Contract

DELIBERAZIONE N. 699 DEL 15/09/2022
OGGETTO: Presa d'atto avvenuta stipula accordo per il trasferimento di materiale tra gli IRCCS Istituti Fisioterapici Ospitalieri -Istituto Nazionale Tumori Regina Elena (IFO-IRE) e IRCCS Ospedale San Xxxxxxxx S.r.l., in qualità di ricevente.
Esercizi/o * Centri/o di costo - - Importo presente Atto: € - - Importo esercizio corrente: € -	STRUTTURA PROPONENTE UOSD Servizio Amministrativo Ricerca Il Dirigente Responsabile Xxxxxxx Xxxxxx
Budget
- Assegnato: € -
- Utilizzato: € -
- Residuo: € -
Autorizzazione n°: -
Servizio Risorse Economiche: Xxxxxxxx Xxxxxxxxxxx	Responsabile del Procedimento Xxxxxxx Xxxxxx
	L’Estensore Lucia D'Auria Proposta n° DL-718-2022
PARERE DEL DIRETTORE SANITARIO	PARERE DEL DIRETTORE AMMINISTRATIVO
Positivo	Positivo
Data 14/09/2022	Data 14/09/2022
IL DIRETTORE SANITARIO Xxxxxx Xxxxx	IL DIRETTORE AMMINISTRATIVO Xxxxx Xxxxxxxxx
Xxxxxx del Direttore Scientifico IRE Segreteria Direzione Scientifica IRE data 05/09/2022 Positivo Parere del Direttore Scientifico ISG Xxxx Xxxxxxx data 05/09/2022 Positivo
La presente deliberazione si compone di n° 6 pagine e dei seguenti allegati che ne formano parte integrante e sostanziale: - All.1 Accordo

Il Dirigente della UOSD Servizio Amministrativo Ricerca

Visto il decreto legislativo 30.12.1992, n. 502 e successive modificazioni ed integrazioni. Visto il decreto legislativo 16.10.2003, n. 288.

Vista la legge regionale 23.01.2006, n. 2.

Visto l’Atto Aziendale adottato con deliberazione n. 153 del 19.02.2019 e approvato dalla Regione Lazio con DCA n. U00248 del 2.07.2019, modificato e integrato con delibe- razioni n. 1254 del 02.12.2020, n. 46 del 21/01/2021 e n. 380 del 25.03.2021, appro- vate dalla Direzione Salute ed Integrazione Sociosanitaria della Regione Lazio, con Determinazione n. G03488 del 30.03.2021.

Visto il Decreto del Presidente della Regione Lazio n. T00200 del 29/10/2021 avente ad oggetto: “Nomina del Direttore Generale dell’IRCCS IFO-Istituti Fisioterapici Ospitalieri.

Vista la deliberazione n.1123 del 2/11/2021 di insediamento ed assunzione in carica del Direttore Generale degli Istituti Fisioterapici Ospitalieri di Roma Dott. ssa Xxxxxx Xxxxxxxx.

Viste le deliberazioni n. 212 del 16/03/2022 e n. 154 del 28/02/2022 con le quali sono stati nominati rispettivamente la Dott. ssa Xxxxx Xxxxxxxxx quale Direttore Xxxxxx- xxxxxxxx ed il Xxxx. Xxxxxx Xxxxx quale Direttore Sanitario degli Istituti Fisioterapici Ospitalieri.

Visto il D.M. del Ministero della Salute del 8 maggio 2020 di conferma del riconoscimen - to del carattere scientifico dell’IRCCS di diritto pubblico a Istituti Fisioterapici Ospitalieri (IFO) relativamente alla disciplina di “oncologia” per l’Istituto Naziona- le Tumori Regina Xxxxx (IRE) e alla disciplina di “dermatologia” per l’Istituto San Gallicano;

Premesso che l’art. 7 del decreto legislativo 16 ottobre 2003 n. 288, contempla le diverse tipolo- gie di ricavi degli IRCCS;

che l’art. 8 del D.Lgs. n. 288/2003 prevede la possibilità per gli IRCCS di stipulare ac- cordi e convenzioni, costituire e/o partecipare a consorzi e attuare misure di collega-

mento e sinergia con altre strutture di ricerca e assistenza sanitaria, pubbliche e private, nonché con le Università, per la realizzazione di comuni progetti di ricerca;

che il vigente Regolamento di Organizzazione e Funzionamento degli IFO definisce, fra l’altro, come missione degli IRCCS la cooperazione con altri enti pubblici di ricer- ca e con altre organizzazioni che operano negli specifici campi, in una logica di com- pletamento di ruoli e di continuità assistenziale;

che con deliberazione n. 146 del 24/02/2022 il Direttore Generale degli IRCCS IFO, in ordine alla stipula degli accordi di riservatezza con i partner degli Istituti, degli MTA e degli MTDA relativi alle sperimentazioni cliniche, agli studi osservazionali, ai brevetti e comunque ai progetti di ricerca ha esteso delega ai Direttori Scientifici IRE e ISG per le attività afferenti i rispettivi Istituti.

