC. Once sorted, the CD8+ and CD4+CD25- cells (non-Treg cells) will be processed for 
RNA extraction and NanoString™ mRNA quantitation. RNA extraction will be performed using the 
MARIS technique to insure RNA quality after sorting (35). We are particularly interested in determining 
gene expression of proteins involved in T cell activation and immune regulation, such as CTLA-4, PD-1, 
OX-40, TIM-3, IL-10, TGF-ß, IL-2, MHC-II and TNF-
The transcriptome data will be processed in 
collaboration with the Biostatistics Division led by Dr. Andrea Troxel at the NYU School of Medicine to 
calculate the relative expression of the key genes of interest previously noted. The NanoString™ 
platform offers the convenience of straightforward high-throughput mRNA quantitation using RNA 
barcodes to probe and count mRNA transcripts of interest (28). Our preliminary analysis will be 
conducted with the pre-designed OncoImmune™ Profile panel, which measures 770 gene targets most 
relevant in tumor immunobiology. 
 
2) Investigate BCI Association with PD1 phenotype of Effector T cells in NSCLC 
 
Research Samples: In addition to peripheral blood specimens as described previously, BAL fluid will 
be collected from regions of the primary tumor and of the distal portion of the same lung. 
Using a subset of samples from enrolled subjects enrolled with sufficient BAL specimens, we will 
perform single- cell RNA sequencing on sorted CD3+ T cells from BAL and PBMCs obtained at the 
time of BCI. This approach will be repeated in PBMC after 7 and 14 days post BCI. The 
percentage of PD-1  expressing T lymphocytes will be determined in CD4+ and CD8+ cells from 
both BAL and PBMC compartments. PD1+ cell percentage and transcriptome will be compared 
between responder categories. RNA sequencing and subsequent data analysis will be performed 
in the Genomic Technology and Bioinformatics Cores of NYU School of Medicine. 

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4.2.3
Sub-Set Analysis and Exploratory Endpoints
 
1) Investigation of changes in peripheral blood CD8+ T cells  displaying a combination of Ki67+,PD-
1+, HLA-DR+, CD38+, and Bcl-2low after BCI at each dose level.  This sub-set analysis will 
investigate if there is an optimal freeze time duration for inducing anti-tumor immune responses.   
 
2) Investigation of changes in peripheral blood CD8+ T cells  displaying a combination of Ki67+,PD-
1+, HLA-DR+, CD38+, and Bcl-2low after BCI at each dose level.  This sub-set analysis will 
investigate if the combination of cryoablation and current active cancer treatment will lead to 
enhanced increases in percent of peripheral blood CD8+ T cell expression of PD-1 and Ki67 
and/or CD8+ T cells displaying an activated effector-like phenotype. 
 
5
Study Enrollment and Withdrawal
5.1
Inclusion Criteria 
In order to be eligible to participate in this study, an individual must meet all of the following criteria: 
 
Biopsy-proven, advanced, inoperable non-small cell lung cancer (stages IIIA/B/C and IVA/B 
based on the 8th edition TNM staging guidelines) or suspected inoperable non-small cell lung 
cancer (stage as defined above) who may undergo on-site pathology review of tissue specimen 
obtained at time of diagnostic and staging bronchoscopy. 
o If on-site pathology review is not feasible or is unable to confirm the diagnosis of non-
small cell lung cancer, patients with high-risk features for the diagnosis of non-small 
cancer including past or current cigarette smoking, history of COPD, spiculated lesion 
with bronchus sign on chest CT, presence of mediastinal/hilar adenopathy or evidence 
of distant metastatic disease on chest CT scan or PET CT scan may be included. 
 
Pre-procedure chest CT scan with the presence of an airway path leading directly to the 
peripheral lung tumor (also known as a “bronchus sign”), and the peripheral lung tumor will need 
to be visualized by radial endobronchial ultrasound at time of bronchoscopy 
 
Undergoing bronchoscopy for diagnostic and/or palliative purpose unrelated to this study 
 
Age of 22 years or older 
 
Ability to provide informed consent  
 
Concomitant chemotherapy, immunotherapy, and/or radiation therapy are allowed 
 
ECOG performance status of 2 
5.2
Exclusion Criteria
An individual who meets any of the following criteria will be excluded from participation in this study: 
 
Pregnancy (negative pregnancy test required for women of child-bearing age)  
 
Currently on a platelet inhibitor (such as clopidogrel) other than aspirin or NSAIDS, or on a 
blood thinner (such as heparin, enoxaparin, or a novel oral anticoagulant), which is unable to be 
held for planned bronchoscopy 
 
INR    1.5 (after correction and only if checked pre-operatively based on clinical indication) 
 
Platelets  100,000 (after correction) 
 
Absence of tissue diagnosis of non-small cell lung cancer either prior to procedure or during on-
site pathology review at time of bronchoscopy in patients without the above specified high-risk 
features 

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Absolute contraindication to bronchoscopy  
5.3
Vulnerable Subjects
Vulnerable subjects will not be included in this study.
5.4
Inclusion of Men and Women  
Men and women of any races and ethnic groups are eligible for this trial. 
5.5
Strategies for Recruitment and Retention
Participants will be recruited from outpatient interventional pulmonary clinic visits and from the inpatient 
setting at NYU Langone Medical Center. Potential participants will be identified based on referrals to 
the interventional pulmonary team for a diagnostic and/or therapeutic bronchoscopy. Patients who have 
been identified as being eligible to participate in this study will be approached in the inpatient setting, in 
the Ambulatory Surgery/Endoscopy unit or during interventional pulmonary clinic visits. 
 
We expect to recruit women and minorities. The number of women and minorities recruited will be 
dependent on the demographics of the lung cancer population at NYULMC. Our recruitment plan will 
not include the use of any NYULMC media services or recruitment material. 
5.5.1 Use of DataCore/Epic Information for Recruitment Purposes 
This study will utilize EPIC to identify subjects. Any recruitment information sent by email will utilize 
Send Safe email. 
Once potential subjects have been identified, the study team will notify the treating physician (TP) that 
they have patients eligible to participate as follows:   
 
Provide TP with names of potential subjects, and discuss directly with TP whether or not 
enrollment of particular patients would be appropriate  
 
Only if TP agrees that particular patient would be appropriate for this study, potential subjects 
will be directly contacted during office clinic visit, on the Ambulatory Sugery/Endoscopy unit, or 
inpatient hospital stay 
 
Once contact is made, approved recruitment language will be used to communicate the reason they are 
being contacted and subjects will be asked if they are interested in participating in this specific study. 
Should the potential subjects agree, the study team will provide the subjects with information regarding 
the next steps for participation. 
 
If a subject requests information regarding opting out of further recruitment for all research, subjects will 
be directed to contact research-contact-optout@nyumc.org or 1-855-777-7858. 
5.6
Duration of Study Participation 
The total duration of study participation is 14 days. BCI will be performed at time of standard of care 
bronchoscopy on day 0. Peripheral blood will be collected by the investigatory team prior to BCI on the 
day of procedure, which will be drawn off the intravenous catheter inserted for the standard of care 
bronchoscopy to avoid a separate blood draw whenever possible. The investigatory team will also 
collect blood samples at approximately 7 days and 14 days after BCI (± 3 days). During each blood 

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draw, the volume of blood collected will be approximately 2 teaspoons (10 ml). This will occur at routine 
standard of care follow up clinic visits whenever possible. Alternatively, the investigatory team will 
collect blood samples at approximately 7 days and 14 days after BCI at the Perlmutter Cancer Center. 
 
Total Number of Participants and Sites 
 
Total number of participants: 15-24 
 
Sites: New York University Langone Medical Center 
5.7
Participant Withdrawal or Termination 
5.7.1
Reasons for Withdrawal or Termination
Participants are free to withdraw from participation in the study at any time upon request. An 
investigator may terminate participation in the study if: 
 
Any clinical adverse event (AE), laboratory abnormality, or other medical condition or situation 
occurs such that continued participation in the study would not be in the best interest of the 
participant 
 
The participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation 
5.7.2 Handling of Participant Withdrawals or Termination 
If participant withdraws after treatment with BCI, the participant will be contacted via phone to monitor 
for any adverse events during the seven day period post-BCI. If participant withdraws after post-BCI 
blood draws have been performed, then their samples will be discarded. 
If participant misses follow up appointment for blood draws on day 7 and 14 post-BCI, the participant 
will be contacted via phone. If participant does not respond to three phone calls, the participant will be 
considered lost to follow up. 
5.8
Premature Termination or Suspension of Study 
This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable 
cause. Written notification, documenting the reason for study suspension or termination, will be 
provided by the suspending or terminating party regulatory authorities. If the study is prematurely 
terminated or suspended, the PI will promptly inform the IRB and will provide the reason(s) for the 
termination or suspension. 
Circumstances that may warrant termination or suspension include, but are not limited to: 
 
Determination of unexpected, significant, or unacceptable risk to participants 
 
Demonstration of efficacy that would warrant stopping 
 
Insufficient compliance with protocol requirements 
 
Data that are not sufficiently complete and/or evaluable 
 
Determination of futility 
 
Study may resume once concerns about safety, protocol compliance, data quality are addressed and 
satisfy the IRB. 

