Phase I Clinical Trial Evaluating the Safety and Feasibility of Bone Marrow Aspiration From Ribs During Thoracic Surgery
Phase: NA
Conditions: Non Small Cell Lung Cancer
Sponsor: Universitair Ziekenhuis Brussel

 
 
 
 
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Study Protocol  
 
Study Title: Phase I clinical trial evaluating the safety and feasibility of bone marrow aspiration from ribs during 
thoracic surgery  
 
Study Acronym: COBM 
Phase of Development: Phase 1 clinical trial 
Protocol Number: 1 
Protocol Version and Date: V2.0 – 26th of October 2023 
 
EudraCT Registry Number: N/A 
ClinicTrials.gov Registry Number: NCT05251805 
Investigational product or Medical Device: N/A 
 
 
 
 
 
 
Sponsor: UZ Brussel 
  
Coordinating/Principal Investigator: Domien Vanhonacker MD 
 
 
 
The information contained in this document is the property of the Coordinating Investigator and may not be reproduced, 
published or disclosed to others without written authorization of the Coordinating Investigator. 
PROTOCOL SIGNATURE PAGE 
Protocol Version and date: 2.0 - 26th of October 2023 
 
Protocol Title: Phase I clinical trial evaluating the safety and feasibility of bone marrow aspiration from ribs 
during thoracic surgery  
 
Sponsor: 
UZ Brussel 
 
 
 
 
 
Principal Investigator:  
Domien Vanhonacker MD 
 
I agree: 
- 
to assume responsibility for the proper conduct of this study 
- 
to conduct the study in compliance with this protocol and any future amendments  
- 
not to implement any deviations from or changes to the protocol without prior review and written approval 
from the Ethics Committee, except where necessary to eliminate an immediate hazard to the subjects, or for 
administrative aspects of the study (where permitted by all applicable regulatory requirements) 
- 
that I am thoroughly familiar with the appropriate use of the investigational drug, as described in this pro-
tocol 
- 
to ensure that all persons assisting me with the study are adequately informed about the investigational drug 
and their study-related duties and functions as described in the protocol 
- 
that I am aware of and will comply with the current good clinical practice (GCP) guidelines and ethical 
principles outlined in the Declaration of Helsinki 
- 
to conduct the study in accordance with all applicable laws and regulations 
 
 
Printed name                                                      Signature                                         Date 
Domien Vanhonacker 
 
 
 
 
 
 
 
 

 
 
 
 
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Table of Contents 
1 
Trial Registration/Protocol Summary ........................................................................................................ 5 
2 
Protocol Version History ......................................................................................................................... 6 
3 
Sponsor/Coordinating Investigator Information .......................................................................................... 6 
4 
List of Abbreviations .............................................................................................................................. 6 
5 
Introduction .......................................................................................................................................... 6 
5.1 
Overview of Disease Pathogenesis ....................................................................................................... 6 
5.2 
Epidemiology ..................................................................................................................................... 7 
5.3 
Current Treatments ............................................................................................................................. 7 
5.4 
Study Rationale and Purpose ............................................................................................................... 7 
5.5 
Rationale for Study Design .................................................................................................................. 8 
5.6 
Rationale for Dose and Regimen/Schedule Selection .............................................................................. 8 
6 
Study Schematic and Schedule of Activities .............................................................................................. 8 
6.1 
Study Schematic .................................................................................................................................. 8 
6.2 
Primary Objective ............................................................................................................................... 8 
6.3 
Secondary Objectives .......................................................................................................................... 9 
6.4 
Endpoints ........................................................................................................................................... 9 
7 
Investigational Plan ................................................................................................................................ 9 
7.1 
Overall Study Design .......................................................................................................................... 9 
7.2 
Study Duration for Subjects ................................................................................................................. 9 
7.2.1 
Screening ....................................................................................................................................... 9 
7.2.2 
Treatment Period ............................................................................................................................ 9 
7.2.3 
Unscheduled Visit(s) ....................................................................................................................... 9 
7.2.4 
Early Study Termination ................................................................................................................. 9 
7.2.5 
End of Study .................................................................................................................................. 9 
8 
Selection of Subjects ............................................................................................................................ 10 
8.1 
Selection of Study Population ............................................................................................................. 10 
8.2 
Inclusion Criteria.............................................................................................................................. 10 
8.3 
Exclusion Criteria ............................................................................................................................. 10 
9 
Screening ............................................................................................................................................ 10 
9.1 
Screening and Enrollment .................................................................................................................. 10 

 
 
 
 
