Understanding Gene ENvironment Interaction in ALcohol-related Hepatocellular Carcinoma
Phase: NA
Conditions: HCC, Genetic Predisposition
Sponsor: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico

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Gene-environment interaction study
in alcohol-related hepatocellular carcinoma
(European Project Title HORIZON-MISS-2021-CANCER- 02-03: <Understanding Gene
ENvironment Interaction in ALcohol-related hepatocellular carcinoma=)
Acronym: GENIAL
Version 2 of May 27, 2024
PROMOTER: IRCCS Ca' Granda Foundation, Maggiore Hospital, Policlinico, via Sforza 29, 20122 Milan, Italy,
COORDINATING CENTER: SC Transfusion Medicine
IRCCS Ca' Granda Foundation Maggiore Hospital Polyclinic, via Sforza 29, 20122 Milan Italy
PRINCIPAL INVESTIGATOR: Dr Serena Pelusi 
Signature
DOCUMENT TYPE: non-pharmacological interventional study protocol
PRIVACY STATEMENT
All information presented in this document will be considered confidential and will remain the exclusive 
property of Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. The use of such confidential 
information must be limited to the recipient for the agreed purpose and must not be disclosed, published or 
otherwise communicated to unauthorized persons, for any reason, in any form without the prior written 
consent of FondazioneIRCCSCa'GrandaOspedaleMaggiorePoliclinico.
Translated from Italian to English - www.onlinedoctranslator.com

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List of abbreviations:
CI: informed consent
EC: Ethics Committee
CRF: case report form
NAFLD: non-alcoholic fatty liver disease. 
ALD: alcoholic liver disease
HCC: hepatocellular carcinoma 
EU: European Union
BMI: body mass index CA: 
abdominal circumference AI: 
artificial intelligence
CAP: controlled attenuation parameter 
LSM: liver stiffness measurement IL-32: 
interleukin 32
Pro-C3: pro-collagen 3 WES: 
whole exome sequencing PRS: 
polygenic-risk score
SNP: single nucleotide polymorphism
Responsibilities (role of the promoter and collaborators)
The Sponsor of the study will be the IRCCS Foundation Ca' Granda Ospedale Maggiore Policlinico of Milan.
The role of coordinating center of the study will be covered by the SC Transfusion Medicine of the IRCCS Foundation Ca' 
Granda Ospedale Maggiore Policlinico of Milan.
The Principal Investigator of the study will be Dr. Serena Pelusi who will be responsible for patient enrollment, collection 
of clinical and genetic data and follow-up visits.
Internal Collaborations
Operational Unit
Participant Name
Role and functions in the study
Complex Structure Transfusion 
Medicine
IRCCS Foundation Ca' Granda Hospital 
Maggiore Policlinico, Milan
Dr. Serena Pelusi
(principal investigator) Dr 
Daniele Prati
Dr. Alessandro Cherubini 
Dr. Luisa Ronzoni
Dr. Vittoria Moretti
Study coordination Genetic 
characterization of patients Clinical 
characterization of patients
External collaborations
Operational Unit
Participant Name
Role and functions in the study
Free University of Brussels_ULB
Prof. Eric Trepo
European Coordinator
University of Milan_UMIL
Professor Luca Valenti
Genetic characterization of patients
Technical University of Dresden_TUD
Dr. Jacob Kather
Integrating genetic and clinical data into 
artificial intelligence algorithms
Institut National de la Santé et de la
Prof. Jessica Zucman-Rossi
Identification of new variants

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Recherche Medicale, Paris_INSERM
genetic predisposition to 
hepatocellular carcinoma
Hospitals of Paris_APHP
Professor Pierre Nahon
Characterization of the role of gene-
environment interaction in the risk of 
hepatocellular carcinoma