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6 Procedural Intervention  
6.1
Description of BCI 
6.1.1
Administration of BCI
This will be an open label, phase I pilot study without a control group. As described in Section 2.2, the
Flexible ERBECRYOTM Probe of 1.9mm outer diameter (ERBE, Inc., Tubingen, Germany) will be 
advanced through a bronchoscope to reach the target peripheral tumor, alternatively the 
ERBECRYOTM2 single-use flexible 1.1mm outer diameter cryoprobe will be used if robotic 
bronchoscopy is being performed . The flexible cryoprobe, which reaches a minimum reported 
temperature of -890C, will be applied to the target tumor. Freeze time will be administered based on a 
dose escalation protocol, as described below. The freeze phase will be followed by a passive thaw 
phase, which will occur until the flexible cryoprobe has separated from the target tissue. The total 
number of freeze-thaw cycles will be performed (depending on tumor size) from distal to proximal within 
the target tumor. For each one centimeter of the longest diameter, one freeze-thaw cycle will be 
performed. The flexible cryoprobe will only be inserted into one airway to facilitate the cryoablation. 
Providers will be instructed to remain greater than 2 cm away from critical structures. The objective will 
not be to completely engulf the peripheral tumor in an ice ball, but to cryoablate a portion of the 
peripheral tumor. 
 
We will evaluate for formation of an “ice ball” with fluoroscopy as well as via bronchoscopic visualization 
(when feasible) as we perform BCI. We will note the presence or absence of discernible ice ball 
formation for each patient at each dose level after completion of the BCI procedure. 
6.1.2 Dose Escalation Protocol  
A standard 3+3 dose escalation strategy will be used. The table below shows the doses that will be 
provided. Three participants will be initially enrolled at Level 1. If none of the first three participants at a 
given dose experiences dose-limiting toxicity (DLT), the dose will be escalated. If two or more of the 
first three participants experience DLT, the prior dose will be deemed the maximum tolerated dose 
(MTD) and the study will be terminated. If one of the first three participants experiences DLT, an 
additional three participants will be enrolled at the same dose.  If among these six, only one 
experiences DLT, the dose will be escalated. If two or more of six experience DLT, the prior dose will 
be deemed the MTD and the study will be terminated. If the MTD is reached and only three participants 
have been treated at that dose, an additional three will be treated to ensure six subjects are treated at 
the MTD. DLT are defined in Section 4.1.1 (Primary Study Endpoints: Feasibility and Safety).
Dose Level 
Freeze Time (per site)
Thaw Time 
Level 1
1 second
Will last until the probe separates from target 
tissue*
Level 2
2 seconds 
Will last until the probe separates from target 
tissue*
Level 3
3 seconds 
Will last until the probe separates from target 
tissue*
Level 4
5 seconds 
Will last until the probe separates from target 
tissue*
Level 5
10 seconds
Will last until the probe separates from target 
tissue*
Level 6
20 seconds
Will last until the probe separates from target 
tissue*

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Level 7
30 seconds
Will last until the probe separates from target 
tissue*
6.1.3 Procedures for Training of Clinicians on Procedural Intervention 
The NYU Interventional Pulmonary service has extensive experience using the flexible cryoprobe for 
debulking of central airway tumors. BCI will be performed by a board-certified, NYU Interventional 
Pulmonologist. 
Only patients with the presence of a bronchus or airway path leading directly to the peripheral lung 
tumor will be eligible to undergo BCI. The peripheral tumor will also need to be identified by radial 
EBUS prior to performing BCI. If the peripheral tumor is unable to be identified by radial ultrasound 
within 20 minutes, then the patient will not be a candidate to undergo BCI. 
6.1.4 Assessment of Compliance with Study Procedural Intervention 
Not applicable 
7 Study Procedures and Schedule 
7.1
Study Procedures/Evaluations 
7.1.1 Study Specific Procedures 
 
Bronchoalveolar lavage from regions of the primary tumor and from the distal portion of the 
same lung on day 0 
 
BCI on day 0 
 
Blood draws on day 0 (prior to BCI) and on days 7 and 14 (± 3 days) (post-BCI) 
7.1.2 Standard of Care Study Procedures 
Pre-BCI screening:  
 
 
Medical history (including cancer and cancer treatment history, co-morbidities, functional status, 
and social history) 
 
Medication and treatment history (specifically if patient is on a platelet inhibitor other than aspirin 
or anticoagulant), and if patient has previously or is currently receiving chemotherapy, radiation 
therapy, or immunotherapy.  
 
Physical examination (vital and full physical exam will be performed by clinical team prior to 
determining patient’s fitness for undergoing bronchoscopy   
 
Chest CT assessment to evaluate for the presence of a bronchus or airway path leading directly 
to the peripheral lung tumor  
 
Screening laboratory evaluation as described in Section 7.2 
 
Pre-BCI blood collection for immune analysis 
 
During bronchoscopy:  
 
Bronchoalveolar lavage (BAL) will be performed in two locations, which are the regions of the 
primary tumor and of the distal portion of the same lung. BAL is a standard of care component 
of routine bronchoscopy for lung cancer. 

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If there is no pre-existing biopsy-proven non-small cell lung cancer diagnosis, on-site pathology 
review of a tissue biopsy obtained during bronchoscopy may be performed, if technically 
feasible or if the patient does not have high risk features for possible non-small cell lung cancer 
(see above).  
 
After the standard-of-care portion of the bronchoscopy is complete, the peripheral tumor will 
need to be identified by radial EBUS. If the peripheral tumor cannot be identified within 20 
minutes, then BCI will not be performed. 
 
BCI will be performed in eligible candidates 
 
Post-BCI: 
 
Monitor for any adverse events from day 0 to day 7 Post-BCI blood collection for immune 
analysis on day 7 ± 3 
 
Post-BCI blood collection for immune analysis on day 14 ± 3 
 
Patients who undergo BCI and are found not to have non-small cell lung cancer will be 
monitored for adverse events from day 0 to day 7 and be followed up at a standard of care 
pose-bronchoscopy visit within 7 ± 3 days. These patients will not have post-BCI blood 
collected. 
7.2
Laboratory Procedures/Evaluations
7.2.1 Clinical Laboratory Evaluations 
Screening Laboratory Evaluation: 
 
Hematology: hemoglobin, hematocrit, white blood cells (WBC) with differential count, platelet 
count. 
 
Biochemistry: basic metabolic panel 
 
Coagulation Panel: INR, PTT (only if deemed necessary by the proceduralist as part of 
standard of care pre-bronchoscopy clinical assessment) 
 
Pregnancy test: In pre-menopausal women (women who have had a menstrual period within 
the past twelve months), a pregnancy test is to be done within 24 hours prior to study 
intervention and results must be available prior to administration of study procedure. 
 
Study Laboratory Evaluation:  
  
 
Pre-BCI blood collection: Two lavender top blood tubes (approximately 10cc) will be collected 
for analysis of peripheral blood mononuclear cells and serum 
 
Bronchoalveolar lavage:  Collected from regions of the primary tumor and of the distal portion 
of the same lung 
 
Post-BCI day 7 blood collection: Two lavender top blood tubes (approximately 10cc) will be 
collected for analysis of peripheral blood mononuclear cells and serum 
 
Post-BCI day 14 blood collection: Two lavender top blood tubes (approximately 10cc) will be 
collected for analysis of peripheral blood mononuclear cells and serum 
 
For further details, pre- and post-BCI peripheral blood immune analysis is described in detail in 
Section 4.1.2 (Secondary Study Endpoints: Immune Analysis). 
7.2.2 Other Assays or Procedures 
None  

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7.2.3
Specimen Preparation, Handling, and Storage
Peripheral blood will be collected by the investigatory team prior to BCI on the day of procedure, which 
will be drawn off the intravenous catheter inserted for the standard of care bronchoscopy to avoid a 
separate blood draw whenever possible. This will occur in pre-op area, bronchoscopy suite, or 
operating room.  Bronchoalveolar fluid will be collected during the bronchoscopy.  The investigatory 
team will also collect blood samples at approximately 7 days and 14 days after BCI.  This will occur at 
routine standard of care follow up clinic visits whenever possible. Alternatively, the investigatory team 
will collect blood samples at approximately 7 days and 14 days after BCI at the Perlmutter Cancer 
Center. 
 
