NEUTROFLOW: Development and Validation of a Pan-cancer Neutrophil Biomarker Test for Predicting Clinical Benefit From Immunotherapy Based on Flow Cytometry Analysis of Blood Samples
Conditions: Non Small Cell Lung Cancer (NSCLC), Melanoma (Skin Cancer), Triple Negative Breast Cancer (TNBC), RCC, Renal Cell Cancer, HNSCC
Sponsor: OncoHost Ltd.

 
NeutroFlow Study Protocol v1.0 
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Development and validation of a pan-cancer neutrophil 
biomarker test for predicting clinical benefit from 
immunotherapy based on flow cytometry analysis of 
blood samples 
 
 
 
 
 
 
 
 
 
OBSERVATIONAL STUDY 
 
 
 
 
 
 
This project has received funding from the European Union Horizon Programme under 
Grant Agreement No 101213660.  

 
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Table of contents 
Table of contents ......................................................................................................................... 2 
Protocol Synopsis ......................................................................................................................... 3 
Version control ............................................................................................................................ 3 
Protocol Signature page ............................................................................................................... 4 
List of Acronyms .......................................................................................................................... 5 
1. Background .............................................................................................................................. 7 
1.1. Description of the clinical study ....................................................................................................... 7 
1.1.1. Study rationale .................................................................................................... 7 
1.1.2. State of the art .................................................................................................... 8 
1.1.3. The NeutroFlow project ...................................................................................... 8 
2. Objectives of the Clinical Study ................................................................................................. 9 
3. Study design ............................................................................................................................ 9 
3.1. Study population .............................................................................................................................. 9 
3.2. Sample size and power calculation ................................................................................................ 11 
3.3. Design of the clinical study ............................................................................................................ 11 
3.4. Type of intervention ...................................................................................................................... 11 
3.5. Description and timing of study procedures ................................................................................. 11 
3.6. Study variables ............................................................................................................................... 12 
4. Preparedness status ............................................................................................................... 14 
4.1. Development of the clinical study protocol ................................................................................... 14 
4.1.1. Scientific advice from regulatory and health technology assessment bodies .. 14 
4.1.2. Clinical efficacy, safety, and methodological guidelines (including guidelines on 
statistics) .................................................................................................................................. 14 
4.1.3. Involvement of citizens / patients, carers in drawing up the clinical study 
protocol .................................................................................................................................... 14 
4.1.4. Regulatory intelligence to ensure timely regulatory approval and ethics 
clearance of the clinical study in all jurisdictions where its implementation is planned ........ 15 
4.2. Ethics and regulatory compliance .................................................................................................. 15 
4.3. Scientific and operational governance .......................................................................................... 16 
5. Operational feasibility ............................................................................................................ 16 
5.1. Availability of resources throughout implementation phase ........................................................ 16 
5.2. Recruitment of study population ................................................................................................... 17 
5.3. Data Management ......................................................................................................................... 18 
5.4. Reporting and monitoring obligations ........................................................................................... 19 
5.5. Study milestones ............................................................................................................................ 19 
6. References ............................................................................................................................. 20 
Annex 1. ICF template ................................................................................................................ 21 
 
 

 
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Protocol Synopsis 
Full Title 
Development and validation of a pan-cancer neutrophil biomarker test for 
predicting clinical benefit from immunotherapy based on flow cytometry 
analysis of blood samples 
Study Code 
NEUTROFLOW 
Study Objectives 
Primary objective 1: 
To develop a kit and flow cytometry assay for detecting and quantifying the level 
of Ly6Ehi neutrophils in blood samples. 
 
Primary objective 2: 
To develop a model for predicting clinical benefit from treatment with PD-(L)1 
inhibitors based on the level of Ly6Ehi neutrophils in patient blood samples. 
Sample Size 
  
A minimum sample size of N=600 was determined with up to 350 NSCLC, 180 
TNBC, 60 RCC, 50 melanoma and 120 HNSCC patients. 
Interventions 
N/A (observational study) 
Number of Sites 
1. Virgen Macarena University Hospital (Clinical coordinator site) 
2. European Institute of Oncology 
3. Heidelberg University Hospital 
Date and version 
12/06/2025 v1.0 
 
 
Version control 
Date 
Version 
Changes 
12/06/2025 
v1.0 
Original study protocol 
 
 
 
 
 

 
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Protocol Signature page 
The present study protocol was subject to critical review and has been approved in the current version 
by the persons undersigned. The information contained is consistent with:  
● the current risk-benefit assessment of the study intervention(s),  
● the moral, ethical, and scientific principles governing clinical research as set out in the latest 
relevant version of Declaration of Helsinki, the principles of the guidelines of International 
Council on Harmonization (ICH) Good Clinical Practice and the applicable legal and regulatory 
requirements.  
 
The investigators will be supplied with details of any significant change of the benefit-risk-assessment 
of the study.  
 
It will be ensured that the first subject is enrolled only after all ethical and regulatory requirements 
are fulfilled. Written informed consent will be obtained from all subjects after detailed oral and 
written information. It will be confirmed that all study subjects are informed about the type of 
encoding of their personal data (pseudonymization) and about who receives or has access to such 
data. Subjects who do not agree to this data encoding and transfer will not be enrolled into the study. 
In this context it will be assured that all investigational sites comply with the local regulatory 
requirements for data protection.  
 
Via current versions of the study protocol, it will be ensured that all principal investigators are 
informed about results regarding the benefits and risks of the study. 
 
Date: 
12/06/2025 
Signature: 
 
Name (Print Name): 
Function:  
 
 
Dr. Alberto Moreno Conde 
Coordinating Investigator 
Date: 
12/06/2025 
Signature: 
 
Name (Print Name): 
Function: 
 
 
Dr. Luis de la Cruz Merino 
Co-Coordinating Investigator 
Date: 
12/06/2025 
Signature: 
 
Name (Print Name): 
Function: 
 
 
Dr. David Vicente Baz 
Co-Coordinating Investigator 
 
Principal Investigators from the participating sites are: 
● Virgen Macarena University Hospital: Dr. Alberto Moreno Conde 
● European Institute of Oncology: Prof. Dr. Francesco Bertolini 
● Heidelberg University Hospital: Prof. Dr. Petros Christopoulos 
 
 