Considerato che l’Ospedale San Xxxxxxxx è un ospedale di ricerca e policlinico universitario fondato nel 1971 per fornire cure specializzate per le condizioni di salute più complesse, rico- nosciuto già nel 1972 come Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS);

che l’IRCCS IRE degli IFO e l’IRCCS Ospedale San Xxxxxxxx e Humanitas Mirasole

S.p.A hanno partecipato ad un progetto nell’ambito delle Reti IRCCS, Alleanza Contro il Cancro – ACC Rete Oncologica degli IRCCS dal titolo “Programma nazionale di on- cologia personalizzata per gli IRCCS della Rete ACC”;

che tra i centri partecipanti rientrava l’Humanitas, il quale ha inteso avviare un proto- collo clinico dal titolo “Genomic Landscape of Early-Onset Cholangiocarcinoma and Its Prognostic Implications” ed in data 3 gennaio 2022, con lettera agli atti della scri- vente, ha formalizzato la Lista dei centri partecipanti avvalendosi dell’IRCCS IRE de- gli IFO e l’ IRCCS Ospedale San Xxxxxxxx per il trasferimento di materiale biologico;

Rilevato che nell’ambito del summenzionato progetto l’IFO IRE è stato identificato come forni- tore, con la collaborazione della dott.ssa Xxxxxxx e il Ricevente è stato identificato nell’ IRCCS Ospedale San Xxxxxxxx la cui finalità comune è l’identificazione e valida- zione di nuovi biomarcatori nell’ambito del colangiocarcinoma secondo quanto previ- sto nel Progetto

che si è ritenuto necessario redigere un accordo per il trasferimento del materiale biolo- gico avente efficacia per tutta la durata del progetto e disciplinante gli aspetti sulla pri- vacy, sulla responsabilità degli enti coinvolti nel trasferimento nonché le modalità di trasferimento del materiale stesso;

Visto il prot. n. 5717 del 2 maggio 2022, agli atti della scrivente, con cui la Direzione Scien- tifica IRE ha trasmesso autorizzazione allo svolgimento delle attività del progetto e bozza dell’accordo per il trasferimento di materiale;

Ritenuto opportuno prendere atto dell’avvenuta stipula dell’Accordo per il trasferimento di materiale, fir- mato in data 30/06/2022 che allegato al presente provvedimento ne forma parte inte- grante e sostanziale;

Attestato che il presente provvedimento, a seguito dell’istruttoria effettuata, nella forma e nella sostanza è totalmente legittimo e utile per il servizio pubblico, ai sensi dell’art. 1 della legge 20 del 14/01/1994 e successive modifiche, nonché alla stregua dei criteri di eco- nomicità e di efficacia di cui all’art. 1, primo comma, della legge 241 del 7/08/1990, come modificata dalla legge 15 del 11/02/2005;

Propone

Per i motivi di cui in narrativa che si intendono integralmente confermati di:

• prendere atto dell’avvenuta stipula dell’Accordo per il trasferimento di materiale, firmato in data 30/06/2022 e allegato al presente provvedimento ne forma parte integrante e sostanziale;

La UOSD Servizio Amministrativo della Ricerca curerà tutti gli adempimenti per l’esecuzione della presente deliberazione.

Il Dirigente della UOSD Servizio Amministrativo Ricerca Xxxxxxx Xxxxxx

Il Direttore Generale f.f.

Visto il decreto legislativo 30.12.1992, n. 502 e successive modificazioni ed integrazioni; Vista la legge regionale 23.01.2006, n. 2;

Visto l’Atto Aziendale adottato con deliberazione n. 153 del 19.02.2019 ed approvato dalla Regione Lazio con DCA n. U00248 del 2.07.2019

In virtù dei poteri conferitigli con decreto del Presidente della Regione Lazio n. T00200 del 29.10.2021.