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10 
Interventions/Treatment ........................................................................................................................ 10 
10.1 
Intervention ...................................................................................................................................... 10 
Randomization and Stratification .................................................................................................................... 11 
10.2 
Prohibited Medication ....................................................................................................................... 11 
10.3 
Known Undesirable Effects of costal rib aspiration.............................................................................. 11 
11 
Study Assessments and Procedures ......................................................................................................... 11 
11.1 
Study Assessments ............................................................................................................................. 11 
11.1.1 
Screening ................................................................................................................................ 11 
11.1.2 
Baseline .................................................................................................................................. 11 
11.1.3 
Treatment Period ...................................................................................................................... 11 
11.1.4 
Follow-Up ............................................................................................................................... 12 
11.1.5 
End of Study ............................................................................................................................ 12 
11.1.6 
Re-screening ............................................................................................................................ 12 
11.1.7 
Early Termination .................................................................................................................... 12 
11.1.8 
Unscheduled Visits ................................................................................................................... 12 
11.2 
Assessment Types .............................................................................................................................. 12 
11.2.1 
Efficacy Assessment ................................................................................................................. 12 
11.2.2 
Safety and Tolerability Assessments .......................................................................................... 12 
11.2.3 
Laboratory Evaluation .............................................................................................................. 13 
12 
Safety Monitoring and Reporting ........................................................................................................... 13 
12.1 
Adverse Events ................................................................................................................................. 13 
12.1.1 
Definitions and Reporting ......................................................................................................... 13 
12.1.2 
Reporting Period ...................................................................................................................... 13 
12.1.3 
Definitions ............................................................................................................................... 13 
12.1.4 
Immediate Reporting ................................................................................................................ 13 
12.2 
Suspected Unexpected Serious Adverse Events (SUSAR) ...................................................................... 14 
12.2.1 
Definitions ............................................................................................................................... 14 
12.2.2 
Reporting ................................................................................................................................ 14 
12.3 
Procedures for Handling Special Situations ........................................................................................ 14 
12.3.1 
Pregnancy ................................................................................................................................ 14 
12.3.2 
Overdose Management: not applicable ....................................................................................... 14 
12.4 
Annual Safety Report ......................................................................................................................... 14 
13 
Data Collection and Management ........................................................................................................... 14 
13.1 
Monitoring ....................................................................................................................................... 14 
13.1.1 
Composition of data monitoring committee ................................................................................ 14 
13.1.2 
Interim analysis ........................................................................................................................ 14 
13.2 
Data Collection ................................................................................................................................ 14 
13.3 
Database Management and Quality Control ........................................................................................ 14 
13.4 
Statistical Considerations and Data Analysis ...................................................................................... 15 

 
 
 
 
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14 
Ethical Considerations .......................................................................................................................... 15 
14.1 
Ethical conduct of the study ............................................................................................................... 15 
14.1.1 
Declaration of Helsinki ............................................................................................................. 15 
14.1.2 
Ethics Committee ..................................................................................................................... 15 
14.2 
Informed Consent.............................................................................................................................. 15 
14.3 
Patient and Study Data Protection ..................................................................................................... 15 
14.4 
Subject Identification......................................................................................................................... 16 
15 
Finance and Insurance .......................................................................................................................... 16 
16 
Reporting and Dissemination ................................................................................................................. 16 
17 
Conflict of Interest Statement ................................................................................................................ 16 
18 
Tables and Figures ............................................................................................................................... 16 
19 
References .......................................................................................................................................... 16 
 
 
 

 
 
 
 
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1 Trial Registration/Protocol Summary 
 
Information 
EudraCT number: 
Date of registration: 
N/A 
ClinicalTrials.gov: 
NCT05251805 
Official Title: 
Phase I clinical trial evaluating the safety and feasibility of bone marrow aspira-
tion from ribs during thoracic surgery 
 
Study Phase/Type: 
Phase I clinical trial 
Condition: 
Lung cancer 
Objectives: 
Evaluate the safety and feasibility of bone marrow aspiration during thoracic surgery 
Investigational Product or 
Medical Device: 
None 
Interventions: 
1. Pre-operative blood sample collection 
2. Costal bone marrow aspiration 
3. Lung tumor and adjacent tissue sampling 
Endpoints: 
1. Safety: the occurrence of complications  
2. Feasibility: Obtain a minimum of 2x106 Lin- CD34+ Hematopoietic Stem and 
Progenitor Cells (HSPCs) to allow single cell RNA sequencing (106), bone mar-
row cultivation in vitro (5x105 cells per culture) and transplantation in immuno-
deficient mice (105 cells per injection).  
Study population: 
Lung cancer patients undergoing thoracic surgery 
Number of patients: 
10  
Overview of  
study design: 
Phase I feasibility trial 
Statistical  
Considerations: 
None 
Sponsor: 
UZ Brussel 
Inclusion Criteria: 
Confirmed or suspected lung cancer patients undergoing thoracic surgery  
Exclusion Criteria: 
1. ASA ≥ 3 
2. Bleeding disorders 
Target Date of first  
enrolment: 
01/10/2022 
Target sample size: 
10 
 
 
 

 
 
 
 
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2 Protocol Version History 
 
Version No. 
Release Date 
Summary of Changes 
V1 
20 April 2022 
- 
V1.1 
28 June 2022 
Upon request of the EC: made some minor changes concerning the reporting of 
SAE’s and added an explanation on the multidisciplinary aspects of the research 
project. 
 