 
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List of Acronyms 
Table 1. List of acronyms 
Acronym 
Definition 
ALT 
Alanine Aminotransferase 
AST 
Aspartate Aminotransferase 
AUC 
Area Under the Curve 
C-index 
Concordance Index 
CB 
Clinical Benefit 
CI 
Confidence Interval 
CRP 
C reactive protein 
EC 
Ethics Committee 
ECOG 
Eastern Cooperative Oncology Group 
eCRF 
Electronic Case Report Form 
EIC 
European Innovation Council 
GDPR 
General Data Protection Regulation 
GEAR 
Gender Equality in Academia and Research 
HIPPA 
Health Insurance Portability and Accountability Act 
HNSCC 
Head and Neck Squamous Cell Carcinoma 
HR 
Hazard Ratio 
ICF 
Informed Consent Form 
ICIs 
Immune Checkpoint Inhibitors 
IEO 
Istituto Europeo di Oncologia 
irAEs 
Immune-related Adverse Events 
IRB 
Institutional Review Board 
KOL 
Key Opinion Leader 
LDH 
Lactate Dehydrogenase 
MSI 
Microsatellite Instability 
NSCLC 
Non-Small Cell Lung Cancer 
NSE 
Neuron-Specific Enolase 
OH 
OncoHost 
OS 
Overall Survival 
PBMC 
Peripheral Blood Mononuclear Cells 
PFS 
Progression Free Survival 
PI 
Principal Investigator 
RBC 
Red Blood Cells 

 
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RCC 
Renal Cell Carcinoma 
RECIST 
Response Evaluation Criteria In Solid Tumors 
RNA-seq 
Ribonucleic Acid sequencing 
ROC 
Receiving Operating Characteristic 
SAS 
Servicio Andaluz de Salud 
SOC 
Standard of Care 
TMB 
Tumour Mutational Burden 
TNBC 
Triple Negative Breast Cancer 
TNM 
TNM Malignancy Classification System 
UKHD 
Universitätsklinikum Heidelberg 
WBC 
White Blood Cells 
 
 
 

 
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1. Background 
1.1. Description of the clinical study 
1.1.1. Study rationale 
Immunotherapy based on immune checkpoint inhibitors (ICIs) has revolutionised the cancer therapy 
field. ICIs harness the patient’s own immune system to fight cancer, producing unprecedented durable 
remissions in a subset of patients. Over the last decade, multiple ICIs in the form of therapeutic 
monoclonal antibodies targeting PD-1 (e.g., Pembrolizumab, Nivolumab, Cemiplimab), PD-L1 (e.g., 
Atezolimumab, Durvalumab, Avelumab) or CTLA-4 (e.g., Ipilimumab) have been approved for treating 
a broad range of solid cancer types. Currently, they are widely used as standard-of-care in first- and 
advanced-line settings (either as monotherapy, ICI-ICI combination, or combined with 
chemotherapy/other agents) for lung cancer, melanoma, head and neck cancer, kidney cancer and 
others [1]. However, despite the remarkable promise of ICIs, several clinical challenges exist: (i) 
Response varies widely between patients, with the majority experiencing resistance to therapy. It is 
estimated that only 10-40% of patients respond to treatment across different cancer types [2,3]; (ii) 
There is a risk of serious side effects known as immune-related adverse events (irAEs) [4]; (iii) 
Treatment costs are high at around €100,000 per patient annually [5]. 
 
Currently, there are three types of clinically endorsed predictive biomarkers to determine eligibility 
for ICI therapies: (i) tumour expression of PD-L1; (ii) tumour mutational burden (TMB); and (iii) tumour 
microsatellite instability (MSI) status [6,7]. Although these biomarkers are routinely used in the clinic 
for multiple cancer types, they are considered imperfect for several reasons. Their predictive 
performance is not optimal, and their utility is limited by tumour heterogeneity, differences in defining 
biomarker thresholds, and variability in sensitivities of detection platforms [8]. Additionally, these 
tests generally require tumour biopsies sampled by an invasive procedure that is not always possible.  
 
Thus, there is an urgent unmet need for novel biomarkers with superior predictive performance. 
Ideally, such biomarkers would be minimally invasive (e.g., measured in a blood sample). Powerful 
predictive biomarkers would expand the benefit of ICIs to more patients, improve therapy outcome 
and quality of life, avoid the risk of irAEs in ineligible patients and reduce economic burden. 
 
Through the proposed EIC Transition project, the consortium aims to develop and validate a novel 
flow cytometry-based diagnostic test (termed NeutroFlow) intended for detecting a specific 
neutrophil subtype in the blood that serves as a predictive biomarker for therapeutic benefit from ICIs 
in patients across different tumour types. The neutrophil biomarker is termed ‘Ly6Ehi neutrophil’. For 
this purpose, a clinical study will be conducted for the collection of pretreatment blood samples and 
clinical data from at least 600 cancer patients across 5 cancer types: non-small cell lung cancer 
(NSCLC); melanoma; head and neck squamous cell carcinoma (HNSCC); triple negative breast cancer 
(TNBC); renal cell carcinoma (RCC).  
 

 
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The blood samples and clinical data will be used to: (i) develop a kit and flow cytometry assay for 
detecting and quantifying Ly6Ehi neutrophils in blood samples; (ii) determine the optimal threshold of 
Ly6Ehi neutrophils that distinguishes between patients with clinical benefit (CB) versus without CB. 
1.1.2. State of the art 
The NeutroFlow diagnostic test (to be developed through the proposed EIC project) will be based on 
a biomarker in the form of a neutrophil subtype termed ‘Ly6Ehi neutrophil’. The discovery of the Ly6Ehi 
neutrophil as a potential pan-cancer biomarker for ICI response stems from a proof-of-concept study 
performed in the laboratory of Prof. Yuval Shaked (Technion, Israel Institute of Technology). The work 
was recently published in Cancer Cell - a leading peer-reviewed scientific journal [9]. The relevant 
markers for the detection of Ly6Ehi neutrophils by flow cytometry were previously defined in the 
proof-of-concept study and are protected by patent application (WO2022/091097). The Ly6Ehi 
neutrophil biomarker has shown strong predictive performance (validated in 109 blood samples from 
NSCLC and melanoma patients, AUC~0.9; validated in 1440 publicly available RNA-seq samples across 
six cancer types) [9]. It thus shows potential for development into a highly accurate clinical test. 
 
The NeutroFlow test will be designed as a simple-to-use, rapid, point-of-care test. To perform the test, 
a blood sample will be drawn from the patient before beginning treatment. Peripheral blood 
mononuclear cells (PBMCs) will be isolated and analysed on-site using the NeutroFlow kit consisting 
of a specific monoclonal antibody panel for the detection of Ly6Ehi neutrophils by flow cytometry. 
 