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INDEX
1. INTRODUCTION
1.1 Background and rationale
2. OBJECTIVE OF THE EXPERIMENTATION
2.1 Primary objective
2.2 Secondary objective(s)
3. STUDY DESIGN
3.1 Study design
3.2 Inclusion criteria
3.3 Exclusion criteria
4. PROCEDURES RELATING TO THE STUDY
4.1 Intervention
4.2 Randomization
4.3 Blindness
5. ENDPOINT
5.1 Primary Endpoint
5.2 Secondary Endpoint
6. DURATION/TIMELINE OF THE STUDY
7. STATISTICAL ANALYSIS
7.1 Sample size
7.2 Data analysis
8. ADVERSE EVENTS
9. RISK/BENEFIT ASSESSMENT
10. STUDIO MANAGEMENT
10.1 Data collection and management
10.2 Regulatory aspects and ethical considerations
10.2.1 Approval by the competent authority
10.2.2 Ethics Committee Approval
10.2.3 Informed consent
10.3 Duties of the experimenter
10.4 Study monitoring
10.5 Study quality assurance
10.6 Closing of the study
10.7 Document storage
10.8 Disclosure of information regarding scientific discovery
10.8.1 Confidentiality
10.8.2 Publications
11. Indemnity and compensation in case of damages 
12 Amendments to the protocol
13 Financial agreements
14 Disclosure on conflicts of interest 15 
References

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16 Appendix

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1. INTRODUCTION
1.1 Background and rationale
Europe has the highest levels of alcohol consumption and burden of alcohol-
attributable disease compared to other regions according to the World Health 
Organization (WHO). In 2019, 6.2% of all deaths in Europe were caused by alcohol. 
Alcohol-attributable deaths were mainly due to cancer (29%) and liver cirrhosis 
(20%).
It has been estimated that alcohol causes approximately 40% of premature liver-
related deaths in Europe each year, although this number is likely underestimated. 
Alcohol-related liver disease (ALD) is the most common cause of liver cirrhosis and 
liver-related death in Europe with the peak age of death occurring in individuals in 
their 40s and 50s.
Thus, it remains a poorly understood disorder with research efforts and 
funding that contrasts sharply with its health relevance in Europe. Liver cancer 
is the second most common cause of cancer-related death (15-20% 5-year 
survival) and the second most common cause of alcohol-attributable cancer 
cases worldwide.
Hepatocellular carcinoma (HCC) accounts for approximately 90% of liver tumors and 
occurs most commonly against the background of ALD in Europe with the majority of 
cases (~90%) developing in patients with liver cirrhosis.
Some clinical features, including older age, male sex, obesity and type 2 
diabetes, have been linked to alcohol-related HCC (ALDHCC) and, together with 
some common genetic determinants, bring alcoholic liver disease and NAFLD 
closer together and they seem to share similar pathogenetic patterns. 
However, the effect of environmental variables including diet (e.g. type of 
alcoholic beverage), lifestyle and sociodemographic characteristics remain 
largely unknown. Both case-control studies and cancer databases have shown 
familial clustering suggesting genetic susceptibility. Liver carcinogenesis is the 
result of a complex multistep process generated by pro-oncogenic genetic 
alterations. These defects may pre-exist in normal cells (i.e. germline 
mutations) or result from DNA replication errors, spontaneous enzymatic 
conversions or DNA damage (i.e. somatic mutations). Recent studies have 
identified genetic variants in the WNT3A-WNT9A region as a novel susceptibility 
locus and confirmed the association of SNPs in PNPLA3 TM6SF2, HSD17B13. 
Note that SNP arrays are unable to detect

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rare variants (allele frequency <1%) that can instead be assessed by whole 
exome sequencing (WES) and whole genome sequencing (WGS).
Like other complex diseases, ALD-HCC results from the interaction between 
environmental determinants and genetic variations but knowledge on gene-environment 
interactions is currently lacking in this area.
Although common genetic variants typically individually have a small effect 
size on disease risk, aggregating multiple common variants into polygenic 
risk scores (PRS) has been shown to successfully identify individuals at risk 
and outperform existing clinical models, especially when combined with 
multiple non-genetic information (e.g., lifestyle, anthropometric factors), 
thus enabling personalized cancer screening recommendations.
Advances in artificial intelligence (AI) algorithms have made it possible to 
extract clinically relevant information from complex and diverse clinical 
datasets of liver diseases and integrate genetic and non-genetic information 
into predictive information. Therefore, characterizing gene-environment 
interactions at the population level is of utmost importance to improve risk 
stratification for ALD-HCC and early diagnosis of the tumor.
This study is part of a larger European programme entitled <Understanding 
Gene ENvironment Interaction in ALcohol-related hepatocellular carcinoma=-
HORIZON-MISS-2021-CANCER- 02-03- PROJECT CODE: GENIAL, whose general 
objective isassess the above needs through a comprehensive assessment of 
gene-environment interactions regarding ALD-HCC.
The project will assist in the identification of individuals at risk of developing alcohol-related 
hepatocellular carcinoma and in the development of strategies to prevent the development of 
alcoholic liver disease.
The GENIAL research project is coordinated by the UNIVERSITE LIBRE DE 
BRUXELLE. Among the various partners involved in the project, it includes 
the University of Milan (as Project Partner) and the Fondazione IRCCS Ca' 
Granda Ospedale Maggiore Policlinico di Milano (as Affiliated Entity).
The GENIAL project has the following objectives:

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- identification of novel genetic variants that modulate the risk of ALD-HCC 
(WP1)
- characterize how these variants are influenced by environmental factors in order to 
clarify the processes of carcinogenesis and identify potential targets for preventive 
therapies (WP2)
- provide a characterization of environmental factors (lifestyle, diet, socio-
demographic factors) related to the development of ALD-HCC and integrate them 
with radiological, histological and genetic information using AI models to predict the 
risk of ALD-HCC (WP3)
- identify the machine learning approach with the greatest potential for 
clinical application in terms of improving diagnosis so as to help healthcare 
professionals provide better and faster recommendations and refine it for 
possible commercial application (WP3)
These evaluations will be carried out in 5 project work packages (WP) 
through a unified coordination, monitoring and evaluation component 
(WP4- WP5).
This study protocol in particular refers to what is foreseen in WP1 and WP3 
which foresees the involvement of the State University of Milan and the IRCCS 
Foundation Ca' Granda Ospedale Maggiore Policlinico di Milano.
In this study we intend to contribute to investigate the genetic predisposition and the 
interaction between genetic and environmental factors in the development and 
progression of liver disease towards HCC. To do so, we will share the clinical and genetic 
data of two cohorts of patients belonging to two previously approved studies (EPIDEMIC-
NAFLD cohort, SERENA study cohort) for the performance of WP1 and WP3 of the GENIAL 
project.
2.OBJECTIVE/S/I HYPOTHESIS OF THE EXPERIMENTATION
2.1 Primary objective
The primary objectives of the study are:

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1) To evaluate whether the rare and common genetic variants identified by the 
Consortium in ALD-HCC patients (ULB and INSERM partners) predispose to the risk of HCC 
both in patients affected by NAFLD and ALD or whether they are ALD specific
2) To assess whether there is an association between the identified genetic variants 
and ALD-HCC within the general population (UK Biobank)
2.2 Secondary objective Secondary 
objectives of the study are:
1) Identify a polygenic risk score (PRS) that predicts HCC risk, combined with 
non-genetic variables.
2) Provide a comprehensive characterization of environmental factors (e.g. 
lifestyle, diet, sociodemographic factors) linked to HCC onset; and how these 
can be integrated with histological, radiological and genetic information and 
biomarkers of liver damage, making the best use of AI models to predict 
individual HCC risk.
Given that alcoholic liver disease and NAFLD share similar pathogenetic 
patterns, we will contribute to achieving these goals by sharing within the 
Consortium the clinical and genetic data and the characterization of 
environmental risk factors of two cohorts of patients already characterized 
from two previously approved studies (EPIDEMIC-NAFLD cohort, SERENA study 
cohort) involving patients affected by metabolic liver disease.
The polygenic risk score (PRS) for HCC risk prediction will be calculated by 
summing the number of steatosis-predisposing alleles in the genesPNPLA3, 
TM6SF2, GCKR, MBOAT7AndHSD17B13,weighted by the size of the effect of 
the genetic factor on liver fat as already described, Dongiovanni 2018). This 
score will be combined with non-genetic data (clinical risk factors for HCC 
such as age, sex, presence of diabetes, presence of severe liver fibrosis) to 
create a combined clinical-genetic score.