 
 
 
3 Sponsor/Coordinating Investigator Information 
Sponsor: UZ Brussel 
Principal Investigator: Domien Vanhonacker MD 
Co-investigators: Prof. Dr. Cleo Govyaerts, Prof. Dr. Dirk Smets and Yanina Jansen MD, PhD 
Additional co-researchers: PhD students Thomas Benoot and Evelyn Calderon Espinosa 
 
 
Statistician: N/A 
 
 
 
 
Laboratory: Laboratory for Molecular and Cellular Therapy (LMCT), VUB 
Pharmacy: N/A 
 
 
 
 
  
Study Coordinator: N/A 
Study sites: UZ Brussel and LMCT, campus Jette, VUB 
4 List of Abbreviations 
ICI: immune checkpoint inhibitors 
NSCL: non-small cell lung cancer 
EU: European Union 
LuCE: Lung Cancer Europe 
PD-L1: programmed death-ligand 1 
TME: tumor micro-environment 
BMN: bone marrow niche 
HSPCs: hematopoietic stem and progenitor cells 
LMCT: Laboratory for Molecular and Cellular Therapy 
PBS: phosphate buffered saline  
5 Introduction 
5.1 
Overview of Disease Pathogenesis 
Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths worldwide, with a staggering 
2.21 million new cases in 2020 alone1. For patients with stage I and stage II disease, surgical resection is the primary 
modality of treatment. However, only 20-30% of lung cancer patients are suitable for curative treatment (surgery versus 
stereotactic radiotherapy) at the time of diagnosis. Moreover, following surgery, the risk of recurrence during the first 4-
year ranges from 6% to 10% per person per year but decreases thereafter to 2%2. Current progress in the field of anti-
tumor immunotherapy is based on the capacity of effector T cells to recognize and kill cancer cells specifically. As 
progressing tumors often master skills to restrict T-cell mediated killing via upregulation of immune checkpoint mole-
cules, immune checkpoint inhibitors (ICIs) were designed to ameliorate antitumor immunity. Today, the latter represents 
a first-line treatment option for advanced NSCLC patients with curative potential3. Unfortunately, up to 75% of patients 
currently do not respond to ICIs, suggesting that lung tumor cells master more skills to mislead effector T cells than 
upregulating immune checkpoint molecules alone, highlighting the ongoing medical need to develop additional/alterna-
tive (immune)therapeutics that enhance the overall survival rate of ICI-treated NSCLC patients. 

 
 
 
 
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5.2 
Epidemiology 
Lung cancer is the fourth most diagnosed cancer, affecting more than 312.000 people every year in the European Union 
(EU4). Cigarette smoking is the major cause of lung cancer, with around 80-90% of all lung cancer attributable to tobacco. 
In men, lung cancer is the second most diagnosed cancer. In women, lung cancer is the third most frequent cancer. How-
ever, this gender gap is closing. Lung cancer survival is poor, although new treatments lead to a slightly better prognosis. 
The overall 5-year survival is around 13% since diagnosis. 
 
5.3 
Current Treatments 
1. Stage I disease: surgery or stereotactic radiotherapy in patient unfit for surgery 
2. Stage II disease: surgery followed by adjuvant chemotherapy 
3. Stage III disease: chemoradiotherapy  
4. Stage IV disease: chemotherapy, targeted therapy, and immunotherapy. Immunotherapy is used as a first-line 
treatment in NSCLC patients without driver mutations whose tumors express PD-L1 (programmed death ligand-
1) at levels of 50% or greater. 
 