The patient blood samples and clinical data (including clinical response data) collected through the 
clinical study will be used for the development and validation of the NeutroFlow test. The flow 
cytometry protocol will be optimised on at least 30 patient samples. A proprietary computational 
model that predicts clinical benefit from ICI therapy based on the level of Ly6Ehi neutrophils in the 
blood will be developed using the flow cytometry results and clinical data from a development cohort 
(~300 patients), and validated in a blinded manner using data from an independent cohort of ~300 
patients. 
1.1.3. The NeutroFlow project 
The recent introduction of cancer immunotherapy based on immune checkpoint inhibitors (ICIs) has 
revolutionised the treatment landscape for a broad range of cancer types. However, response to ICIs 
varies widely between patients, with the majority experiencing resistance to therapy. Moreover, the 
increasing use of these costly drugs coupled with management of ICI-related toxicities creates a 
substantial economic burden. Current biomarker tests for determining eligibility for ICIs have limited 
predictive performance, and many require invasive tumour biopsies. Thus, novel (and preferentially 
non-invasive) biomarkers for predicting ICI clinical benefit are desperately needed for better guiding 
clinical decisions.  
 
The NeutroFlow project directly addresses this unmet need. We build on comprehensive academic 
research describing a flow cytometry assay for measuring a novel predictive biomarker in the blood – 
Ly6Ehi neutrophil – that accurately predicts therapeutic benefit from ICIs, outperforming the approved 

 
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PD-L1 biomarker. The NeutroFlow project aims to upgrade the research-level assay to an industry-
level, point-of-care prototype test based on a kit consisting of an antibody panel for biomarker 
detection by standard flow cytometry (FC). Specifically, we will: (i) develop a FC assay according to 
clinical standards; (ii) develop and clinically validate a computational model that translates FC readout 
into a prediction of clinical benefit; (iii) evaluate analytical validity of the prototype across three 
medical centres (i.e., at the point-of-care); and (iv) prepare for future market introduction. The future 
product represents a minimally invasive, simple, rapid, and cost-effective test performed using 
standard hospital equipment.  
 
As a pan-cancer blood test for accurately predicting ICI therapeutic benefit, the product will potentially 
expand the benefit of ICIs to more patients, improve quality of life, limit the risk of ICI-related toxicities 
and reduce economic burden. 
 
2. Objectives of the Clinical Study 
Primary objective 1: 
To develop a kit and flow cytometry assay for detecting and quantifying the level of Ly6Ehi neutrophils 
in blood samples. 
 
Primary objective 2: 
To develop a model for predicting clinical benefit from treatment with PD-(L)1 inhibitors based on the 
level of Ly6Ehi neutrophils in patient blood samples. 
 
Secondary objectives: 
1. Evaluate the capability of the model to predict clinical benefit from treatment with PD-(L)1 
inhibitors for each of the investigated indications: non-small cell lung cancer (NSCLC), 
melanoma, head and neck squamous cell carcinoma (HNSCC), renal cell carcinoma (RCC), 
triple-negative breast cancer (TNBC). 
2. Evaluate the capability of the model to predict the efficacy difference between different 
approved PD-1/PD-L1 inhibitor-based treatments. 
3. Evaluate the correlation between the level of Ly6Ehi neutrophils in the blood to PD-L1 staining 
(TPS / CPS). 
4. Evaluate the correlation between the level of Ly6Ehi neutrophils in the blood to other clinical 
parameters associated with response to PD-(L)1 inhibitors. 
 
 
3. Study design 
3.1. Study population 
At least 600 treatment-naïve patients due to be treated with a first line regimen that includes PD- 
1/PD-L1 inhibitors as shown in  Table 2 with the following diagnoses are planned to be enrolled in the 
study over a period of 3 years or as needed: 

 
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1. Stage IV NSCLC or Stage III-unresectable NSCLC 
2. Stage IV or Stage IIIb-d malignant melanoma 
3. Stage IV HNSCC 
4. Stage IV RCC 
5. Stage IV TNBC 
 
Table 2. Approved first-line treatments based on PD-1/PD-L1 inhibitors 
Indication 
PD-1/PD-L1 inhibitors - monotherapy 
PD-1/PD-L1 inhibitors with another agent 
NSCLC 
Pembrolizumab 
Atezolizumab 
Cemiplimab 
Pembrolizumab + Chemotherapy 
Nivolumab + Ipilimumab 
Cemiplimab + Chemotherapy 
Atezolizumab + Chemotherapy + Bevacizumab 
Melanoma Nivolumab 
Pembrolizumab 
Nivolumab + Ipilimumab 
Nivolumab + Relatlimab 
HNSCC 
Pembrolizumab 
Cemiplimab 
Pembrolizumab + Chemotherapy 
RCC 
N/A 
Nivolumab + Ipilimumab; 
Nivolumab + Cabozantinib 
Pembrolizumab + Lenvatinib or Axitinib 
Avelumab + Axitinib 
TNBC 
N/A 
Pembrolizumab + Chemotherapy 
 
Inclusion criteria: 
1. Patients newly diagnosed with advanced-stage/metastatic NSCLC, melanoma, HNSCC, RCC, or 
TNBC, who are due to be treated with first-line PD-(L)1 inhibitors (as monotherapy or 
combined with other agents, as listed in Table 2). 
2. Male or female aged at least 18 years. 
3. ECOG PS: 0/1-2. 
4. Normal hematologic, renal and liver function. 
5. Absolute neutrophil count > 1500/mm3, platelets > 100,000/mm3, hemoglobin > 9 g/dL. 
6. Creatinine concentration ≤ 1.4 mg/dL, or creatinine clearance > 40 mL/min. 
7. Total bilirubin < 1.5 mg/dL, ALT+ AST levels ≤ 3 times above the upper normal limit. 
 
Exclusion criteria: 
1. Any concurrent and/or other active malignancy that has required systemic treatment within 
2 years of the first dose of treatment. 
2. For the NSCLC: presence of activating EGFR, ALK, ROS1, RET, NTRK alterations linked to an 
approved first-line targeted drug. 
 
Participation in NeutroFlow study will be compatible with other research studies as long as the 
inclusion and exclusion criteria described above are met. 
 

 
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The study is neither gender-blinded nor gender-biased (as defined by the GEAR tool). The gender 
dimension is considered in the trial’s selection criteria for patient recruitment. Since breast cancer 
mostly affects women, recruitment of participants for this indication is expected to be focused on 
biological sex. However, a balanced cohort with respect to gender and sex will be recruited for all 
other cancer indications. 
3.2. Sample size and power calculation 
A sample size of N=600 was determined with up to 350 NSCLC, 180 TNBC, 60 RCC, 50 melanoma and 
120 HNSCC patients. 
 