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The combined risk score was developed considering acquired and genetic risk 
factors to predict HCC using the following formula as described elsewhere (Donati 
2017): 1/ (1 + e2 ((212.588 + (0.162 * age) + (0.404 * Sex: 1 if male, 21 if female) + 
(0.259 * Obesity: 1 present, 21 absent)+(0.587 * T2DM: 1 present, 21 absent)+(1.299 
* Severe fibrosis: 1 yes, 21 no)+(0.442 * number of risk alleles among common 
genetic factors associated with liver fat:inPNPLA3, TM6SF2, GCKR, MBOAT7And
HSD17B13).
3. STUDY DESIGN
3.1 Study design
Interventional, with collection of biological, non-pharmacological, monocentric, 
retrospective-prospective material.
3.2 Inclusion criteria
Patients enrolled in the EPIDEMIC (approval no. 1822 of 27 August 2013) and SERENA 
(approval of latest amendment no. 1151_2021 of 9 November 2021) studies, already 
approved by the CE Milano Area 2, will be included.
That is, subjects with the following characteristics:
1.Diagnosis of NAFLD or cryptogenic liver disease, allowing a more liberal 
alcohol intake limit (<60/40 g/day in M/F), so as to also include subjects 
with a moderate alcohol component of the liver disease, an important 
factor given the high epidemiological burden of this group
2.Age between 45 and 75 years
3.Any of the following criteria:
3.a.
F3-F4 fibrosis, determined histologically, or by non-invasive 
techniques (stiffness > 7.9 kPa on Fibroscan and positive NAFLD fibrosis 
score or APRI or FIB4), or evidence of cirrhosis resulting from 
biochemical tests or imaging techniques;
3.b.
Family history of primary liver cancer in first degree of kinship, or 
carrier status of rare mutations associated with the development of HCC 
(such as mutations in APOB and TERT)
3.c.
Male patient with type 2 diabetes or obesity carrying at least 
three genetic variants in PNPLA3, TM6SF2, MBOAT7.

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4.Willingness to sign informed consent.
3.3 Exclusion criteria
The exclusion criteria are those already foreseen for the two studies mentioned above, that is, they will 
beexcluded from the study patients with:
1.Alcohol intake >60/40 g/day in M/F
2.Chronic viral or autoimmune hepatitis
3.Any previously diagnosed genetic liver disease associated with increased risk of 
HCC (such as hereditary haemochromatosis, Wilson's disease, Alpha-1 
Antitrypsin deficiency)
4.Use of drugs known to induce steatosis and liver disease
5.HCC diagnosed prior to study start date.
6.Other medical conditions with a prognosis of less than two years.
4. PROCEDURES RELATING TO THE STUDY
4.1 Intervention
In the context of this study, patients already included in the above-mentioned studies will 
be enrolled at the Foundation and the enrollment of 200 patients within the SERENA 
study will be continued.
Patients who agree to be included in the GENIAL study, as already foreseen in 
the context of the SERENA and EPIDEMIC studies, will be subjected (if 
prospectively enrolled) to the collection of the following data useful for the 
characterization of clinical risk factors: biohumoral parameters, histological 
parameters relating to liver biopsy, lifestyle and metabolic factors (body mass 
index BMI, abdominal circumference (AC), smoking, alcohol, diet, ongoing 
medical therapy), liver fat content and liver damage (by abdominal ultrasound 
and measurement of liver stiffness measurement (LSM) and controlled 
attenuation parameter (CAP) by Fibroscan), as indicated by clinical practice in 
these subjects.
Upon enrollment, patients will be subjected (if prospectively enrolled) to 
blood sampling with collection of a 6 ml EDTA tube and a 6 ml serum tube, 
respectively for DNA extraction and the dosage of some biomarkers 
associated with liver damage (IL-32, Pro-C3) (this last sampling is

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repeated annually ad hoc for the study). Finally, as already foreseen by clinical 
practice, patients will be followed every six months for hepatological follow-up.
Any residual biological material not used by the end of the study will be 
biobanked, subject to the patient's consent to biobanking, for future 
research purposes in the same pathology at the Foundation's Biobank.
At the end of theprimary objectivesof the study, DNA samples from 
prospectively enrolled patients will be subjected to targeted sequencing to 
identify rare genetic variants and genotyping through SNP arrays to identify 
common variants.
DNA samples already collected in the context of the SERENA study that have 
not yet been sequenced will instead be analyzed, as per protocol, by whole-
exome sequencing (WES).
In general, DNA will be extracted from peripheral blood using the automatic 
platform of the Fondazione IRCCS Ca' Granda. Quality control will be performed 
by evaluating the absorbance ratio 260/280 nM and by gel electrophoresis. The 
enrichment of the DNA library for exome sequencing will be performed using 
SureSelect Human All Exon v6 (Agilent). Sequencing will then be performed on 
the HiSeq 4000 platform (Illumina). This procedure will ensure an average 
coverage of 70-80X of the target region.
The obtained raw data will be analyzed using standard bioinformatics pipelines, 
including alignment, variant annotation, and function annotation. Functional 
analysis of mutations will focus on integrating the data by performing different 
analyses to comprehensively characterize variants in known genes associated with 
liver disease, as well as to search for novel genes and genetic risk factors 
potentially involved in HCC progression.
Multiple available resources such as genetic variant frequency data (ExAC, ESP, 
1000Genomes), pathogenic variants (ClinVar) and insilico predictors of damage 
(SIFT, PolyPhen, CADD) will be used to prioritize candidate variants and genes 
involved in liver disease predisposition.