5.4 
Study Rationale and Purpose 
In general, antitumor immunotherapy tends to spark the patients’ own tumor-specific effector T cells to multiply, recog-
nize and kill tumor cells. Yet, a typical characteristic of immunotherapy-resistant (lung) tumors’ deceptive gambit, is their 
molding of an effector T-cell hostile tumor microenvironment (TME). The latter comprises cellular, molecular, vascu-
lar, and physical barriers for effector T cells which hamper effective tumor cell killing. With regard to the cellular 
barriers, we and others have appointed a leading role to the immunosuppressive myeloid cells5,6. These myeloid cells 
represent a heterogeneous group of subsets with embryonic origin (macrophages) and/or hematopoietic origin (dendritic 
cells, macrophages, granulocytes, mast cells, and platelets), suggesting that the bone marrow niche (BMN) is involved 
in the creation of an immunosuppressive T-cell hostile TME. Moreover, this niche has been shown to aid in pre-metastatic 
niche formation while it has also been proposed as a supportive hideout for disseminated cancer cells, as reported by the 
detection of Epcam+ CD45- disseminated cancer cells in 43,5% of sternal aspirated BM samples from 62 NSCLC pa-
tients7. Nevertheless, as BM aspiration is a painful process and therefore, unlike blood drawing not routinely performed, 
it remains a tremendously understudied niche in solid cancers.  
 
Next to the fundamental information that BMN investigation would deliver to ameliorate our understanding of NSCLC 
progression and resistance to (immuno)therapy, bone marrow represents an extremely valuable biomaterial for preclinical 
onco-immunology research. More specifically, the Lin- CD34+ hematopoietic stem (and progenitor) cells or HSPCs have 
been used to generate humanized mice by transplanting total body irradiated mice with human bone marrow8–10. Anti-
tumor immunotherapy (and research thereof) relies on histocompatible cell-to-cell interactions between the immune sys-
tem, tumor cells and their TME. Hence, humanized patient-derived xenograft models in which the immune system and 
tumor are derived from the same patient, a.k.a. ‘murinized patients’, represent the ultimate model to study human tumor 
immunology and evaluate innovative immunotherapies.   
 
To study the BMN and autologous tumor-bone marrow interactions, we want to collect blood, bone marrow and lung 
tissue of 10 lung cancer patients. 
 
1) This pilot trial is designed to evaluate the feasibility and safety of obtaining bone marrow upon costal aspiration 
during surgery. The rationale for using rib bone marrow aspiration is the easily accessible localization due to trocar 
placement (in minimally invasive surgery) or during thoracotomy. Previous trials demonstrated the feasibility of micro-
metastasis detection in rib marrow aspiration11–13. This procedure was safe without any documented adverse events for 
patients. We hypothesize that we can use this technique to obtain sufficient bone marrow-derived HSPCs (which represent 
only 0,1% of the total bone marrow fraction) for ex vivo single-cell evaluation14, optimization of a 3D BMN platform15 
and preclinical ex/in vivo assessment in patient-derived organoids16,17 and murine xenograft models18. The use of rib 
marrow eliminates the need for extra incision as incisions used for trocar placement or thoracotomy will be used. We 

 
 
 
 
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hypothesize that the patient will not experience extra discomfort from this technique since every patient undergoing tho-
racic surgery is treated according to the ERAS protocols with a major emphasis on pain control.  
 
2) If the feasibility and safety of bone marrow aspiration are confirmed by this pilot trial, we plan to conduct a large-scale 
study in which we will collect and bank blood, bone marrow and tumor per NSCLC patient. With this unique autologous 
biological material collection, we aspire to conduct innovative research into the current resistance mechanisms of NSCLC 
to immunotherapy via at least two ongoing research projects: 
 
a) X-talk project with the hypothesis: ‘The bone marrow niche of NSCLC patients withholds invaluable infor-
mation about tumor-installed resistance mechanisms to immunotherapy15. By deciphering this information, we 
aspire to serve the diagnostic and therapeutic field.’ 
b) RADar project with the aim to answer the following research questions: 1) can we efficiently deliver RADars 
specifically to human lung tumor cells? and 2) will RADar delivery result in tumor cells’ sensitivity to and killing 
by patient-specific human T cells? To be able to answer these questions in a relevant way, we opt for two me-
ticulous human(ized) immunological models namely: 
- an in vitro Predixoid platform in which NSCLC organoids are grown as TME-on-a-chip19,20.  
- ‘murinized patients’, in which patient-derived NSCLC cells or organoids are engrafted intrathoracic 
into immunodeficient mice to generate orthotopic lung cancer patient xenografts8,9. Next, we will op-
timize the generation of humanized mice, transplanted with NSCLC patient-derived HSPCs and subse-
quent autologous and orthotopic NSCLC tumor engraftment to generate ‘murinized patients. 
 
Hence, by exploiting patient-derived material, we aspire to perform more clinically relevant fundamental and trans-
lation research, especially as the use of both platforms will allow us to take inter-patient-heterogeneity into account 
(unlike immortalized lung tumor cell lines).  
 