Minimal sample size per indication was determined assuming predictive capability of AUC ≥ 0.7 per 
indication. Under this assumption, and in order to have 95% power to detect a similar AUC at an alpha 
of 0.05, a cohort of at least 50 patients per indication is required. 
 
Sample size per largest indication (i.e., NSCLC) was determined assuming an Overall Survival Hazard 
Ratio of 0.5 between patients with positive to negative NeutroFlow prediction. Under this assumption, 
and assuming a 55% event rate, a validation cohort of 166 patients is required to have 90% power to 
detect a similar (0.5) HR at an alpha of 0.05. Further assuming a 1:1 ratio between development and 
validation cohort, a total sample size of 332 patients is required. 
 
Sample size calculations were conducted using pROC package V1.18.4 for R. 
3.3. Design of the clinical study 
This is an uncontrolled (observational) clinical study. The study will prospectively collect patient blood 
samples and clinical data from patients diagnosed with NSCLC, melanoma, HNSCC, RCC or TNBC for 
the development of an in vitro diagnostic medical device. 
3.4. Type of intervention 
Study is a non-interventional. The patients are treated with the standard of care treatment for their 
disease and stage. The study serves to collect patient blood samples and clinical data for the 
development of an in vitro diagnostic medical device. 
3.5. Description and timing of study procedures 
Patients will participate in the study for at least 2 years. The study schedule per patient is described 
below: 
1. Patient consent for participation in the clinical study (after diagnosis and before 
commencement of treatment). 
2. Blood withdrawal at baseline before treatment initiation (any time from 1 month before 
treatment up to the day of first treatment). 
3. Blood sample analysis at the point of care (i.e., flow cytometry assay to measure the level of 
Ly6Ehi neutrophils in the blood sample). 

 
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4. Up to 2 years of follow up, monitoring disease progression, death events and adverse events. 
5. Statistical analysis of interim results once every 4 months. 
 
Table 3. Schedule of procedures and follow-ups 
Procedure 
Screening / Before 
1st treatment 
Every 3 months following 
treatment or according to 
the SOC 
End of Study (up to 
2 years from 
screening) 
Clinical data collection 
X 
X 
X 
Blood collection 
X 
- 
- 
Blood sample analysis 
(flow cytometry) 
X 
- 
- 
Response assessment 
by RECIST 1.1  
- 
X 
- 
Total volume of blood 
collected 
up to 34 ml 
- 
- 
 
3.6. Study variables 
Data will be collected by a qualified personnel into a secure, de-identified (without personal 
identifying details) electronic database as Electronic Case Report Forms (eCRF) and will include the 
following information (according to the time points): 
 
Screening/Prior to the first blood collection: 
● Demographic information (ethnicity, year of birth, gender) 
● Medical history (including smoking history, hypertension, diabetes, ischemic heart disease, 
renal insufficiency, chronic obstructive pulmonary disease, asthma, liver insufficiency, 
Inflammatory Bowel Disease, autoimmune diseases, endocrine diseases, and others) 
● Concomitant medication (prescribed and non-prescribed, including vitamins and herbal 
supplements) taken at the time of blood sampling (name and dosage) 
● Hematology (RBC, WBC, neutrophils count, platelets count, lymphocytes count, % 
lymphocytes, Absolute eosinophils count, % eosinophils, Monocyte count, % monocytes, 
hemoglobin) 
● Renal function (Creatinine concentration or creatinine clearance) 
● Liver function (Total bilirubin, ALT, AST) 
● Specific biomarkers (LDH, CRP, albumin, alkaline phosphatase, NSE where applicable) 
● Chemistry (Calcium, Sodium) 
● Cancer information: Date of cancer diagnosis, stage, primary cancer type and location, 
molecular type (if exists), history of the cancer treatments (if relevant), histology (if exists), 
Biomarkers (including mutation if exist) including PD-L1 status, MSI (Microsatellite Instability) 
and TMB (Tumor Mutational Burden), TNM stage, assessment of measurable lesions (at least 

 
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one), metastasis location, date of metastatic disease diagnosis (if relevant), site of metastasis 
(if relevant, local, distant: liver, lung, brain, bone, lymph nodes, other).  
● Intended treatment protocol (drug/treatment and dosages, regimens) 
● ECOG status  
● Vital signs (Temperature, Pulse, Weight, Height, blood pressure) 
● Date, time, and volume of sample collection 
● Date and time of first treatment 
 
Clinical Benefit (CB) evaluation (Every 3 months or according to SOC until progression): 
● CB using the imaging modality of RECIST 1.1 to assess response. If imaging is used, it should 
be the same modality used to assess the measurable lesion at screening. 
● Date of CB evaluation 
● Compliance with the planned treatment (which is defined as compliance with the treatment 
up to the point of current CB evaluation) 
● Current anti-cancer treatment and the date and reason of change or stop, if relevant 
● Data on patients’ outcome (including first event of recurrence/progression, survival status and 
OS date, PFS date, date of last treatment given) 
● In case of patient death - cause of death 
● Side effects to the cancer treatment, and treatment given to treat these side effects 
● Received concomitant medications 
● In case of stopping/changing the treatment protocol - the proposed treatment protocol and 
the reason for changing 
● Adverse events related to the study procedures 
 
End of study (2 years after screening or earlier in case of death or early termination): 
● Data on completion or reason for early termination of the study, if relevant. 
● Data on patients’ outcome (survival status and OS or last follow-up date, PFS date, date of last 
treatment given) 
● In case of patient death - cause of death 
 
4. Preparedness status 
4.1. Development of the clinical study protocol 
4.1.1. Scientific advice from regulatory and health technology assessment 
bodies 
This clinical study protocol has been established by the NeutroFlow project consortium made up of 
four members: 
1. OncoHost Ltd (OH) 
2. European Institute of Oncology – Istituto Europeo di Oncologia SRL (IEO) 
3. Heidelberg University Hospital – Universitätsklinikum Heidelberg (UKHD) 

 
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4. Virgen Macarena University Hospital – Servicio Andaluz de Salud (SAS) 
 