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To complement these analyses, a genome-wide association study (GWAS) will also 
be conducted to evaluate genetic variants associated with severe liver disease and 
HCC. The GWAS study aimed at identifying genetic variants associated with severe 
liver disease and HCC will specifically include approximately n~1000 cases with 
severe liver disease related to NAFLD from the EPIDEMIC and SERENA studies vs 
n~10,000 controls with uncomplicated fatty liver whose genetic data are already 
available (public data - UK Bionbank)).
As explained in thesecondary objectives,a polygenic risk score (PRS) will also be 
created that predicts the risk of HCC. This score will then be combined with 
non-genetic data (clinical risk factors for HCC such as age, sex, presence of 
diabetes, presence of severe liver fibrosis) to create a combined clinical-genetic 
score.
In summary, the clinical and genetic data described above, whose collection and analysis 
is already foreseen for the EPIDEMIC and SERENA studies, will be shared in 
pseudonymised form within the GENIAL consortium in order to:
1) identify new genetic variants associated with HCC (this will be carried out in 
collaboration with the Institut National de la Santé et de la Recherche Medicale, Paris 
and the Universite Libre De Bruxelles under the responsibility of Prof Jessica Zucman-
Rossi and Prof. Eric Trepo respectively);
2) evaluate the interaction between genetic, clinical and biohumoral factors in 
modulating the risk of HCC (at Hopitaux de Paris under the responsibility of Prof 
Pierre Nahon);
3) train an AI model to predict the risk of HCC (this algorithm will be developed at 
the Technical University of Dresden under the responsibility of Dr Jacob Kather) 
and identify hypotheses of clinical recommendations that healthcare professionals 
can use in the future in the diagnosis and follow up of ALD and NAFLD-related liver 
disease.
Finally, within the scope of the study proposal and in reference to the objectives of 
WP3, the genetic data resulting from the study will be sent to allow Start

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up Stratipath Research and development with registered office at Nanna Svartz väg 4° 
Solna, Sweden to create a predictive model to reach the diagnosis of HCC.
5. ENDPOINT
5.1 Primary Endpoints
1) Identification of novel genetic variants associated with HCC (ALD and NAFLD 
related)
2) Association between identified genetic variants and ALD-HCC within the 
general population
5.2 Secondary Endpoints
1) Creation of polygenic-clinical risk scores for the development of HCC
2) Integration of genetic and non-genetic information into AI algorithms with the 
aim of deriving predictive information.
6. DURATION / TIMELINE OF THE STUDY
Gantt Chart
0-6
month
7-12
month
13-18
month
19-24
month
25-36
month
37-42
month
43-48
month
49-54
month
55-60
month
Screening criteria
inclusion/exclusion
X
X
X
X
X
Explanation of the study and 
signing of the informed consent
X
X
X
X
X
Anamnesis collection and
parameters (clinical risk 
factors, lifestyle and metabolic 
factors (body mass index BMI, 
abdominal circumference (AC), 
smoking, alcohol, diet, ongoing 
medical therapy, presence of 
diabetes)
X
X
X
X
X
Blood tests (indicators of 
necrosis and liver damage: AST, 
ALT, GGT, ferritin,
X
X
X
X
X