The PI of this feasibility trial, Domien Vanhonacker (MD), is an anesthesiologist. He has written the study protocol in 
close cooperation with Yanina Jansen (MD, PhD) and Cleo Goyvaerts (PhD). In conjunction with Dirk Smets (MD) he 
will include the patients in this trial, attend to the patient during and after the surgical procedure and perform the bone 
marrow aspiration. 
 
5.5 
Rationale for Study Design 
This trial is a feasibility trial evaluating the possibility to obtain bone marrow-derived HSPCs by bone marrow aspiration 
from ribs. 
5.6 
Rationale for Dose and Regimen/Schedule Selection 
Not applicable 
 
6 Study Schematic and Schedule of Activities 
6.1 
Study Schematic 
1. Patient screening and informed consent 
2. Collection of samples: 
a. 
Blood sample collection during pre-operative blood collection 
b. Bone marrow aspiration during thoracic surgery 
c. 
Lung tumor and adjacent tissue true cut on removed lung tissue 
3. Observation of adverse events during the first 30 days following surgery 
4. Processing of obtained tissue at LMCT   
6.2 
Primary Objective 
The safety and feasibility of rib bone marrow aspiration during thoracic surgery 

 
 
 
 
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6.3 
Secondary Objectives 
None 
6.4 
Endpoints 
1. 
Safety: the occurrence of complications 
2. 
Feasibility: Obtain a minimum of 2x106 Lin- CD34+ HSPCs to allow single cell sequencing (106), bone mar-
row cultivation (5x105 cells per culture) and transplantation in immunodeficient mice (105 cells per injection).  
7 Investigational Plan 
7.1 
Overall Study Design 
1) This trial is a phase I feasibility trial. It is designed to evaluate the feasibility and safety of obtaining bone marrow 
upon costal aspiration during surgery.  
2) If feasibility and safety of rib bone marrow aspiration are confirmed in this pilot trial, a large-scale study will be started 
to optimize and conduct fundamental and translational research via the X-talk project and RADar project (see details 
under 5.4, page 9). 
 
7.2 
Study Duration for Subjects 
7.2.1 
Screening 
All patients undergoing thoracic surgery (by minimally invasive technique or by thoracotomy) will be screened for their 
eligibility by the principal investigator.  
7.2.2 
Treatment Period 
a. 
Blood sample collection during pre-operative blood collection 
b. Bone marrow aspiration during thoracic surgery – no additional incisions will be made 
c. 
Lung tumor collection – only tumor not used for diagnostic purpose 
 
7.2.3 
Unscheduled Visit(s) 
All patients will be hospitalized the day before surgery and between 5-7 days following surgery. This is standard practice 
for all patients undergoing thoracic surgery in our department. No unscheduled visits will be necessary 
7.2.4 
Early Study Termination 
The study will be closed prematurely if 2 patients experience a grade 3 adverse event directly related to the bone marrow 
aspiration 
7.2.5 
End of Study 
The study will end once 10 study patients have been included.We forsee that the end will be scheduled for 31st of Decem-
ber 2025.  

 
 
 
 
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8 Selection of Subjects 
8.1 
Selection of Study Population 
All patients undergoing thoracic surgery for a confirmed or suspected lung cancer. 
8.2 
Inclusion Criteria  
Patient undergoing thoracic surgery for a confirmed or suspected lung cancer. All patients eligible for major surgery, as 
defined by a thoracic procedure, are considered eligible for participation in this trial. 
 
8.3 
Exclusion Criteria 
1. Uncertainty of pre-operative diagnosis, exception is per-op frozen section analysis confirming malignity 
2. ASA ≥ 3 
3. Bleeding disorders 
4. Contraception/Pregnancy Avoidance 
 
The aspiration of bone marrow will not impact contraception/pregnancy avoidance. 
9 Screening  
9.1 
Screening and Enrollment 
All patients undergoing thoracic surgery will be screened pre-operative by the principal investigator. When eligible, pa-
tients will be asked for their participation and included after signing the informed consent.  
10 Interventions/Treatment 
10.1 
Intervention 
a. 
Blood sample collection during pre-operative blood collection 
A total of 12 ml will be collected 
b. Bone marrow aspiration during thoracic surgery 
 
Following the surgical intervention, bone marrow aspiration will be performed through the incisions 
made for the thoracic procedure. The ribs lying directly above and below the trocar incision will be 
exposed. A bone marrow needle will be advanced into the periost until the needle tip reaches the bone 
marrow. After removing the stylet a 2ml syringe will be attached to the aspiration needle and 0.5ml to 
2ml of bone marrow will be aspirated before removing the needle. This procedure will be repeated 
twice for every trocar site, for a maximum of 3 trocar sites, or until 5ml of bone marrow aspirate is 
obtained. This part of the trial is experimental. Literature on rib bone marrow aspiration is scares but 
good results are obtained. The procedures does not seem to be correlated with major AE11,12,21. 
c. 
Lung tumor and adjacent healthy tissue collection – only tumor not used for diagnostic purposes.   
All surgical samples will be analyzed by our anatomopathological department. Viable and fresh mate-
rial, obtained via true cute biopsy on the excised tissue and not used for the diagnostic purposes, will 
be collected by the LMCT. 
 