The consortium has consulted regulatory consultants regarding regulatory issues (e.g., GDPR 
requirements, ethics), and a KOL committee established from representatives of each clinical partner 
involved in the project regarding clinical issues (e.g., response labelling, time-points per indication). 
4.1.2. Clinical efficacy, safety, and methodological guidelines (including 
guidelines on statistics) 
As the study is non-interventional, efficacy and safety considerations have a limited effect on the study 
design. Dr. Anat Reiner Ben-Naim (Department of Public Health, Ben Gurion University, Israel) will 
supervise statistical analysis through the study. Depending on the statistical and clinical characteristics 
of the cohort and the study objective, the following metrics may be used to evaluate the model 
predictive capability: 
a. Area Under Curve (AUC) of the Receiving Operating Characteristic (ROC) curve.  
b. R2 (goodness-of-fit). 
c. Concordance Index (C-index). 
d. Hazard Ratio (HR) of Overall Survival and Progression Free Survival (PFS) distribution between 
different treatment modalities. 
e. Statistical significance, as expressed by Cox-regression p-value, and 95% Confidence Interval 
(CI). 
4.1.3. Involvement of citizens / patients, carers in drawing up the clinical study 
protocol 
Citizens and patients are not involved in the clinical study protocol design. Physicians from SAS, IEO 
and UKHD, serving as the principal investigators, have participated in the protocol design. A KOL 
committee established from representatives of each clinical partner have been consulted regarding 
clinical issues (e.g., response labelling, time-points per indication). 
4.1.4. Regulatory intelligence to ensure timely regulatory approval and ethics 
clearance of the clinical study in all jurisdictions where its implementation is 
planned 
For each participating site, an experienced investigator (who has experience with initiation and 
approval of clinical trials in the specific site) will be assigned to support regulatory approval and ethics 
clearance. Three clinical sites will participate in the study: IEO, UKHD, SAS. 
 
Dr. A. Moreno (SAS) will lead the clinical trial and co-ordinate between all participants involved (IEO, 
UKHD, SAS). Dr. D. Vicente-Baz, Coordinator of the Andalusian Integral Oncology Plan at SAS, will lead 
NSCLC & HNSCC indications. Dr. L. de la Cruz-Merino, Director of the Clinical Oncology Unit at SAS, will 
lead TNBC & Melanoma indications. Dr. M. Colleoni will lead breast cancer patient recruitment at IEO.  
 

 
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The assigned investigator will submit the study protocol, informed consent form (ICF) and any required 
study documents to the Ethics Committee (EC) / Institutional Review Board (IRB) for review and 
approval. Any amendment to the protocol will require review and approval by the EC/IRB before the 
changes are implemented to the study. All changes to the consent form will be EC/IRB approved. An 
assessment will be conducted to decide if re-consent from participants is required. 
4.2. Ethics and regulatory compliance 
Regulatory requirements in all jurisdictions will be learned and applied. If necessary, the consortium 
will contact the regulatory bodies for clarifications or will use regulatory advisors. The study will be 
conducted in compliance with the HIPPA (Health Insurance Portability and Accountability Act) 
legislation and the GDPR (General Data Protection Regulation). Only the PIs, site personnel and study 
monitors will have access to the medical records of the patient. In order to maintain confidentiality of 
the patients, patients will be identified using a designated code, and the key that relates the code with 
the subject will be kept by the PI. Clinical information will be entered to the eCRFs using the coded 
subject number. The ICF will incorporate wording that complies with relevant data protection and 
privacy legislation. All relevant software that may contain patient’s information will be compliant with 
HIPPA/GDPR. 
 
The study will be performed in accordance with ethical principles that have their origin in the 
Declaration of Helsinki and are consistent with ICH E6 (GCP), and in full conformity with the regulations 
for the protection of human subjects of research codified in 45CFR part 46, 21CFR part 50, 21CFR part 
56, and applicable local regulatory requirements whichever provides most protection to the human 
subjects. 
 
Ethical requirements in all jurisdictions will be learned and applied to the study proceedings and 
documentation. In case of misunderstanding, the consortium will contact the ethics committee or an 
advisor for clarification. 
4.3. Scientific and operational governance 
The proposed NeutroFlow project will be conducted by a consortium made up of four members: 
● OncoHost LTD (OH) – SME, Israel 
● European Institute of Oncology (IEO) – hospital, Italy 
● Heidelberg University Hospital (UKHD) – hospital, Germany 
● Virgen Macarena University Hospital – Servicio Andaluz de Salud (SAS) – hospital, Spain 
 
The clinical trial (which is an integral part of the NeutroFlow project) will be led by SAS. 
 
OH will be responsible for: 
(i) Overall EIC project coordination. 

 
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(ii) Development and clinical validation of a prototype NeutroFlow test including: Diagnostic-grade kit; 
Flow cytometry assay protocol; Computational predictive model that translates flow cytometry 
readout into a prediction of durable clinical benefit.  
(iii) Business development activities. 
 
IEO will be responsible for: 
(i) Leading flow cytometry-related activities and analytical validation of the prototype to ensure 
reproducibility, standardisation, precision and accuracy. 
(ii) Providing blood samples and clinical data from ~250 patients (NSCLC, TNBC). 
(iii) On-site flow cytometry analysis. 
 
UKDH will be responsible for: 
(i) Leading communication and dissemination activities. 
(ii) Providing blood samples and clinical data from ~250 patients (NSCLC, RCC, HNSCC). 
(iii) On-site flow cytometry analysis. 
 
SAS will be responsible for: 
(i) Leading the collection of patient blood samples and clinical data. 
(ii) Providing blood samples and clinical data from ~250 patients (NSCLC, TNBC, melanoma, HNSCC). 
(iii) On-site flow cytometry analysis. 
 
5. Operational feasibility 
5.1. Availability of resources throughout implementation phase 
The NeutroFlow prototype kit for the flow cytometry assay will comprise a panel of fluorophore- 
conjugated monoclonal antibodies, each specifically directed to a marker expressed on the surface of 
Ly6Ehi neutrophils (CD45, HLA-DR, Lin-CD3/14/16/19/20/56, CD11b, CD33, CD14, CD15, Ly6E). All 
antibodies will be GMP-compliant, diagnostic-grade monoclonal antibodies that comply with 
requirements for in vitro diagnostic (IVD) device components. The relevant antibodies for the above- 
listed markers (excluding Ly6E) are commercially available as IVD-grade off-the-shelf products, and 
will be acquired from specialist providers (e.g., BD Biosciences). The Ly6E antibody will be available as 
a proprietary, monoclonal Ly6E antibody that will be custom produced by a contractor specialising 
in clinical products. All antibodies will be shipped frozen to each medical centre. 
 