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blood count, blood sugar,
glycated hemoglobin,
cholesterol, triglycerides)
Blood sampling for
genetics*
X
X
X
X
X
Non-invasive assessment 
of liver damage (LSM) and 
liver fat (CAP)
using Fibroscan and
abdominal ultrasound
X
X
X
X
X
X
Annual blood sampling for 
serum biomarker assay 
(interleukin 32, IL-32; 
procollagen 3; pro-C3)*
X
X
X
X
X
Hepatological follow-up
X
X
X
X
X
X
X
Data Analysis
X
X
X
X
X
* Ad hoc for the study
Study start: December 2023 
Enrollment end: December 2026 
Study end: December 2028 The 
study will last 60 months.
7. STATISTICAL ANALYSIS
7.1 Sample size
The EPIDEMIC-NAFLD cohort includes approximately 2800 subjects with NAFLD (n=347, 11.6% with 
advanced fibrosis, n=251, 8.9% with HCC). The SERENA cohort includes approximately 400 patients 
with advanced NAFLD.
Based on the hypothesis based on previous literature that we will observe a 1.5% 
HCC incidence rate in the entire cohort over a mean follow-up of 5 years, and that 
based on data generated in a cross-sectional cohort (Donati, Scientific Reports 
2017 and Cancer Medicine 2017) we will be able to identify at least 30% of 
individuals with at least a threefold increased risk of HCC, for a type I error rate of 
5%, we planned to enroll 375 patients to have a statistical power of 80% to detect a 
significant association of genetic score with HCC risk. However, we estimated the 
need to increase the number of enrollees to account for patients

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potentially censured due to the occurrence of other major clinical events 
prior to the onset of HCC (death, cancer, major cardiovascular events, and 
liver failure) and a small group lost to follow-up. Therefore, we intend to 
enroll at least 600 patients to complete the study. We then expect to 
prospectively enroll another 200 patients over the next 3 years.
The GWAS study aimed at identifying genetic variants associated with severe liver 
disease will include approximately n~1000 cases with severe NAFLD-related liver 
disease from the EPIDEMIC and SERENA studies vs n~10,000 controls with 
uncomplicated fatty liver disease whose genetic data are already available (public 
data - UK Biobank).
7.2 Data analysis
Time to progression to HCC will be estimated by Kaplan-Meier estimates of 
cumulative incidence rates. Survival distributions among genotypes will be 
compared using Cox proportional hazard models. Hazard ratios and P values   
will be adjusted for confounding factors. In the analysis, risk genotypes will be 
coded using additive genetic models.
We will evaluate the impact of genetic risk variants on HCC risk, using Cox 
regression models adjusted for classical HCC risk factors (age, sex, BMI, type 
2 diabetes, advanced fibrosis, smoking, alcohol intake).
As a secondary outcome, we will also evaluate the interaction between genetic 
and acquired risk factors in the pathogenesis of HCC. As a secondary objective of 
the study, we will develop a combined risk score to predict the incidence of HCC, 
which will be cross-validated within the cohort. Statistical analyses will be 
performed using JMP 16.0 Pro (SAS Institute) and version 3.3.2 of the statistical 
analysis software R (http://www.Rproject . org/). P-values   <0.05 will be considered 
statistically significant.
P-values   <0.05 (two-tailed test) will be considered statistically significant.
8. ADVERSE EVENTS

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The project does not involve the administration of drugs or other substances or invasive 
clinical practices other than those normally foreseen in clinical practice. Therefore, no adverse 
events are expected.
9. RISK/BENEFIT ASSESSMENT
We expect to define whether a genetic HCC risk score can accurately predict 
the onset of NAFLD-HCC in a high-risk cohort. This <Precision Medicine= 
approach would have an important clinical implication, allowing to identify 
subjects for whom ultrasound screening for HCC could be cost-effective with 
potential clinical benefits for patients and savings for the health system.
Validation of genetic variants associated with ALD-related HCC within our 
cohort will also help to elucidate the pathogenic mechanisms of alcoholic 
and non-alcoholic liver disease, possibly demonstrating how these 
conditions share similar processes.
10. STUDIO MANAGEMENT
10.1 Data collection and management
Company software designed to manage outpatient visits and laboratory 
tests will be used to collect data.
Each participant, at the time of enrollment, will be assigned a unique code. The de-
identification of the data will be done in such a way that the people who access the 
database will not be able to trace the identity of the subjects in any way. Only local 
experimenters will be able to trace the identity of the enrolled subjects.
The data needed for the study will be recorded in a specific eCRF in a Data 
Management System validated according to national regulations, provided by 
the Scientific Direction of the Foundation. The platform used will be RedCap 
(Research Electronic Data Capture).
The REDCap Consortium is composed of >1000 institutional partners worldwide 
(research institutes, universities, ministries etc). The consortium supports a secure 
web application (REDCap) designed exclusively to support data acquisition for 
research studies. The REDCap application allows users to create and manage 
online databases quickly and securely, and is currently in use for more than