The investigators will store bronchoalveolar lavage fluid and blood samples on ice, and transport 
samples to the William N. Rom Environmental Lung Disease Laboratory at NYU/Bellevue Medical 
Center or the Segal Translational Lung Biology Laboratory at NYU Medical Center. The investigators 
will then process specimens. After isolated peripheral mononuclear cells undergo FACS, if additional 
processing is not to occur immediately, samples will be stored in a -80C freezer in the locked William N. 
Rom Environmental Lung Disease Laboratory at NYU/Bellevue Medical Center or the Segal 
Translational Lung Biology Laboratory at NYU Medical Center. 
7.2.4 Specimen Shipment 
Not applicable  
7.3
Study Schedule
7.3.1 Screening  
Screening Visit (Day -28 to 0) 
 
Review medical history, medications, and chest CT scan. 
 
Determine eligibility based on inclusion/exclusion criteria. 
 
Obtain informed consent of potential participant verified by signature on written informed 
consent form. 
 
Collect screening labs (if not already collected by clinical team): basic metabolic panel, 
complete blood count, and coagulation panel (if deemed necessary by the proceduralist). 
 
Schedule study visits for participants for day 7 and day 14 post-BCI (will attempt to coincide with 
scheduled office visit if possible). 
7.3.2 Enrollment 
Enrollment (Day -28 to Day 0) 
 
Obtain informed consent of potential participant (if not collected above) verified by signature on 
study informed consent form. 
 
Verify inclusion/exclusion criteria. 
 
Obtain urine pregnancy test (if applicable). 
 
Confirm demographic information, medical history, medication history, alcohol and tobacco use 
history. 
 
Record vital signs, results of examinations, other assessments. 
 
Collect pre-BCI peripheral blood sample  
 
Perform bronchoalveolar lavage during standard-of-care bronchoscopy 
 
Perform BCI 

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7.3.3
Study Visits
7.3.3.1 
Visit 1: Day 7 (± 3) post-BCI 
 
Vital signs and physical exam 
 
Record adverse events as reported by participant or observed by investigator 
 
Collect blood for immune analysis 
7.3.3.2 
Visit 2: Day 14 (± 3) post-BCI 
 
Vital signs and physical exam 
 
Record adverse events as reported by participant or observed by investigator 
 
Collect blood for immune analysis 
7.3.4 Data Collection 
 
Data on patients’ pathologic diagnosis, cancer surveillance imaging results, demographics, smoking 
history, cancer history, and medical history will be collected by researchers.  Tumor sizes on any available 
cancer surveillance imaging, cancer treatment history, and mortality data will also be collected for up to 
one-year post-bronchoscopic cryo-immunotherapy by researchers.
7.3.5 Withdrawal/Early Termination Visit 
If the subject withdraws or if early termination occurs, the participant will not be asked to provide further 
blood specimens. 
7.3.6 Unscheduled Visit 
Unscheduled visits will not occur. In the event of an adverse event requiring medical attention, the 
participant will be directed to seek immediate attention from their clinical team or to the emergency 
department. 
7.4
Concomitant Medications, Treatments, and Procedures
All concomitant prescription medications taken during study participation will be recorded on the case 
report forms (CRFs). For this protocol, a prescription medication is defined as a medication that can be 
prescribed only by a properly authorized/licensed clinician. Medications to be reported in the CRF are 
concomitant prescription medications, over-the-counter medications and non-prescription medications. 
 
In treatment naïve patients undergoing a diagnostic bronchoscopy, enrollment in this study will not lead 
to a delay in appropriate, standard of care treatment.  Involvement in this study is not anticipated to 
preclude enrollment in any other ongoing clinical trial.  In patients undergoing a diagnostic or 
therapeutic bronchoscopy who are already receiving treatment with chemotherapy, radiation, or 
immunotherapy, combination therapy with BCI could theoretically affect anti-tumor immune responses. 
7.5
Justification for Sensitive Procedures
Not applicable  
7.5.1 Precautionary Medications, Treatments, and Procedures 
Not applicable  

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7.6
Prohibited Medications, Treatments, and Procedures 
Patients currently on a platelet inhibitor (such as clopidogrel) other than aspirin or NSAIDs, or on a 
blood thinner (such as heparin, enoxaparin, or a novel oral anticoagulant) may be at increased risk for 
bleeding during their standard of care bronchoscopy or due to BCI.  These medications will need to be 
held prior to planned bronchoscopy at the discretion of the proceduralist. If prohibited medications are 
not discontinued, then the patient will not be a candidate for BCI. 
7.7
Prophylactic Medications, Treatments, and Procedures
None 
7.8
Rescue Medications, Treatments, and Procedures
In the event of dose-limiting toxicity, rescue medication, treatments, and procedures may be necessary.  
 
 
Grade 2 bleeding may be treated with iced saline, topical epinephrine, or placement of an 
endobronchial blocker 
 
Grade 3 bleeding may require bronchial artery embolization or surgical intervention  
 
Pneumothorax may require chest tube placement based on size and other clinical features 
7.9
Participant Access to Study Agent at Study Closure 
BCI will only be performed once at time of standard of care bronchoscopy. 
8 Assessment of Safety 
8.1
Specification of Safety Parameters  
Dose-limiting toxicities (DLT) will be used to guide the dose-escalation procedure and determine the 
maximum tolerated dose. A DLT is defined as any of the following occurring up to 7 days post-BCI:  
 
 
Grade 2 or 3 bleeding, based on the following scale (34): 
a) Grade 0: absence of bleeding 
b) Grade 1: bleeding requiring suctioning to clear 
c) Grade 2: bleeding requiring an endoscopic procedure (iced saline, 1:20,000 topical 
epinephrine, or bronchial occlusion) 
d) Grade 3:  severe bleeding (>100cc total volume).  Severe bleeding will also be defined as 
bleeding which cannot be controlled endoscopically, requires ICU admission or 
interventional radiology/ surgical intervention, or results in respiratory or hemodynamic 
instability (34) 
 
pneumothorax necessitating tube thoracostomy; recorded features of pneumothorax to include: 
a) size 
b) associated symptomatology 
c) respiratory impairment 
d) need for chest tube placement 
 
any NCI CTCAE (33) Grade 3, 4, or 5 adverse events, which may be possibly, probably, or 
definitely related to BCI 

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8.1.1
Definition of Adverse Events (AE)
An adverse event (AE) is any symptom, sign, illness or experience that develops or worsens in 
severity during the course of the study.  Intercurrent illnesses or injuries should be regarded as adverse 
events.  Abnormal results of diagnostic procedures are considered to be adverse events if the 
abnormality: 
 results in study withdrawal 
is associated with a serious adverse event
 is associated with clinical signs or symptoms 
leads to additional treatment or to further diagnostic tests
 is considered by the investigator to be of clinical significance 
8.1.2 Definition of Serious Adverse Events (SAE) 
Serious Adverse Event
Adverse events are classified as serious or non-serious. A serious adverse event is any AE that is:  
 fatal 
 life-threatening 
 requires or prolongs hospital stay 
 results in persistent or significant disability or incapacity 
 a congenital anomaly or birth defect 
 an important medical event 
 
Important medical events are those that may not be immediately life threatening, but are clearly of 
major clinical significance. They may jeopardize the subject, and may require intervention to prevent 
one of the other serious outcomes noted above. For example, drug overdose or abuse, a seizure that 
did not result in in-patient hospitalization, or intensive treatment of bronchospasm in an emergency 
department would typically be considered serious.  
 
All adverse events that do not meet any of the criteria for serious should be regarded as non-serious 
adverse events.  
8.1.3 Definition of Unanticipated Problems (UP) 
Unanticipated Problems Involving Risk to Subjects or Others
Any incident, experience, or outcome that meets all of the following criteria:  
 Unexpected in nature, severity, or frequency  (i.e. not described in study-related documents such 
as the IRB-approved protocol or consent form, the investigators brochure, etc) 
 Related or possibly related to participation in the research (i.e. possibly related means there is a 
reasonable possibility that the incident experience, or outcome may have been caused by the 
procedures involved in the research) 
 Suggests that the research places subjects or others at greater risk of harm (including physical, 
psychological, economic, or social harm). 
This definition could include an unanticipated adverse device effect, any serious adverse effect on 
health or safety or any life-threatening problem or death caused by, or associated with, a device, if that 
effect, problem, or death was not previously identified in nature, severity, or degree of incidence in the 
investigational plan or application (including a supplementary plan or application), or any other 
unanticipated serious problem associated with a device that relates to the rights, safety, or welfare of 
subjects (21 CFR 812.3(s)). 