All samples (blood, bone marrow, and fresh tumor tissue), will be transferred to the LMCT for immediate processing of 
viable cells (within 2 hours after isolation) and subsequent cultivation and/or cryopreservation for later usage. 
 

 
 
 
 
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Randomization and Stratification 
No randomization or stratification will be performed. All patients will be included in a consecutive manner. 
 
10.2 
Prohibited Medication 
All anti-coagulants should be interrupted before the procedure as is standard practice for thoracic surgery. 
10.3 
Known Undesirable Effects of costal rib aspiration 
 
Pain 
 
Bleeding 
 
Infection 
 
In a survey of members of the British Society of Haematology reported in 2003, there were 26 adverse events, including 
one fatality, out of an estimated total of 55,000 bone marrow biopsy procedures (0.05 percent) 22. A follow-up survey of 
this group, reported in 2006 involving 20,323 procedures, resulted in the reporting of 15 adverse events (0.07 percent) 13. 
Previous research evaluating the feasibility to detect disseminated tumor cells in bone marrow aspirate from the ribs 
couldn’t identify any adverse events related to the bone marrow aspiration. 
11 Study Assessments and Procedures 
11.1 
Study Assessments 
11.1.1 
Screening 
All patients undergoing thoracic surgery for lung cancer will be screened by the principal investigator for inclusion in 
this trail. 
 
11.1.2 
Baseline 
Baseline screening consists of the pre-operative assessment for all patients undergoing thoracic surgery: blood tests (in-
cluding coagulation testing), CT or PET CT, as well as consulting an anesthesiologist. No additional tests are required 
for this trial. 
11.1.3 
Treatment Period 
a. 
Blood sample collection during pre-operative blood collection 
A total of 12ml (3 EDTA tubes) will be collected 
b. Bone marrow aspiration during thoracic surgery  
Following the surgical intervention, a bone marrow aspiration will be performed through the incisions 
made for the thoracic procedure. The ribs lying directly above and below the trocar incision will be 
exposed. A bone marrow needle will be advanced into the periost until the needle tip reaches the bone 
marrow. After removing the stylet, a 2ml syringe will be attached to the aspiration needle and 0.5ml to 
2ml bone marrow will be aspirated before removing the needle. This procedure will be repeated twice 
for every trocar site, for a maximum of 3 trocar sites, or until 5ml of bone marrow aspirate is obtained. 
c. 
Lung tumor collection – only tumor not used for diagnostic purposes.   
All surgical samples will be analyzed by our anatomopathological department. Viable and fresh mate-
rial, obtained via true cute biopsy on the excised tissue and not used for the diagnostic purposes, will 
be collected by the LMCT. 
 

 
 
 
 
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11.1.4 
Follow-Up 
All patients are hospitalized one day prior to surgery. After surgery, patients will stay in the post-anesthesia care unit 
(PACU) for one night. Following surgery, the patients will stay in the hospital for 5-7 days. All patients will be seen by 
their surgeon at the consultation 2 weeks following surgery. We don’t expect a prolonged hospital stay or additional 
hospital visits will be necessary for the patients partaking in the trial. 
11.1.5 
End of Study 
Adverse events will be followed up to four weeks postoperatively. 
11.1.6 
Re-screening 
No 
11.1.7 
Early Termination 
The trial will be ended prematurely if grade 3 (or higher) adverse events occur in 2 patients 
11.1.8 
Unscheduled Visits 
Not expected 
11.2 
Assessment Types  
11.2.1 
Efficacy Assessment 
In accordance with a recent single-cell analysis study on patient-derived bone marrow14, we will process blood and bone 
marrow specimens from patients within two hours of collection. The bone marrow mononuclear cells (BM-MNCs) will 
subsequently be isolated by density centrifugation using LSM Lymphocyte Separation Medium (#50494X, MPbio). In 
short, the bone marrow will be diluted twofold using phosphate-buffered saline (PBS), layered on top of 1 volume LSM 
Lymphocyte Separation Medium in a 50-ml Falcon tube, and centrifuged at 2300 rpm for 25 minutes at room temperature 
without brake. Next, the MNC layer will be isolated and washed with PBS after lysis of red blood cells with RBC lysing 
buffer. To count and purify the Lin- CD34+ HSPCs per patient using our BD FACSAria™ III Cell Sorter, BM-MNCs 
will be stained with monoclonal antibodies for 30 minutes on ice: anti-Lineage antibody, anti-CD34 antibody, anti-CD45 
antibody, and anti-Epcam antibody to divide each bone marrow sample over HSPCs (Lin/CD45/Epcam- CD34+), im-
mune cells (Lin/CD34/Epcam- CD45+), disseminated cancer cells (CD45/CD34- Epcam+) and the rest fraction.  
 