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110,000 projects with approximately 150,000 users covering numerous areas of research interest 
across the consortium.
Through REDCap, for this study the following will be implemented: a) user-
level identification, with specific restrictions based on the role in the study b) 
real-time data integrity validation and control c) patient de-identification 
before data export d) centralized data storage with daily backup, a secure 
server within the Foundation's IT structure.
10.2 Regulatory aspects and ethical considerations
10.2.1 Approval by the Competent Authority
In accordance with applicable regulations, the principal investigator must obtain 
approval from the appropriate Competent Authority prior to initiating the clinical 
study.
This study will be conducted in accordance with ICH/GCP (International 
Conference of Harmonization/Good Clinical Practice) rules and all applicable 
laws, including the Declaration of Helsinki.
of June 1964, as last amended by the World Medical Association General 
Assembly in Seoul, 2008.
10.2.2 Ethics Committee Approval
The investigator must ensure that the protocol has been reviewed and approved by the 
local independent Ethics Committee (EC) before starting the study.
The CE must also review and approve the informed consent (IC) form and all written 
information received from the patient prior to enrollment in the study. Should it be 
necessary to modify the protocol and/or the IC during the study, the investigator will 
be the guarantor and therefore the person responsible for ensuring the review and 
approval of such modified document as requested by the CE.
The content of these changes will be implemented only after the CE has 
approved them. Until then, it will be necessary to refer to the previous version 
of the document already approved.
10.2.3 Informed consent (IC)
The investigator or other personnel designated by him have the task of informing the 
subjects about all aspects and procedures of the study.

Department of Services Area SC 
Transfusion Medicine Tel. 02 
5503.4687
E-mail:daniele.prati@policlinico.mi.it
- Director: Dr. Daniele Prati
The informed consent of the participants will be collected by the physician in charge of the 
clinical and metabolic evaluation during the visit.
The process for obtaining informed consent must comply with applicable 
regulatory procedures. The investigator (or designated collaborator) and the 
subject must date and sign the informed consent form before the patient 
begins any study-related procedures.
The subject will receive a copy of the CI dated and signed by both parties; the 
original copy will be kept in the designated study archives. Neither the 
investigator nor designated personnel should in any way exert any coercion or 
influence on a subject to induce him or her to participate or continue to 
participate in the study. A subject's decision to participate in the study must be 
completely voluntary. The investigator and designated personnel should 
emphasize to the subject that he or she may withdraw consent at any time 
without penalty or loss of any benefits to which he or she may be entitled.
Written or oral information about the study, including the written consent 
form, must not contain any language that forces the subject to waive (even 
apparently) his or her legal rights, or that would exonerate the investigator, 
agency, or sponsor from liability for negligence.
The time that the subject will have to dedicate to participating in the study does not exceed 
that normally foreseen by normal clinical practice for the treatment of the condition from 
which he/she suffers.
10.3 Duties of the experimenter
In accordance with applicable local regulations, the investigator must 
submit periodic reports regarding the progress of the study at his/her site 
to the CE and notify the CE of study closure. Periodic reports and closure 
notification are part of the investigator's responsibilities.
10.4 Study monitoring
In accordance with applicable regulations and good clinical practice (GCP), the 
monitor must visit or contact the center periodically. The duration, nature and 
frequency of such visits / contacts depend on the recruitment frequency, the quality 
of the documents held by the center and their adherence to the protocol. Through 
such contacts, the monitor must:
" monitor and evaluate the progress of the study