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8.2
Classification of an Adverse Event
8.2.1
Severity of Event
For AEs not included in the protocol defined grading system, the following guidelines will be used to 
describe severity. 
 Mild – Events require minimal or no treatment and do not interfere with the participant’s daily 
activities. 
 Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
 Severe – Events interrupt a participant’s usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating. 
8.2.2 Relationship to BCI 
The clinician’s assessment of an AE's relationship to BCI is part of the documentation process, but it is 
not a factor in determining what is or is not reported in the study. If there is any doubt as to whether a 
clinical observation is an AE, the event should be reported. All AEs must have their relationship to study 
agent assessed. In a clinical trial, the study product must always be suspect. To help assess, the 
following guidelines are used. 
 Related – The AE is known to occur with cryoablation, there is a reasonable possibility that the 
study agent caused the AE, or there is a temporal relationship between the study agent and 
event. Reasonable possibility means that there is evidence to suggest a causal relationship 
between the study agent and the AE. 
 Not Related – There is not a reasonable possibility that BCI caused the event, there is no 
temporal relationship between the study agent and event onset, or an alternate etiology has 
been established. 
 
For all collected AEs, the clinician who examines and evaluates the participant will determine the AE’s 
causality based on temporal relationship and his/her clinical judgment. The degree of certainty about 
causality will be graded using the categories below. 
 
 Definitely Related – There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out. The clinical event, including an abnormal laboratory test 
result, occurs in a plausible time relationship to drug administration and cannot be explained by 
concurrent disease or other drugs or chemicals. The response to withdrawal of the drug 
(dechallenge) should be clinically plausible. The event must be pharmacologically or 
phenomenologically definitive, with use of a satisfactory rechallenge procedure if necessary. 
 Probably Related – There is evidence to suggest a causal relationship, and the influence of 
other factors is unlikely. The clinical event, including an abnormal laboratory test result, occurs 
within a reasonable time after BCI, is unlikely to be attributed to concurrent disease or other 
drugs or chemicals. 
 Possibly Related – There is some evidence to suggest a causal relationship (e.g., the event 
occurred within a reasonable time after BCI). However, other factors may have contributed to the 
event (e.g., the participant’s clinical condition, other concomitant events). Although an AE may 
rate only as “possibly related” soon after discovery, it can be flagged as requiring more 
information and later be upgraded to “probably related” or “definitely related,” as appropriate. 

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Unlikely to be related – A clinical event, including an abnormal laboratory test result, whose 
temporal relationship to BCI makes a causal relationship improbable (e.g., the event did not 
occur within a reasonable time after administration of the trial medication) and in which other 
drugs or chemicals or underlying disease provides plausible explanations (e.g., the participant’s 
clinical condition, other concomitant treatments). 
Not Related – The AE is completely independent of BCI and/or evidence exists that the event is 
definitely related to another etiology. There must be an alternative, definitive etiology 
documented by the clinician. 
8.2.3 Expectedness 
Dr. Jun-Chieh Tsay (Assistant Professor, Division of Pulmonary, Critical Care, and Sleep Medicine) will 
be the study’s designated medical monitor who will be responsible for determining whether an AE is 
expected or unexpected. An AE will be considered unexpected if the nature, severity, or frequency of 
the event is not consistent with the risk information previously described for the study agent. 
8.2.4 Time Period and Frequency for Event Assessment 
The occurrence of an AE or SAE may come to the attention of study personnel during study visits and 
interviews of a study participant presenting for medical care, or upon review by a study monitor. All AEs 
including local and systemic reactions not meeting the criteria for SAEs will be captured on the 
appropriate RF. Information to be collected includes event description, time of onset, clinician’s 
assessment of severity, relationship to study product (assessed only by those with the training and 
authority to make a diagnosis), and time of resolution/stabilization of the event. All AEs occurring while 
on study must be documented appropriately regardless of relationship. All AEs will be followed to 
adequate resolution. 
Any medical condition that is present at the time that the participant is screened will be considered as 
baseline and not reported as an AE. However, if the study participant’s condition deteriorates at any 
time during the study, it will be recorded as an AE. UPs will be recorded in the data collection system 
throughout the study. 
Changes in the severity of an AE will be documented to allow an assessment of the duration of the 
event at each level of severity to be performed. AEs characterized as intermittent require 
documentation of onset and duration of each episode. 
The PI will record all reportable events with start dates occurring any time after informed consent is 
obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last day of study participation. At 
each study visit, the investigator will inquire about the occurrence of AE/SAEs since the last visit. 
Events will be followed for outcome information until resolution or stabilization.  
All unresolved adverse events should be followed by the investigator until the events are resolved, the 
subject is lost to follow-up, or the adverse event is otherwise explained.  At the last scheduled visit, the 
investigator should instruct each subject to report any subsequent event(s) that the subject, or the 
subject’s personal physician, believes might reasonably be related to participation in this study.  The 
investigator should notify the study sponsor of any death or adverse event occurring at any time after a 
subject has discontinued or terminated study participation that may reasonably be related to this study.  
The sponsor should also be notified if the investigator should become aware of the development of 
cancer or of a congenital anomaly in a subsequently conceived offspring of a subject that has 
participated in this study. 

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8.3
Reporting Procedures
8.3.1
Adverse Event Reporting
Any adverse events or serious adverse events possibly, probably, or definitely related to BCI will be 
reported in real-time notification by the clinical investigators to the Data and Safety Monitoring Board 
and Data Coordinating Center.  In accordance with 21 CFR 812.150(a)(1), the clinical investigators will 
also report to the IRB as soon as possible and within 10 working days. 
 
In the event of any unexpected fatal or life-threatening suspected adverse reaction, the Data and Safety 
Monitoring Board and IRB will be notified as soon as possible and within 2 working days. 
8.3.2 Unanticipated Problem Reporting 
Incidents or events that meet the OHRP criteria for UPs require the creation and completion of an UP 
report form. It is the site investigator’s responsibility to report UPs to their IRB.  The UP report will 
include the following information: 
 Protocol identifying information: protocol title and number, PI’s name, and the IRB project 
number; 
 A detailed description of the event, incident, experience, or outcome; 
 An explanation of the basis for determining that the event, incident, experience, or outcome 
represents an UP; 
 A description of any changes to the protocol or other corrective actions that have been taken or 
are proposed in response to the UP. 
 
To satisfy the requirement for prompt reporting, UPs will be reported using the following timeline: 
 
 UPs that are SAEs will be reported to the IRB within 3 days of the investigator becoming aware 
of the event. 
 Any other UP will be reported to the IRB within 3 days of the investigator becoming aware of the 
problem. 
 All UPs should be reported to appropriate institutional officials (as required by an institution’s 
written reporting procedures) within 3 days of the IR’s receipt of the report of the problem from 
the investigator. 
8.3.3 Reporting of Pregnancy 
Pre-menopausal persons of child-bearing age will be screened with a pregnancy test prior to 
enrollment. Pregnant people will be excluded from enrollment in this study. However, if an enrolled 
participant becomes pregnant after BCI is performed, they will be eligible to remain in the study without 
any modifications to study procedures.  
8.4 Study Halting Rules
If 2 or more participants experience DLT at any dose level, then the prior dose will be determined to be 
the MTD and the study will be terminated.  Participants already enrolled in the study will be followed post 
procedure as per protocol. The study will be terminated in the event of death, massive hemoptysis, or 
acute respiratory distress syndrome (ARDS). 
 

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Additionally, the study will be suspended when three grade 3 AEs determined to be “probably related.”. 
The medical monitor will notify the investigators immediately when the third grade 3 event is reported 
and enrollment screens will stop accepting new study participants. The investigators will inform the 
DSMB members within 24 hours of this occurrence and will provide the DSMB with AE listing reports. 
The DSMB will convene an ad hoc meeting by teleconference or in writing as soon as possible. The 
DSMB will provide recommendations for proceeding or halting the study.  
8.5
Safety Oversight: 
8.5.1 Data Safety Monitoring Board (DSMB)  
Safety oversight will be under the direction of a Data Safety Monitoring Board (DSMB) composed of Dr. 
John Hay (Associate Chief of Staff for research at the VA NY Harbor Healthcare System and Associate 
Professor in the Division of Pulmonary, Critical Care, and Sleep Medicine) and Dr. Jeffrey Weber 
(Deputy Director of the Perlmutter Cancer Center and Professor of Oncology). 
 