The endpoint will be the determination of viable HSPC numbers per sample as well as detection of disseminated Epcam+ 
NSCLC cells. For future experiments we aim at a minimum of 2x10E6 Lin- CD34+ HSPCs to allow single-cell sequenc-
ing (106), bone marrow cultivation (5x105 cells per culture), and transplantation in immunodeficient mice (105 cells per 
injection).  
11.2.2 
Safety and Tolerability Assessments 
 
NRS pain score: The NRS pain score will be used to evaluate postoperative pain. Bone marrow aspiration will 
be performed at a maximum of 3 trocar sites. Thoracic procedures are known as painful procedures mandating 
multimodal pain treatment. Bone marrow aspiration is not expected to add to the pain experience. 
 
Bleeding: in all patients undergoing a thoracic procedure a thoracic drainage system is used and all patients will 
stay at the recovery ward following surgery. Since the bone marrow aspiration is done under visual control we 
don’t expect any significant postoperative bleeding. 
 
Infection: the bone marrow aspiration is done under sterile conditions, using the incisions made to introduce the 
trocars. Follow-up is provided 14 days following surgery.  

 
 
 
 
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11.2.3 
Laboratory Evaluation  
Hematology: a preoperative coagulation screening will be performed for all patients; this is the standard procedure for all 
patients undergoing thoracic surgery.  
12 Safety Monitoring and Reporting  
12.1 
Adverse Events  
12.1.1 
Definitions and Reporting 
All Adverse events will be graded according to the Clavien Dindo classification system: 
 
 
Clavien Dindo classification system 
12.1.2 
Reporting Period 
All adverse events related to the bone marrow aspiration will be evaluated and classified by their treating physician. 
Adverse events occurring during the first postoperative month (30 days) will be registered. 
 
Serious Adverse Events  
12.1.3 
Definitions  
All adverse events of grade 3 or higher (according to the Clavien Dindo classification) will be reported as serious adverse 
events. 
12.1.4 
Immediate Reporting  
In the occurrence of adverse events of grade 3 or higher related to the trial procedure, the ethical committee will be 
contacted.  In the occurrence of 2 adverse events of grade 3 or higher the trial will be prematurely discontinued. 

 
 
 
 
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12.2 
Suspected Unexpected Serious Adverse Events (SUSAR) 
12.2.1 
Definitions 
All adverse events of grade 3 or higher (according to the Clavien Dindo classification) that might be related to a trial 
related procedure will be reported as a serious adverse event 
 
12.2.2 
Reporting 
In the occurrence of adverse events of grade 3 or higher related to the trial procedure, the ethical committee will be 
contacted. In the occurrence of 2 adverse events of grade 3 or higher the trial will be prematurely discontinued and the 
ethical committee will be notified. 
 
12.3 
Procedures for Handling Special Situations 
12.3.1 
Pregnancy 
Pregnancy is no contra-indication for participating in the trial, however due to the context of the underlying disease we 
don’t expect to include any pregnant patients. 
12.3.2 
Overdose Management: not applicable 
12.4 
Annual Safety Report 
A yearly safety report will be submitted to the ethical committee up to 1 year following recruitment of the final study 
patient 
13 Data Collection and Management 
13.1 
Monitoring  
13.1.1 
Composition of data monitoring committee 
Given the low number of included patients, the low risk of serious adverse events, and the single-center set-up, no external 
data monitoring committee is foreseen. 
During patient recruitment and up to 3 months following the inclusion of the last study patient, the principal investigator 
and the co-investigators will review and confirm all data on a 3-monthly basis during a teleconference. All data (outcome, 
duration of hospital stay, complications) will be gathered on red-cap. No statistical analyses will be performed.  
13.1.2 
Interim analysis 
After the inclusion of the first 5 patients, an interim analysis will be performed to confirm whether the size of the blood 
sample is adequate, how much bone marrow is extracted and whether this is adequate to do the lab analysis. 
13.2 
Data Collection 
Redcap will be used to collect clinical data and translational data. 
13.3 
Database Management and Quality Control  
Database management and quality control will be performed by the principal investigator and co-investigators. All 
study-related documents will be collected and kept in a closed location. 