Department of Services Area SC 
Transfusion Medicine Tel. 02 
5503.4687
E-mail:daniele.prati@policlinico.mi.it
- Director: Dr. Daniele Prati
" examine the collected data
" conduct source document verification
" identify each problem and related solutions
The purposes of the monitoring activity are to verify that:
" the rights and well-being of the subject are respected
" the study data are accurate, complete and verifiable from the original documents
" the study is conducted in accordance with the protocol and any approved 
amendments, GCP and
applicable regulations 
The experimenter must:
" give the monitor direct access to all relevant documentation
" dedicate part of his time and his staff to the monitor to discuss the monitoring 
results and any other possible aspects.
The monitor must also contact the center prior to the start of the study to discuss 
the protocol and data collection procedures with the staff.
10.5 Study quality assurance
As the sponsor, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico 
may, at its discretion, carry out a quality control of the study. In this case, the 
investigator must allow the monitor direct access to all relevant 
documentation and dedicate part of his or her time and personnel to the 
reviewer to discuss the results of the monitoring and any other aspects of 
the study.
In addition, Regulatory Authorities may conduct inspections. In this case, the 
investigator must allow the inspector direct access to all relevant 
documentation, and dedicate part of his time and personnel to the inspector 
to discuss the monitoring results and any other aspects of the study.
10.6 Closing of the study
At the time of study closure, the monitor and the experimenter must 
activate a series of procedures:
" review all study documentation
" reconcile study data
" reconcile all clarifying reports.

Department of Services Area SC 
Transfusion Medicine Tel. 02 
5503.4687
E-mail:daniele.prati@policlinico.mi.it
- Director: Dr. Daniele Prati
10.7 Document storage
In accordance with current national regulations, the investigator must keep 
a copy of all documentation and store it in a dry and safe place after the 
study has been closed.
10.8 Disclosure of information regarding scientific discovery
10.8.1 Confidentiality
The investigator and other personnel involved in the study must handle all 
information relating to the study (including the protocol, data obtained and all 
documentation generated during the study) and must not use such information, 
data or reports for purposes other than those described in the protocol. These 
restrictions do not apply to:
1) information that becomes publicly available, not due to negligence on the 
part of the investigator or his staff;
2) information that requires confidential disclosure to CE for the sole purpose of 
evaluating the study;
3) information that must be disclosed in order to obtain appropriate medical 
care for a study subject.
10.8.2 Publications
Each Party is the owner of the intellectual and industrial property rights relating to 
its Background. Furthermore, the Affiliated Entity will grant the Beneficiary access 
rights to the Background and the Results, if necessary for the implementation and 
exploitation of the results, in order to fulfill its obligations and perform its tasks as 
described in the Grant agreement and in the Affiliated Entity Agreement stipulated 
between the Parties. This provision applies vice versa to the Affiliated Entity if it 
requests Access for the performance of its tasks and/or for the valorization of its 
Results.
The results are owned by the Party that generates them. If the Parties have jointly 
produced results and it is not possible to establish their respective contributions 
or separate them for protection, the Parties automatically become co-owners. In 
case of co-ownership of the Results, the ownership share of the co-owners will be 
proportional to the intellectual contribution invested in generating that specific 
Result. The co-owners agree on the ownership shares, all protection measures and 
the distribution of related costs in a co-ownership agreement.

Department of Services Area SC 
Transfusion Medicine Tel. 02 
5503.4687
E-mail:daniele.prati@policlinico.mi.it
- Director: Dr. Daniele Prati
co-ownership to be negotiated in advance and in any case before any 
commercial exploitation of the joint results.
In any case, the Parties undertake to adequately protect their Results taking 
into account all relevant considerations, including the prospects of 
commercial exploitation.
For further information, please refer to the Affiliated Entity Agreement.
11. Indemnity and compensation in case of damages
In case of unwanted events or any damages that may arise from 
participation in research, our Institute's Insurance Policy also extends to 
cover subjects participating in research projects.
12. Amendments to the Protocol
Communications regarding significant changes to the protocol will be the 
responsibility of the researchers involved in the study and will be under the 
responsibility of the principal investigator.
13. Financial Agreements
The interventions planned within the study are partly foreseen within 
normal clinical practice.
The costs of study procedures exceeding normal clinical practice 
(genotyping, genetic analysis and biomarkers) will be covered by the 
following funding:
The European Union, HORIZON-MISS-2021-CANCER-02-03 program <Genial= under 
grant agreement <101096312=
14. Disclosure on conflicts of interest
The experimenters declare no conflict of interest.
15. References
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World Health Organization. Status report on alcohol consumption, harm and 
policy responses in 30 European countries 2019. (2019).
World Health Organization. https://www.who.int/publications/i/item/
9789241565639 (2018). GBD 2019 Diseases and Injuries Collaborators. The 
Lancet. 396, (2020).
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Department of Services Area SC 
Transfusion Medicine Tel. 02 
5503.4687
E-mail:daniele.prati@policlinico.mi.it
- Director: Dr. Daniele Prati
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16. Appendix