The members of the DSMB have appropriate medical and research expertise, and are not involved in 
this study. The DSMB will meet at least semiannually to assess safety and efficacy data on each 
research subject undergoing BCI. The primary responsibilities of the DSMB will be to periodically 
review and evaluate the accumulated study data for participant safety, study conduct and progress, and 
make recommendations concerning the continuation, modification, or termination of the trial.  Pre-
defined stopping rules are described in Section 8.4: Study Halting Rules. The outcome of each DSMB 
review and decision made (eg, continuing the study, continuing with modifications, suspending the 
study, or terminating the study) will be submitted to the IRB. 
Dr. Jun-Chieh Tsay will serve as a medical monitor who be responsible for overseeing participant 
safety. Dr. Tsay has extensive expertise in both bronchoscopy and translational research. He will 1) 
review all AEs (including DLT and unanticipated events) on a regular basis throughout the trial; 2) be 
available to advise the investigators on trial-related medical questions or problems, and 3) evaluate 
cumulative participant safety data and make recommendations regarding the safe continuation of the 
study. He will identify/evaluate AE/SAEs, determine seriousness, severity, causality, and monitor and 
follow subjects to resolution. He will report the events and will notify the IRB. In the event of DLT, the 
IRB will be notified as soon as possible and within three days. 
9 Clinical Monitoring
Clinical site monitoring is conducted to ensure that the rights and well-being of human subjects are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct of the 
trial is in compliance with the currently approved protocol/amendment(s), with GCP, and with applicable 
regulatory requirement(s). 
 
 Monitoring for this study will be performed by an assigned medical monitor 
 On-site monitoring will take place throughout the study, which will include a comprehensive 
review of all data and safety  
 Details of clinical site monitoring are documented in a clinical monitoring plan. This describes in 
detail who will conduct the monitoring, at what frequency monitoring will be done, at what level of 
detail monitoring will be performed, and the distribution of monitoring reports. 

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10 Statistical Considerations 
10.1 Statistical and Analytical Plans 
All dose-limiting toxicities will be tabulated by type of DLT and dose level. 
In addition to tabulating toxicities, we will record data on pre- and post-BCI peripheral blood biomarkers. 
For the continuous immunologic outcomes, we will calculate means by sampling time and perform 
exploratory graphical analyses, but with the small sample sizes and potentially irregular lengths of the 
series of observations it will not be possible to perform formal longitudinal analysis. We will apply non-
parametric tests to evaluate changes in immunologic outcomes before and after BCI. Details are 
provided below. 
10.2 Statistical Hypotheses 
1) BCI will be feasible and safe, with a maximum tolerated dose to be determined in this trial.
2) Post-BCI peripheral blood will demonstrate an increase in the percent of CD8+ T cells expressing 
a combination of Ki67+,PD-1+, HLA-DR+, CD38+, and Bcl-2low compared to pre-BCI peripheral 
blood CD8+ T cells. 
3) CD8+ T cell responses to BCI will be greater among subjects with high PD-1 expressing T cells in 
BAL effluent. 
10.3 Analysis Datasets
All participants who were treated with BCI will be included. 
10.4 Description of Statistical Methods
10.4.1 General Approach 
 
Continuous data will be summarized with means and standard deviations. 
 
Categorical data will be summarized with frequencies and percentages.   
 
P values will be considered statistically significant at the 0.05 level.  Note that the study is 
designed to determine the MTD, and thus is not powered to detect statistically significant 
differences in any of the secondary outcomes. 
10.4.2 Analysis of the Primary Endpoint: Feasibility  
The proportion of patients in whom BCI is successfully completed will be estimated, along with its 95% 
exact confidence interval.   
10.4.3 Analysis of the Primary Endpoint: Safety  
Safety endpoints will be descriptive in nature, and any possible, probable, or definite adverse event will 
be counted only once for a given participant.  Frequency and severity of all adverse events will be 
reported, both in the total study population and stratified by dose level.   
 
Dose-limiting toxicities will be tabulated in the whole population and by dose level.  Bleeding severity 
will be graded based on severity scale as described in Section 4.1.1 (Primary Study Endpoints: 
Feasibility and Safety).  Pneumothoraces will be reported and described based on size, associated 

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symptomatology and respiratory impairment, and need for chest tube placement.  Length of time to 
perform BCI and length of time of fluoroscopy exposure during BCI will be reported in minutes.  Other 
adverse events will be recorded and categorized as per the NCI Common Terminology Criteria for 
Adverse Events (CTCAE) (33). 
 
The maximum tolerated dose (MTD) will be determined based on the dose escalation rules described 
below and in Section 6.1.1. The MTD is the dose at which fewer than 1 of 3 or at most 1 of 6 
participants on that dose experience a DLT. 
10.4.4 Analysis of the Secondary Endpoint(s) 
Wilcoxon signed-rank tests will be used to assess paired pre- and post-BCI peripheral blood 
measurements, and to assess paired post-BCI peripheral blood CD8+ T cell responses and T cell PD-1 
expression in BAL effluent. 
10.4.5 Baseline Descriptive Statistics 
All demographic and clinical characteristics of participants and tumor will be summarized and tabulated.  
Pre-BCI peripheral blood T cell levels and expression of surface and intracellular markers will be 
measured. Continuous data such as measured levels of T cell subsets will be presented with means 
and standard deviations. Categorical data such as presence of an activated effector-like phenotype will 
be presented with frequencies and percentages. 
10.4.6 Planned Interim Analysis 
10.4.6.1 Safety Review 
 
Ongoing safety review will occur as part of the 3+3 dose escalation scheme, which requires evaluation 
of dose-limiting toxicities in order to escalate to the next dose or de-escalate to a lower dose. 
10.4.6.2 Efficacy Review 
Our primary focus will be on safety and feasibility.  Formal interim analyses to review for efficacy will 
not be performed. 
10.4.7 Additional Sub-Group Analyses 
Sub-group analyses as described below will employ Wilcoxon signed-rank tests as described above for 
secondary analyses. In particular: 
1) We will compare changes in pre- and post BCI peripheral blood CD8+ T cells expressing a 
combination of Ki67+,PD-1+, HLA-DR+, CD38+, and Bcl-2low at each dose level.   
 
2) In the subset of participants receiving active treatment (radiation therapy, chemotherapy, and/or 
immunotherapy in the past 30 days) for non-small cell lung cancer, we will investigate if the 
combination of cryoablation and his/her current treatment will lead to enhanced increases in the 
percent of CD8+ T cells expressing a combination of Ki67+,PD-1+, HLA-DR+, CD38+, and Bcl-
2low compared to participants treated with BCI alone. 
10.4.8 Tabulation of Individual Response Data 
Individual participant data will be listed by time points of peripheral blood collection day. 

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10.5 Sample Size
We will aim to enroll approximately 24 patients. The exact number will be determined based on our 
dose escalation protocol (as described in section 6.1.2). 
 
The table below gives the operating characteristics of this design, presenting the likelihood of 
escalation when the true DLT rate takes various values.  For example, if the true DLT rate is 60%, 
the probability of escalation is only 0.08.  If the true DLT rate is 10%, the probability of escalation is 
0.91. 
 
True DLT Rate
10%
20%
30%
40%
50%
60%
70%
80%
90%
Probability of Escalation 
0.91
0.71
0.49
0.31
0.17
0.08
0.03
<0.001 <0.001
The small sample size will not provide sufficient power to definitively address the secondary endpoint of 
measuring BCI induced anti-tumor immune responses. For example, 15 subjects provide approximately 
80% power to detect a difference of about 0.75 standard deviations between pre- and post-BCI 
peripheral blood CD8+ T cell expression of PD-1 and Ki67, conservatively assuming a within-subject 
correlation between assessments of 0.5. However, our results will be critical for informing the design of 
further studies aimed at estimating response and survival rates with BCI combined with anti-PD1-
therapy for the treatment of advanced non-small cell lung cancer. 
10.6 Measures to Minimize Bias 
10.6.1 Enrollment/Randomization/Masking Procedures 
Not applicable  
10.6.2 Evaluation of Success of Blinding 
Not applicable  
10.6.3 Breaking the Study Blind/Participant Code 
Not applicable 
11 Source Documents and Access to Source Data 
Source data is all information, original records of clinical findings, observations, or other activities in a 
clinical trial necessary for the reconstruction and evaluation of the trial.  Source data are contained in 
source documents. Examples of these original documents, and data records include: hospital records, 
clinical and office charts, laboratory notes, memoranda, subjects’ diaries or evaluation checklists, 
pharmacy dispensing records, recorded data from automated instruments, copies or transcriptions 
certified after verification as being accurate and complete, microfiches, photographic negatives, 
microfilm or magnetic media, x-rays, subject files, and records kept at the pharmacy, at the 
laboratories, and at medico-technical departments involved in the clinical trial. It is acceptable to use 
CRFs as source documents. If this is the case, it should be stated in this section what data will be 
collected on CRFs and what data will be collected from other sources. 