Patient blood samples will be analysed on-site by the clinical flow cytometry facility available at each 
medical centre. Disposable and non-disposable equipment required for blood collection, PBMC 
isolation and storage will be provided by each of the participating clinical centres. All antibodies 
required for the flow cytometry assay will be shipped frozen to the analysis sites. All clinical data will 
be documented in a secure, de-identified (without any personal identifying details) electronic 
database as Electronic Case Report Forms (eCRF) hosted by SAS and made accessible to the rest of 
recruitment sites. 

 
NeutroFlow Study Protocol v1.0 
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5.2. Recruitment of study population 
PI and sub-investigators from SAS, IEO and UKHD will identify potentially eligible patients from their 
practices. They will discuss participation in the study with patients and their families during the 
consent discussion and address any questions or concerns they may have about the study. Informed 
consent will be obtained from all patients being considered for this study prior to the initiation of any 
study-specific procedure, including screening. Only patients meeting the eligibility criteria will 
participate in the study. 
 
During the consent discussion, all potential risks and benefits associated with this study will be 
explained to the patient in their language as objectively as possible by one of the study investigators, 
and an Informed Consent Form approved by the Institutional Review Board / Ethics Committee will be 
provided. In the informed consent form, the patient will be required to provide consent for the 
collection of biological samples and clinical information. Potential subjects will be given an opportunity 
to review the consent form at their leisure. Once all their questions have been addressed and the 
individual is willing to participate in the study, informed consent to proceed with the study procedures 
will be obtained from the patient using the IRB-approved form (a template of the ICF to be translated 
and adapted for each clinical site is provided in Annex 1) and co-signed by one of the study 
investigators. 
 
Recruitment progress will be monitored to identify delays or underperformance. We have set goals of 
reaching interim targets of 75 recruited patients by M12, 250 patients by M18, and a final target of 
600 patients by M30. The targets are in line with the timeline of the EIC project as a whole (specified 
in clause 5.5), ensuring successful completion of the project. 
 
Three clinical sites will contribute to patient recruitment – IEO (Italy), UKHD (Germany) and SAS 
(Spain). These clinical centres are part of the NeutroFlow consortium applying to the EIC transition 
program. All clinical centres have on-site flow cytometry facilities required for sample analysis. 
 
Patient recruitment will not be of competitive nature. The estimated number of recruited patients per 
centre is summarised in Table 4. Numbers are based on patient throughput per centre in recent years. 
Samples will be collected over 2 years (M6 to M30, allowing at least 6-month follow up by project end 
at M36). 
 
Table 4. Estimated number of recruited patients 
 
SAS 
UKHD 
IEO 
Total 
NSCLC 
150 
100 
100 
350 
TNBC 
40 
- 
140 
180 
RCC 
- 
60 
- 
60 
Melanoma 
50 
- 
- 
50 
HNSCC 
16 
100 
- 
116 

 
NeutroFlow Study Protocol v1.0 
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Total 
256 
260 
240 
756 
 
Mitigation of underperformance: 
The trial recruitment potential is 25% higher than the requested number of participants (n=600). We 
have set a conservative end goal of reaching 600 samples by M30, and interim targets of 75 samples 
by M12, and 250 samples by M18. If we do not reach the interim targets, we will purchase 
retrospective, frozen samples (in accordance with trial protocol and inclusion/exclusion criteria) from 
blood banks to further increase the sample size. In case of major underperformance, additional 
medical centres will be approached. 
 
As this is an uncontrolled study, the recruitment capability is estimated based on the average annual 
number of patients (per indication) that are treated in each of the participating sites, and an 
estimation of the percentage of patients that will provide their consent for participation (based on 
performance in previous trials). 
5.3. Data Management 
The study will be conducted in compliance with the HIPPA (Health Insurance Portability and 
Accountability Act) legislation and the GDPR (General Data Protection Regulation). Patient clinical data 
will be collected by qualified personnel into a secure, de-identified (without personal identifying 
details) electronic database as Electronic Case Report Forms (eCRF) hosted by SAS and made 
accessible to the rest of recruitment sites. 
 
Data integrity, accuracy, and completeness will be verified in comparison to the source data by a 
qualified person. All project-related data (including clinical data and flow cytometry data) will be 
stored in an encrypted database restricted to the data owners on SAS’ secure IT infrastructure with 
daily back-ups. Access to the database will not be made public, and all connections to the database 
will be performed via VPN connection. No information concerning the study, collected data or 
generated data will be released to any unauthorized third party without prior written approval from 
study PIs. 
5.4. Reporting and monitoring obligations 
Reporting and monitoring obligations will be met by the consortium clinical partners (UKHD, IEO, SAS). 
Study monitoring will be performed in compliance with recognized Good Clinical Practices. The 
monitor’s duties include on-site or remote visits and review of study documents. Monitoring visits are 
intended to assess the investigators’ adherence to the investigational plan and study protocol, 
maintenance of the study records, and review of the source documents for accuracy, completeness 
and legibility. During these visits, the monitor will assess the progress of the study towards meeting 
the study objectives and identify any concerns that stem from reviewing of the patient records, study 
management documents and informed consent documents. The monitor will report the findings of 
each visit to the study personnel. The final monitoring visit is intended to assure that all the data have 
been properly completed, clinical study documentation is complete, and to perform a closing meeting 

 
NeutroFlow Study Protocol v1.0 
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with the investigator and the staff members. Reports of the visits will be written by the monitor and 
should include resolution of concerns, completion of follow up activities, completion of assigned tasks 
and any necessary corrective actions. At study completion, the monitor will prepare a final report. 
 
The study protocol, documentation, data and all other information generated will be held in strict 
confidence. Only the study monitors, other authorized representatives of the clinical sites or 
representatives of the EC/IRB/competent authority may inspect documents and records required to 
be maintained by the investigator. No information concerning the study, or the data will be released 
to any unauthorized third party without prior written approval of the study PIs. 
 
All regulatory requirements, clinical site monitoring and data management activities will be answered 
directly by the consortium partners (OH, UKHD, IEO, SAS). No responsibilities will be supported by 
entities that are not part of the NeutroFlow consortium partners. 
5.5. Study milestones 
The study schedule will follow the timeline (evaluating time in months from day of proposal approval): 
1. Compilation of required regulatory and ethics submission package – M1-M3 
2. Receipt of regulatory and ethics approval - M6 
3. Initiation of clinical sites – M6 
4. Recruitment of the first 200 patients - M15 
5. Clinical response data (6 months clinical benefit) collected for 50 patients - M15 
6. Mid-term recruitment report (300 recruited patients) – M21 
7. Blood samples collected from a total of 600 patients across 5 tumour types – M30 
8. Final assessment of all study participants – M36 
9. Analysis and reporting of study results (including flow cytometry data analysis and 
performance of final predictive model) – M36 
10. Completion of last patient follow-up (post-project) – M54 
 
The plan is based on previous experience of the involved parties (OH, UKHD, IEO, SAS) in the initiation 
and execution of multicentre observational trials. 
 