 
 
 
 
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13.4 
Statistical Considerations and Data Analysis 
No statistical analysis will be done in this feasibility trail 
14 Ethical Considerations 
14.1 
Ethical conduct of the study 
14.1.1 
Declaration of Helsinki 
The Investigator shall be responsible for ensuring that the clinical study is performed in accordance with the protocol, the 
ethical principles that have their origin in the Declaration of Helsinki (as amended by the 59th WMA General Assembly, 
Seoul, October 2008, to be found at www.wma.net) as well as with the ICH Note for GCP (ICH, Topic E6, 1995) and 
applicable regulatory requirements. These documents state that the informed consent of subjects is an essential precondi-
tion for participation in the clinical study. 
 
14.1.2 
Ethics Committee 
Prior to commencement of the study, the study protocol will be submitted together with its associated documents (in-
formed consent, IB, insurance policy and insurance conditions, and all written information regarding this study to be 
provided to the subject or the subject’s legal guardian) to the relevant IRB/EC for their favorable opinion/approval. The 
favorable opinion/approval of the IRB/EC will be filed in the Investigator Site File and a copy in the Study Master File 
by the Sponsor. 
The study will only commence following provision of a written favorable opinion/approval. The date of the meeting, 
constitution of the committee, and voting members present at the meeting will be documented. Written evidence that 
clearly identifies the study, protocol version, and consent documents reviewed is required. Where possible, copies of the 
minutes of this meeting will be obtained. 
 
14.2 
Informed Consent 
Each subject will sign an informed consent for participation in the clinical part of this study.  
The written informed consent of the subject must be obtained before any study related activities are carried out. It must 
be signed and personally dated by the subject and by the Investigator/person designated by the Investigator to conduct 
the informed consent discussion.  
The subject will be given sufficient time to read the information and the opportunity to ask questions and to request 
additional information and clarification about the study. The subject information sheet will be revised whenever important 
new information becomes available that may be relevant to the consent of subjects. Subjects with legal incapacity or 
limited legal capacity should be excluded from participation in the clinical study. 
When informed consent is obtained, this will be documented in the CRF by the Investigator. The signed and dated dec-
laration of informed consent will remain at the Investigator's site and must be safely archived by the Investigator so that 
the forms can be retrieved at any time for monitoring, auditing and inspection purposes. A copy of the signed and dated 
information and consent forms should be provided to the subject prior to participation. 
 
14.3 
Patient and Study Data Protection  
A unique subject number will be assigned to a subject at inclusion, immediately after informed consent has been obtained. 
This number serves as the subject’s identifier in the trial as well as in the clinical trial database. The subject’s data col-
lected in the trial will be stored under this number. Only the Investigator will be able to link the subject’s trial data to the 
subject via an identification list kept at the site. 

 
 
 
 
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The subject’s original medical data reviewed at the site during source data verification by the monitor, audits, and author-
ity inspections, will be kept strictly confidential. 
Data protection and privacy regulations will be observed in capturing, forwarding, processing, and storing subject data. 
Subjects will be informed accordingly and will be requested to give their consent on data handling procedures in accord-
ance with national regulations. 
 
14.4 
Subject Identification  
All patients undergoing thoracic surgery for lung cancer will be screened by the principal investigator for inclusion in 
this trial. 
15 Finance and Insurance 
For all subjects enrolled in the study, appropriate insurance coverage is provided in line with legal requirements and GCP 
guidance. Details can be asked for at the investigational site (certificates and conditions in the Investigator Site File). 
Insurance coverage shall be provided by a Global Insurance Provider. Since no additional visits are mandatory for this 
trial, no financial compensation is provided for the patients taking part in the trial.  
16 Reporting and Dissemination 
Valuable data will be disseminated via national (e.g. ORC, BACR, etc.) and international conferences (e.g. CIMT, SITC, 
IASLC, ESMO, etc.). Moreover, the obtained results of the trial will be summarized for publication in a peer-reviewed 
journal with impact factor > 5. Finally, the co-investigator Prof. Dr. Cleo Goyvaerts will disseminate valuable results in 
layman’s’ terms as she gives seminars on a monthly basis to explain the concepts of cancer and immunotherapy to a broad 
audience. 
17 Conflict of Interest Statement 
Domien Vanhonacker MD has received financial compensations for talks on advanced hemodynamic management and 
proctoring sessions from Edwards Lifesciences. 
Yanina Jansen MD, PhD: travel, accommodation, and expenses— Bristol-Myers Squibb, MSD, Pfizer 
18 Tables and Figures 
Not applicable 
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Remon, J. et al. Immune Checkpoint Inhibitors in Thoracic Malignancies: Review of the Ex-
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