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The study case report form (CRF) is the primary data collection instrument for the study. All data 
requested on the CRF must be recorded.  All missing data must be explained. If a space on the CRF is 
left blank because the procedure was not done or the question was not asked, write “N/D”.  If the item 
is not applicable to the individual case, write “N/A”. All entries should be printed legibly in black ink.  If 
any entry error has been made, to correct such an error, draw a single straight line through the 
incorrect entry and enter the correct data above it. All such changes must be initialed and dated.  DO 
NOT ERASE OR WHITE OUT ERRORS. For clarification of illegible or uncertain entries, print the 
clarification above the item, then initial and date it. 
Access to study records will be limited to IRB-approved members of the study team. The investigator 
will permit study-related monitoring, audits, and inspections by the IRB/EC, the sponsor, government 
regulatory bodies, and University compliance and quality assurance groups of all study related 
documents (e.g. source documents, regulatory documents, data collection instruments, study data 
etc.). The investigator will ensure the capability for inspections of applicable study-related facilities (e.g. 
pharmacy, diagnostic laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential inspection by government 
regulatory authorities and applicable University compliance and quality assurance offices. 
12 Quality Assurance and Quality Control 
QC procedures will be implemented beginning with the data entry system and data QC checks that will 
be run on the database will be generated. Any missing data or data anomalies will be communicated to 
the site(s) for clarification/resolution. 
Following written SOPs, the monitors will verify that the clinical trial is conducted and data are 
generated, documented (recorded), and reported in compliance with the protocol, GCP, and the 
applicable regulatory requirements (e.g., Good Laboratory Practices (GLP), Good Manufacturing 
Practices (GMP)). 
The investigational site will provide direct access to all trial related sites, source data/documents, and 
reports for the purpose of monitoring and auditing by the sponsor, and inspection by local and 
regulatory authorities. 
13 Ethics/Protection of Human Subjects
13.1 Ethical Standard
The investigator will ensure that this study is conducted in full conformity with Regulations for the 
Protection of Human Subjects of Research codified in 45 CFR Part 46, 21 CFR Part 50, 21 CFR Part 
56, and/or the ICH E6. 
13.2 Institutional Review Board 
The protocol, informed consent form(s), recruitment materials, and all participant materials will be 
submitted to the IRB for review and approval. Approval of both the protocol and the consent form must 
be obtained before any participant is enrolled. Any amendment to the protocol will require review and 
approval by the IRB before the changes are implemented to the study. All changes to the consent form 
will be IRB approved; a determination will be made regarding whether previously consented 
participants need to be re-consented. 

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writing by the study sponsor. 
13.3 Informed Consent Process 
13.3.1 Consent Form Provided to Participants
Informed consent form describing BCI, pre-BCI bronchoalveolar lavage, and pre- and post-BCI blood 
draws in detail including risks, potential benefits, and alternatives are given to the participant. Written 
documentation of informed consent is required prior to undergoing BCI. A detailed informed consent 
form has been submitted with this protocol to the IRB. 
13.3.2 Consent Procedures and Documentation 
 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the study 
and continues throughout the individual’s study participation. Extensive discussion of risks and possible 
benefits of participation will be provided to the participants and their families. Consent forms will be 
IRB-approved and the participant will be asked to read and review the document. The investigator will 
explain the research study to the participant and answer any questions that may arise. All participants 
will receive a verbal explanation in terms suited to their comprehension of the purposes, procedures, 
and potential risks of the study and of their rights as research participants. Participants will have the 
opportunity to carefully review the written consent form and ask questions prior to signing. The 
participants should have the opportunity to discuss the study with their surrogates or think about it prior 
to agreeing to participate. The participant will sign the informed consent document prior to any 
procedures being done specifically for the study. The participants may withdraw consent at any time 
throughout the course of the trial. A copy of the signed informed consent document will be given to the 
participants for their records. The rights and welfare of the participants will be protected by emphasizing 
to them that the quality of their medical care will not be adversely affected if they decline to participate 
in this study. 
 
A copy of the signed informed consent document will be stored in the subject’s research record. The 
consent process, including the name of the individual obtaining consent, will be thoroughly documented 
in the subject’s research record. Any alteration to the standard consent process (e.g. use of a 
translator, consent from a legally authorized representative, consent document presented orally, etc.) 
and the justification for such alteration will likewise be documented.  
13.4 Participant and Data Confidentiality
Information about study subjects will be kept confidential and managed according to the requirements 
of the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  Those regulations require a 
signed subject authorization informing the subject of the following:  
 What protected health information (PHI) will be collected from subjects in this study 
 Who will have access to that information and why 
 Who will use or disclose that information  
 The rights of a research subject to revoke their authorization for use of their PHI.  
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by regulation, 
retains the ability to use all information collected prior to the revocation of subject authorization.  For 
subjects that have revoked authorization to collect or use PHI, attempts should be made to obtain 
permission to collect at least vital status (i.e. that the subject is alive) at the end of their scheduled study 
period. 
 
Participant confidentiality is strictly held in trust by the participating investigators, their staff, and the 
sponsor(s) and their agents. This confidentiality is extended to cover testing of biological samples and 

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writing by the study sponsor. 
genetic tests in addition to the clinical information relating to participants. Therefore, the study protocol, 
documentation, data, and all other information generated will be held in strict confidence. No 
information concerning the study or the data will be released to any unauthorized third party without 
prior written approval of the sponsor. 
The study monitor, other authorized representatives of the sponsor, representatives of the IRB or 
pharmaceutical company supplying study product may inspect all documents and records required to 
be maintained by the investigator, including but not limited to, medical records (office, clinic, or hospital) 
and pharmacy records for the participants in this study. The clinical study site will permit access to such 
records. 
 
The study participant’s contact information will be securely stored at each clinical site for internal use 
during the study. At the end of the study, all records will continue to be kept in a secure location for as 
long a period as dictated by local IRB and Institutional regulations. 
Study participant research data, which is for purposes of statistical analysis and scientific reporting, will 
be transmitted to and stored at NYU Langone Medical Center. This will not include the participant’s 
contact or identifying information. Rather, individual participants and their research data will be 
identified by a unique study identification number. The study data entry and study management 
systems used by clinical sites and by NYU Langone Medical Center research staff will be secured and 
password protected. At the end of the study, all study databases will be de-identified and archived at 
the NYU Langone Medical Center. 
13.4.1 Research Use of Stored Human Samples, Specimens, or Data 
 Intended Use: Samples and data collected under this protocol may be used to study the immune 
response to BCI in non-small lung cancer. No genetic testing will be performed. 
 Storage: Access to stored samples will be limited to the research investigator and his research 
assistants. Samples and data will be stored using codes assigned by the investigators. Data will 
be kept in password-protected computers. Only investigators will have access to the samples 
and data. 
 Tracking: Data will be tracked using RedCap. 
o Disposition at the completion of the study: All stored samples will be discarded. Study 
participants who request destruction of samples will be notified of compliance with such 
request and all supporting details will be maintained for tracking. 
13.5 Optional Future Use of Stored Specimens
Data collected for this study will be analyzed and stored at the William Rom Laboratory at 
NYU/Bellevue Medical Center.  After the study is completed, the de-identified, archived data will be 
transmitted to and stored at the William Rom Laboratory Data Repository, under the supervision of Dr. 
Daniel Sterman, for use by other researchers including those outside of the study. Permission to 
transmit data to the William Rom Laboratory Data Repository will be included in the informed consent. 
The only PHI maintained in any research records are the research subjects’ medical record number 
and date of birth, and these are stored only on the secure NYU server. Only investigators with 
approved permission (those on the IR B protocol who have undergone CITI training), will have 
access to the data and banked samples, and appropriate steps to maintain confidentiality of the data 
will be taken, including coding data with an anonymous identifying number unique to each subject. 

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writing by the study sponsor. 
Samples will be labelled with coding data using permanent marker. Samples will be banked for up to 
five years. 
 
With the participant’s approval and as approved by the IRB, de-identified biological samples will be 
stored at the William Rom laboratory and/or the Segal Translational Lung Biology Laboratory with the 
same goal as the sharing of data with the William Rom Laboratory Data Repository. These samples 
could be used for research into the causes of lung cancer, its complications and other conditions for 
which individuals with lung cancer are at increased risk, and to improve treatment. Genetic testing will 
not be done on the samples to identify risks for other conditions. The William Rom Laboratory Data 
Repository will also be provided with a code-link that will allow linking the biological specimens with the 
phenotypic data from each participant, maintaining the masking of the identity of the participant. The PI 
will maintain the link between the sample code and the identity of the participant. 
 