6. References 
[1] Alturki, N. A. (2023). Review of the immune checkpoint inhibitors in the context of cancer 
treatment. Journal of Clinical Medicine, 12(13), 4301. 
[2] Haslam, A., Gill, J., & Prasad, V. (2020). Estimation of the percentage of US patients with cancer 
who are eligible for immune checkpoint inhibitor drugs. JAMA network Open, 3(3), e200423-
e200423. 
[3] Man, J., Millican, J., Mulvey, A., Gebski, V., & Hui, R. (2021). Response rate and survival at key 
timepoints with PD-1 blockade vs chemotherapy in PD-L1 subgroups: meta-analysis of 
metastatic NSCLC trials. JNCI Cancer Spectrum, 5(3), pkab012. 

 
NeutroFlow Study Protocol v1.0 
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[4] Martins, F., Sofiya, L., Sykiotis, G. P., Lamine, F., Maillard, M., Fraga, M., ... & Obeid, M. (2019). 
Adverse effects of immune-checkpoint inhibitors: epidemiology, management and 
surveillance. Nature reviews Clinical oncology, 16(9), 563-580. 
[5] Hurwitz, J. T., Vaffis, S., Grizzle, A. J., Nielsen, S., Dodson, A., & Parry, S. (2022). Cost-
Effectiveness of PD-L1 Testing in Non-Small Cell Lung Cancer (NSCLC) Using In Vitro Diagnostic 
(IVD) Versus Laboratory-Developed Test (LDT). Oncology and therapy, 10(2), 391-409. 
[6] Lei, Y., Li, X., Huang, Q., Zheng, X., & Liu, M. (2021). Progress and challenges of predictive 
biomarkers for immune checkpoint blockade. Frontiers in Oncology, 11, 617335. 
[7] Arora, S., Velichinskii, R., Lesh, R. W., Ali, U., Kubiak, M., Bansal, P., ... & Boumber, Y. (2019). 
Existing and emerging biomarkers for immune checkpoint immunotherapy in solid tumors. 
Advances in therapy, 36, 2638-2678. 
[8] McKean, W. B., Moser, J. C., Rimm, D., & Hu-Lieskovan, S. (2020, May). Biomarkers in Precision 
Cancer Immunotherapy: Promise and Challenges. In American Society of Clinical Oncology 
Educational book. American Society of Clinical Oncology. Annual Meeting (Vol. 40, pp. e275-
e291). 
[9] Benguigui, M., Cooper, T. J., Kalkar, P., Schif-Zuck, S., Halaban, R., Bacchiocchi, A., ... & Shaked, 
Y. (2024). Interferon-stimulated neutrophils as a predictor of immunotherapy response. 
Cancer Cell, 42(2), 253-265. 
 
 

 
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Annex 1. ICF template 
 
PATIENT INFORMATION SHEET 
 
Title: Development and validation of a pan-cancer neutrophil biomarker test for predicting clinical 
benefit from immunotherapy based on flow cytometry analysis of blood samples (NeutroFlow) 
 
This form is part of the informed consent process. You do not have to take part in this study if you do not 
want to, and your medical care will not depend on whether or not you take part. This study may not directly 
help you, but we hope that it will allow us to expand our knowledge so that we can help other patients in 
the future.  
 
In accordance with the provisions of the General Data Protection Regulation and applicable regulations for 
clinical and biomedical research, we ask you to take the necessary time to read this document, consult with 
your relatives and clarify with your doctor any possible doubts you may have in this regard. This research 
study fully respects your data-related rights, such as access, rectification, cancellation, the right to 
limitation of processing, portability, the right to withdraw consent to data processing and the right to 
complain to your data privacy agency. 
 
GENERAL INFORMATION 
We are reaching out to you to inform you about a research study in which you are invited to participate. 
You are being invited to participate in this research study because you have been diagnosed with any of 
the following diseases: Stage IV Non-Small Cell Lung Cancer or Stage III-unresectable Non-Small Cell Lung 
Cancer; Stage IV or Stage IIIb-d malignant melanoma; Stage IV Head and neck squamous cell carcinoma; 
Stage IV Renal Cell Carcinoma; or Stage IV Triple-Negative Breast Cancer; and your doctor considers that 
you are eligible for this research study. Your inclusion in the study is subject to the screening procedures 
described below, and other entry criteria. Before you can take part in this study, it is important that you 
understand what is involved. Please read this document carefully and ask any questions you may have, and 
consult with others as you feel appropriate. The study has been approved by the relevant Clinical Research 
Ethics Committee. 
 
OBJECTIVE 
The study objective is two-folded: 
1. To develop a kit and flow cytometry assay for detecting and quantifying the level of Ly6Ehi 
neutrophils in blood samples. 
2. To develop a model for predicting clinical benefit from treatment with PD-(L)1 inhibitors based on 
the level of Ly6Ehi neutrophils, a specific type of white blood cells in patient blood samples. 
 
In this study, the Ly6Ehi neutrophils will be assessed as potential biomarkers to predict whether a type of 
treatment (Immunotherapy based on immune checkpoint inhibitors) will have a clinical benefit in patients 
like you. 
 
 
 
 

 
NeutroFlow Study Protocol v1.0 
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VOLUNTARY NATURE OF PARTICIPATION AND THE POSSIBILITY OF WITHDRAWAL 
Your participation in this study is voluntary. If you decide to participate, but later change your mind, you 
are free to do so without explanation. You may withdraw from the study at any time without explanation 
and without affecting your medical care.  
 