During the conduct of the study, an individual participant can choose to withdraw consent to have 
biological specimens stored for future research. When the study is completed, access to study data 
and/or samples will be provided through the William Rom Laboratory Data Repository. 
14 Data Handling and Record Keeping 
14.1 Data Collection and Management Responsibilities 
Data collection is the responsibility of the clinical trial staff at the site under the supervision of the site 
PI. The investigator is responsible for ensuring the accuracy, completeness, legibility, and timeliness of 
the data reported. 
All source documents should be completed in a neat, legible manner to ensure accurate interpretation 
of data. Black ink is required to ensure clarity of reproduced copies. When making changes or 
corrections, cross out the original entry with a single line, and initial and date the change. 
 
Copies of the electronic CRF (eCRF) will be provided for use as source documents and maintained for 
recording data for each participant enrolled in the study. Data reported in the eCRF derived from source 
documents should be consistent with the source documents or the discrepancies should be explained 
and captured in a progress note and maintained in the participant’s official electronic study record. 
 
Clinical data (including AEs, concomitant medications, and expected adverse reactions data) and 
clinical laboratory data will be entered into RedCap, a 21 CFR Part 11-compliant data capture system. 
The data system includes password protection and internal quality checks, such as automatic range 
checks, to identify data that appear inconsistent, incomplete, or inaccurate. Clinical data will be entered 
directly from the source documents. 
 
Subject’s research records will be confidential. Subject’s blood specimens will be banked for future 
research without personal identifiers. Any identifying information that could link a subject to his or her 
blood sample by name will be stored in a locked cabinet by the investigators separate from any medical 
records. Subjects will be identified by code. Subjects will not be personally identified in any publication.   
14.2 Study Records Retention 
Study documents will be retained for the longer of 3 years after close-out or 5 years after final 
reporting/publication. No records will be destroyed without the written consent of the sponsor, if 
applicable. It is the responsibility of the sponsor to inform the investigator when these documents no 
longer need to be retained. 

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writing by the study sponsor. 
14.3 Protocol Deviations
A protocol deviation is any noncompliance with the clinical trial protocol, GCP, or Manual of Procedures 
(MOP) requirements. The noncompliance may be either on the part of the participant, the investigator, 
or the study site staff. As a result of deviations, corrective actions are to be developed by the site and 
implemented promptly. 
These practices are consistent with ICH E6: 
 4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3 
 5.1 Quality Assurance and Quality Control, section 5.1.1 
 5.20 Noncompliance, sections 5.20.1, and 5.20.2. 
 
It is the responsibility of the site PI/study staff to use continuous vigilance to identify and report 
deviations within 1 working day of identification of the protocol deviation, or within 1 working days of the 
scheduled protocol-required activity. 
 
All protocol deviations must be addressed in study source documents, reported to the IRB.   
 
Protocol deviations must be reported to the local IRB per their guidelines. The site PI/study staff is 
responsible for knowing and adhering to their IRB requirements. Further details about the handling of 
protocol deviations will be included in the MOP. 
14.4 Publication and Data Sharing Policy
This study will comply with the NIH Public Access Policy, which ensures that the public has access to 
the published results of NIH funded research. It requires scientists to submit final peer-reviewed journal 
manuscripts that arise from NIH funds to the digital archive PubMed Central upon acceptance for 
publication. 
The International Committee of Medical Journal Editors (ICMJE) member journals have adopted a 
clinical trials registration policy as a condition for publication. The ICMJE defines a clinical trial as any 
research project that prospectively assigns human subjects to intervention or concurrent comparison or 
control groups to study the cause-and-effect relationship between a medical intervention and a health 
outcome. Medical interventions include drugs, surgical procedures, devices, behavioral treatments, 
process-of-care changes, and the like. Health outcomes include any biomedical or health-related 
measures obtained in patients or participants, including pharmacokinetic measures and adverse 
events. The ICMJE policy, and the Section 801 of the Food and Drug Administration Amendments Act 
of 2007, requires that all clinical trials be registered in a public trials registry such as ClinicalTrials.gov, 
which is sponsored by the National Library of Medicine. Other biomedical journals are considering 
adopting similar policies. For interventional clinical trials performed under NIH IC grants and 
cooperative agreements, it is the grantee’s responsibility to register the trial in an acceptable registry, 
so the research results may be considered for publication in ICMJE member journals. The ICMJE does 
not review specific studies to determine whether registration is necessary; instead, the committee 
recommends that researchers who have questions about the need to register err on the side of 
registration or consult the editorial office of the journal in which they wish to publish. 
 
FDAAA mandates that a "responsible party" (i.e., the sponsor or designated principal investigator) 
register and report results of certain "applicable clinical trials": 
 
 Trials of Drugs and Biologics: Controlled, clinical investigations, other than Phase I investigations 
of a product subject to FDA regulation; 

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writing by the study sponsor. 
Trials of Devices: Controlled trials with health outcomes of a product subject to FDA regulation 
(other than small feasibility studies) and pediatric postmarket surveillance studies. 
 NIH grantees must take specific steps to ensure compliance with NIH implementation of FDAAA. 
15 Study Finances
15.1 Funding Source 
The principal investigator, Dr. Daniel Sterman, has received a NIH Exploratory/Developmental 
Research Grant (R21CA229714-01A1). 
15.2 Costs to the Participant 
Bronchoscopic cryo-immunotherapy and pre- and post-blood draws and analysis are being supported 
by external grant funding, which will cover study-related costs. 
 
Please note that the standard of care portion of the bronchoscopy, including the cost of anesthesia, and 
pathological evaluation of clinically indicated specimens obtained at bronchoscopy will be billed to the 
participant’s insurance, with co-payments for these services as per standard protocol. Additionally, the 
cost of any standard of care follow-up blood work or radiographic imaging will also be billed to the 
participant’s insurance, with co-payments for these services as per standard protocol. 
15.3 Participant Reimbursements or Payments 
There is no payment for participation in this study.   
16 Conflict of Interest Policy 
The independence of this study from any actual or perceived influence, such as by the pharmaceutical 
industry, is critical. Therefore any actual conflict of interest of persons who have a role in the design, 
conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, 
persons who have a perceived conflict of interest will be required to have such conflicts managed in a 
way that is appropriate to their participation in the trial. The study leadership in conjunction with the 
NYU School of Medicine has established policies and procedures for all study group members to 
disclose all conflicts of interest and will establish a mechanism for the management of all reported 
dualities of interest. 
 
Any investigator who has a conflict of interest with this study (patent ownership, royalties, or financial 
gain greater than the minimum allowable by their institution, etc.) must have the conflict reviewed by the 
NYU Langone Conflict of Interest Management Unit (CIMU) with a Committee-sanctioned conflict 
management plan that has been reviewed and approved by the study sponsor prior to participation in 
this study. All NYULMC investigators will follow the applicable conflict of interest policies. 

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writing by the study sponsor. 
17 References 
1. 
DiBardino DM, Lanfranco AR, Haas AR. Bronchoscopic Cryotherapy. Clinical Applications of the 
Cryoprobe, Cryospray, and Cryoadhesion. Annals of the American Thoracic Society. 2016;13(8):1405-15. 
2. 
Sidana A. Cancer immunotherapy using tumor cryoablation. Immunotherapy. 2014;6(1):85-93. 
3. 
Chu KF, Dupuy DE. Thermal ablation of tumours: biological mechanisms and advances in therapy. 
Nature reviews Cancer. 2014;14(3):199-208. 
4. 
Forest V, Peoc'h M, Campos L, Guyotat D, Vergnon JM. Effects of cryotherapy or chemotherapy on 
apoptosis in a non-small-cell lung cancer xenografted into SCID mice. Cryobiology. 2005;50(1):29-37. 
5. 
Seijo LM, Sterman DH. Interventional pulmonology. The New England journal of medicine. 
2001;344(10):740-9. 
6. 
Maiwand MO, Asimakopoulos G. Cryosurgery for lung cancer: clinical results and technical aspects. 
Technology in cancer research & treatment. 2004;3(2):143-50. 
7. 
Wang H, Littrup PJ, Duan Y, Zhang Y, Feng H, Nie Z. Thoracic masses treated with percutaneous 
cryotherapy: initial experience with more than 200 procedures. Radiology. 2005;235(1):289-98. 
8. 
Ernst A, Silvestri GA, Johnstone D, American College of Chest P. Interventional pulmonary procedures: 
Guidelines from the American College of Chest Physicians. Chest. 2003;123(5):1693-717. 
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Bolliger CT, Mathur PN, Beamis JF, Becker HD, Cavaliere S, Colt H, et al. ERS/ATS statement on 
interventional pulmonology. European Respiratory Society/American Thoracic Society. The European respiratory 
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10. 
Inoue M, Nakatsuka S, Jinzaki M. Cryoablation of early-stage primary lung cancer. BioMed research 
international. 2014;2014:521691. 
11. 
Inoue M, Nakatsuka S, Yashiro H, Ito N, Izumi Y, Yamauchi Y, et al. Percutaneous cryoablation of lung 
tumors: feasibility and safety. Journal of vascular and interventional radiology : JVIR. 2012;23(3):295-302; quiz 5. 
12. 
Lee KS, Erinjeri JP. Decision Making in Interventional Oncology: Ablative Options in the Lung. Seminars 
in interventional radiology. 2017;34(2):176-81. 
13. 
Gershman E, Fruchter O, Benjamin F, Nader AR, Rosengarten D, Rusanov V, et al. Safety of Cryo-
Transbronchial Biopsy in Diffuse Lung Diseases: Analysis of Three Hundred Cases. Respiration; international 
review of thoracic diseases. 2015;90(1):40-6. 
14. 
DiBardino DM, Haas AR, Lanfranco AR, Litzky LA, Sterman D, Bessich JL. High Complication Rate after 
Introduction of Transbronchial Cryobiopsy into Clinical Practice at an Academic Medical Center. Annals of the 
American Thoracic Society. 2017;14(6):851-7. 
15. 
Lentz RJ, Argento AC, Colby TV, Rickman OB, Maldonado F. Transbronchial cryobiopsy for diffuse 
parenchymal lung disease: a state-of-the-art review of procedural techniques, current evidence, and future 
challenges. Journal of thoracic disease. 2017;9(7):2186-203. 
16. 
Schuhmann M, Bostanci K, Bugalho A, Warth A, Schnabel PA, Herth FJ, et al. Endobronchial ultrasound-
guided cryobiopsies in peripheral pulmonary lesions: a feasibility study. The European respiratory journal. 
2014;43(1):233-9. 
17. 
Takahashi Y, Izumi Y, Matsutani N, Dejima H, Nakayama T, Okamura R, et al. Optimized magnitude of 
cryosurgery facilitating anti-tumor immunoreaction in a mouse model of Lewis lung cancer. Cancer immunology, 
immunotherapy : CII. 2016;65(8):973-82. 
18. 
Alteber Z, Azulay M, Cafri G, Vadai E, Tzehoval E, Eisenbach L. Cryoimmunotherapy with local co-
administration of ex vivo generated dendritic cells and CpG-ODN immune adjuvant, elicits a specific antitumor 
immunity. Cancer immunology, immunotherapy : CII. 2014;63(4):369-80. 
19. 
Gu XY, Jiang Z, Fang W. Cryoablation combined with molecular target therapy improves the curative 
effect in patients with advanced non-small cell lung cancer. The Journal of international medical research. 
2011;39(5):1736-43. 
20. 
Lin M, Liang SZ, Wang XH, Liang YQ, Zhang MJ, Niu LZ, et al. Clinical efficacy of percutaneous 
cryoablation combined with allogenic NK cell immunotherapy for advanced non-small cell lung cancer. 
Immunologic research. 2017. 
21. 
Yuanying Y, Lizhi N, Feng M, Xiaohua W, Jianying Z, Fei Y, et al. Therapeutic outcomes of combining 
cryotherapy, chemotherapy and DC-CIK immunotherapy in the treatment of metastatic non-small cell lung cancer. 
Cryobiology. 2013;67(2):235-40. 
22. 
Reck M, Rodriguez-Abreu D, Robinson AG, Hui R, Csoszi T, Fulop A, et al. Pembrolizumab versus 
Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer. The New England journal of medicine. 
2016;375(19):1823-33. 

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writing by the study sponsor. 
23.
Lievense LA, Sterman DH, Cornelissen R, Aerts JG. Checkpoint Blockade in Lung Cancer and 
Mesothelioma. American journal of respiratory and critical care medicine. 2017;196(3):274-82. 
24. 
Moon EK, Langer CJ, Albelda SM. The Era of Checkpoint Blockade in Lung Cancer: Taking the Brakes 
Off the Immune System. Annals of the American Thoracic Society. 2017;14(8):1248-60. 
25. 
Lin M, Liang S, Wang X, Liang Y, Zhang M, Chen J, et al. Cryoablation combined with allogenic natural 
killer cell immunotherapy improves the curative effect in patients with advanced hepatocellular cancer. 
Oncotarget. 2017. 
26. 
Miller JD, van der Most RG, Akondy RS, Glidewell JT, Albott S, Masopust D, et al. Human effector and 
memory CD8+ T cell responses to smallpox and yellow fever vaccines. Immunity. 2008;28(5):710-22. 
27. 
Kamphorst AO, Pillai RN, Yang S, Nasti TH, Akondy RS, Wieland A, et al. Proliferation of PD-1+ CD8 T 
cells in peripheral blood after PD-1-targeted therapy in lung cancer patients. Proceedings of the National 
Academy of Sciences of the United States of America. 2017;114(19):4993-8. 
28. 
Payton JE, Grieselhuber NR, Chang LW, Murakami M, Geiss GK, Link DC, et al. High throughput digital 
quantification of mRNA abundance in primary human acute myeloid leukemia samples. The Journal of clinical 
investigation. 2009;119(6):1714-26. 
29. 
Kellar A, Egan C, Morris D. Preclinical Murine Models for Lung Cancer: Clinical Trial Applications. BioMed 
research international. 2015;2015:621324. 
30. 
Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological 
tolerance to self and non-self. Nature immunology. 2005;6(4):345-52. 
31. 
Waitz R, Solomon SB, Petre EN, Trumble AE, Fasso M, Norton L, et al. Potent induction of tumor 
immunity by combining tumor cryoablation with anti-CTLA-4 therapy. Cancer research. 2012;72(2):430-9. 
32. 
Takaki H, Imai N, Thomas CT, Yamakado K, Yarmohammadi H, Ziv E, et al. Changes in peripheral blood 
T-cell balance after percutaneous tumor ablation. Minimally invasive therapy & allied technologies : MITAT : 
official journal of the Society for Minimally Invasive Therapy. 2017:1-7. 
33. 
Bennett AV, Dueck AC, Mitchell SA, Mendoza TR, Reeve BB, Atkinson TM, et al. Mode equivalence and 
acceptability of tablet computer-, interactive voice response system-, and paper-based administration of the U.S. 
National Cancer Institute's Patient-Reported Outcomes version of the Common Terminology Criteria for Adverse 
Events (PRO-CTCAE). Health and quality of life outcomes. 2016;14:24. 
34. 
Pajares V, Puzo C, Castillo D, Lerma E, Montero MA, Ramos-Barbon D, et al. Diagnostic yield of 
transbronchial cryobiopsy in interstitial lung disease: a randomized trial. Respirology. 2014;19(6):900-6. 
35. 
Hrvatin S, Deng F, O'Donnell CW, Gifford DK, Melton DA. MARIS: method for analyzing RNA following 
intracellular sorting. PloS one. 2014;9(3):e89459. 
36. 
Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response 
evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). European journal of cancer. 
2009;45(2):228-47. 
37. 
Oberg C, Lau RP, Folch EE, He T, Ronaghi R, Channick CL, et al. Novel robotic-assisted cryobiopsy for 
peripheral pulmonary lesions, a pilot study. Am J Respir Crit Care Med 2022;205:A5556 
38.  
Patel V. Cryobiopsy with Ion robotic bronchoscopy, initial case series findings on safety and efficacy: first 
known case series to look at further future applications. Chest 2022;161(6):suppA406   
 
 
 
 
 
 
 
18  Attachments 
These documents are relevant to the protocol, but they are not considered part of the protocol.  They 
are stored and modified separately. As such, modifications to these documents do not require protocol 
amendments. 
Informed Consent Form  
ERBOKRYO® CA – Cryosugical Unit with Flexible ERBECRYOTM Probe FDA 510K Approval Form 

Study number: S17-00812 
 
  
 
 
 
Page 44  
 
Updated 10/7/19 
 
CONFIDENTIAL 
This material is the property of the NYU School of Medicine and Langone Medical Center.  Do not disclose or use except as authorized in writing by the study sponsor
19 Schedule of Events
 
                             
 
Activity 
Enrollment 
(Day -28 to 0) 
Pre-BCI Peripheral 
Blood Collection 
(Day 0) 
BCI 
(Day 0) 
Post-BCI Peripheral 
Blood Collection 
(Day 7 ± 3) 
Post-BCI Peripheral 
Blood Collection 
(Day 14 ± 3) 
Study Team Procedures 
 
  
  
  
  
Consent 
X 
Medical history 
X 
 
 
 
 
 
 
 
X
X
X
Physical exam
X
X
X
X
Bronchoalveolar lavage 
X 
BCI 
 
 
X 
 
 
Laboratory Assessments 
 
 
 
 
 
Complete blood count
X
 
 
 
 
Chemistry panel
X
Pregnancy test (if childbearing potential)
X
Immunologic analysis
X
X
X
Imaging Assessments
Review Chest CT scan
X