STUDY PROCEDURES 
● 
To take part in this study, we’ll need to take a sample of your blood of up to 34 ml to analyse your 
Ly6Ehi neutrophils level. This sample will be taken before you start your treatment, and will be 
followed by standard procedures for blood extraction in your clinical center.  
● 
During the study you will receive the standard of care treatment from your doctor, and no 
additional study-related extra tests beyond those considered necessary by your clinician to 
monitor and follow-up your health status will be performed. 
● 
In addition to the blood sample, we ask you to give us permission to collect information from your 
health record, such as demographic information; medical history, concomitant medication, lab 
tests, and specific information about your disease. In addition, throughout the next 2 years, we 
will also collect information about your evolution, treatment efficacy, and potential adverse events 
that you may experience. 
● 
The clinical information and results generated as part of the study may be shared with other 
researchers and doctors, national and international regulatory agencies as well as in scientific 
repositories, always ensuring the necessary conditions of quality, security and confidentiality. In 
case of transfer of your data to third countries, the Research Group will ensure that a level of data 
protection at least equivalent to that granted by Regulation (EU) 2016/679 of the European 
Parliament and of the Council of 27 April 2016 on Data Protection (GDPR) is maintained. The 
Research Group is obliged to retain the data collected for the study for at least 25 years after its 
completion. Thereafter, your personal information will only be retained by the centre for your 
health care and by the sponsor for other scientific research purposes if you have given your 
consent to do so, and if permitted by applicable law and ethical requirements. 
 
POTENTIAL BENEFITS 
There may be no potential benefit to you from participating in this study. However, a better understanding 
of the potential clinical benefit related to immunotherapy based on immune checkpoint inhibitors and the 
role that Ly6Ehi neutrophils may have in this regards may make it easier to provide individualised treatment 
for each patient in the future, minimising the impact of side effects and increasing the likelihood of 
treatment success. 
 
POTENTIAL HAZARDS 
To participate in the study we need you to allow us to obtain peripheral blood samples which will be 
collected in two EDTA tubes. Drawing blood is a routine medical procedure, but like any procedure, it may 
involve some risks and discomfort. These may include: 
● 
Mild pain or discomfort: You may feel a brief pinch or stinging sensation when the needle is 
inserted. 
● 
Bruising or swelling: It is common to experience some bruising, swelling, or redness around the 
site where the blood is drawn. 
● 
Bleeding: Occasionally, there may be slight bleeding at the puncture site. Rarely, some people may 
experience more prolonged bleeding, especially if they have a bleeding disorder or take blood-
thinning medications. 

 
NeutroFlow Study Protocol v1.0 
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● 
Fainting or dizziness: Some individuals may feel lightheaded or faint during or shortly after the 
procedure. 
● 
Infection: There is a small risk of infection any time the skin is broken. All necessary precautions 
are taken to keep this risk very low. 
● 
Nerve injury: Rarely, the needle may come close to a nerve, which can cause temporary pain or 
tingling. 
All these side effects are usually minor and go away on their own. 
 
COMMERCIAL INTERESTS AND PATENTS DERIVED FROM THE STUDY 
OncoHost, Ltd., an SME based in Binyamina, Israel, coordinates the NeutroFlow project, and it has declared 
its interest in the commercialization of the results derived from the research study. You will not receive 
any benefit derived from any potential commercial and/or industrial patent derived from this research 
study. 
 
PERSONAL DATA PROCESSING AND CONFIDENTIALITY 
Your personal data, as well as clinical data and any data obtained from the analysis of samples, will be 
processed in accordance with the General Data Protection Regulation (GDPR) 2016/679 on the protection 
of personal data and any other applicable regulation. 
 
In accordance with current law, you have the right to access your personal data, and to request its 
rectification, cancellation, or objection. You may exercise these rights at any time by contacting your 
attending physician or the principal investigator of this study. If you choose to withdraw from the study, 
the research team will continue to use any data already collected about you, unless you specifically request 
that such data not be used. 
 
You may also exercise your rights to limit processing, withdraw consent (exclusively in relation to the 
processing of personal data), and file complaints by contacting the designated data protection agency. 
 
Samples will be coded and identified using a combination of numbers and letters, which will not allow your 
identification except by the principal investigator of the study. Your name, initials, or medical record 
number will not appear in the databases generated by the study. However, if the information obtained in 
the study is deemed by your physician to be relevant to your health or that of your biological relatives, you 
may be informed of this if you so wish. In any case, communication will be limited strictly to the data 
necessary for these purposes. 
 
USE OF BIOMEDICAL SAMPLES 
Your blood samples and associated data will be used only by the researchers of the project. The data and 
samples will be used only for the purposes of the study. After the end of the project, surplus samples may 
be stored in the Biobank if you give your express consent. In this case, you will sign the biobank consent 
form in force at the time. Otherwise, the samples will be stored until the project has been completely 
concluded and will be subsequently destroyed in accordance with the regulations applicable in the centre.  
 
Should you have further doubts or questions about your participation in the study and your rights, please 
contact with __________________________________ phone number: __________ 
 
 
 

 
NeutroFlow Study Protocol v1.0 
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INFORMED CONSENT FORM (Date: 12/06/2025; VERSION: v1.0) 
 
Title: Development and validation of a pan-cancer neutrophil biomarker test for predicting clinical 
benefit from immunotherapy based on flow cytometry analysis of blood samples (NeutroFlow) 
 
I, ____________________________________________________ 
 
Hereby declare that: 
YES ▢ NO ▢ 
I’ve read the patient information sheet attached to this consent form. 
YES ▢ NO ▢ 
I’ve been able to ask questions and have received enough information about the 
NeutroFlow study. 
YES ▢ NO ▢ 
I understand that my participation in the study involves giving blood sample and 
information from my clinical records. 
YES ▢ NO ▢ 
I accept that my blood samples can be analysed for the purposes described in the 
information sheet. 
YES ▢ NO ▢ 
I understand that I participate on a voluntary basis and that I’m free to participate or 
not in the study, being able to withdraw whenever I want, without further explanations, 
and that this will not affect my medical care. 
YES ▢ NO ▢ 
I authorize that the results obtained from this research study can be reused in future 
research projects. 
YES ▢ NO ▢ 
I authorize that the surplus of my biological samples, data, and any derivatives from this 
project, if any, may be stored in the Biobank, to be used in future projects within the 
same line of Oncology research. 
YES ▢ NO ▢ 
I wish to be informed of any personal information obtained during the course of the 
research, including any unexpected findings that may arise, provided that this 
information is necessary to prevent serious harm to my health or that of my biological 
relatives. 
YES ▢ NO ▢ 
I freely give my consent for my data to be transferred to other public or private 
institutions, individuals and legal entities, within the European Union or outside it, that 
will use them in studies aimed at acquiring or expanding knowledge related to cancer 
treatment and research. 
I freely give my consent to participate in the study entitled: Development and validation of a pan-cancer 
neutrophil biomarker test for predicting clinical benefit from immunotherapy based on flow cytometry 
analysis of blood samples (NeutroFlow). 
 
Signed in _______________________ , at __________ 
 
 
 
 
Study participant 
Informing clinician 
Legal representative  
(when needed)