Preso atto che il Dirigente proponente il presente provvedimento, sottoscrivendolo, attesta che lo stesso a seguito dell’istruttoria effettuata, nella forma e nella sostanza è totalmente legittimo e utile per il servizio pubblico, ai sensi dell’art. 1 della legge 20/94 e s.m., nonché alla stregua dei criteri di economicità e di efficacia di cui all’art. 1, primo comma, della legge 241/90, come modificata dalla legge 15/2005.

Visto il parere favorevole del Direttore Amministrativo e del Direttore Sanitario Aziendale;

ritenuto di dover procedere;

Delibera

di approvare la proposta così formulata concernente “Presa d'atto avvenuta stipula accordo per il trasferi- mento di materiale tra gli IRCCS Istituti Fisioterapici Ospitalieri -Istituto Nazionale Tumori Regina Elena (IFO-IRE) e IRCCS Ospedale San Xxxxxxxx S.r.l., in qualità di ricevente.” e di renderla disposta.

Il Direttore Generale F.F. Dott.ssa Xxxxx Xxxxxxxxx

Documento firmato digitalmente ai sensi del D.Lgs 82/2005 s.m.i. e norme collegate

Data: 30/06/2022

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Genomic Landscape of Early-Onset Cholangiocarcinoma and Its Prognostic Implications

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PROTOCOL NAME

Genomic Landscape of Early-Onset Cholangiocarcinoma and Its Prognostic Implications

PROTOCOL VERSION DATE v 1.0 7.1.2021

Protocol Number ONC/OSS-05/2021

Coordinating Physician: Xxxxxx Xxxxxxxxx, MD

Assistant Professor of Medical Oncology | Humanitas University

Medical Oncology and Hematology Unit| Humanitas Research Hospital

Address: Xxx Xxxxxxx, 00 - 00000 Xxxxxxx - Xxxxx, Xxxxx

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Summary

Title Genomic Landscape of Early-Onset Cholangiocarcinoma and Its Prognostic Implications

Investigator sponsor Xxxxxx Xxxxxxxxx, MD

Protocol identifying number ONC/OSS-05/2021

Protocol version date V 1.0 7.1.2021

Background and rationale

Previous studies investigating the genomic alterations in a variety of BTC types have found commonalities, such as TP53, KRAS and SMAD4 mutations, with a second tier of less frequently mutated genes including XXXX0X, XXXX0X, XXX0, XXX0 xxx XXX0XX that were not seen in all studies. Molecular differences between the subtypes have tended to be in the frequency of mutations in certain genes, rather than different sets of genes being mutated. Recently, a Chinese cohort study (based on a targeted gene sequencing) that included 26 patients aged ≤45 years with iCCA or extrahepatic CCA [35] found an increased frequency of Additional sex xxxxx like 1 (ASXL1) (p

= 0.02) and KMT2C (p = 0.02) mutations compared to their older counterparts.

Here, we hypothesize that a more comprehensive analysis using whole-exome sequencing (WES) may identify additional potentially actionable genomic alterations. XXX was recently reported to allow the detection of additional variants in relevant genes in 38% of patients. Mutation detection sensitivity of WES was 95% compared with targeted genome sequencing.

Population and patient selection criteria

This study will include:

a) Patients <45 years old, with a diagnosis of cholangiocarcinoma and available FFPE or fresh- frozen tumor tissue will be considered eligible for this retrospective study. If available, non-tumor tissue will be collected for germline DNA analyses. Similarly, matching patients ≥45 years old will also be collected. Frozen tissue samples will be obtained from the institutional biobank. FFPE samples from the anatomy pathology archive may also be used.

b) Availability of clinical and survival data.

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Study design and study duration

This is a retrospective study and no formal hypothesis for sample size has been postulated. All consecutive patients who meet inclusion and exclusion criteria will be included in this analysis.

Objectives

1. To report the mutation frequency and significantly mutated genes in CCA and matched non-tumor liver tissue, and to describe their prognostic impact.

2. To perform a pathway analysis and subtype enriched features according to anatomic location.

4. To establish whether a link exist between germline structural variants in known cancer susceptibility or DNA repair genes to the somatic mutation processes.

Ethical considerations

Given the retrospective, observational, non-interventional nature of this study, data will be collected anonymously. Recorded data will be protected by an encryption key to avoid any chance to get back to patients’ identity. The study does not have any influence on the patient's course of treatment. All subjects whose samples will be used for this study have signed the institutional biobank informed consent.

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BACKGROUND AND SIGNIFICANCE

Biliary tract cancers (BTC) represent a heterogeneous group of cancers arising from the bile ducts. Based on anatomical location, BTC are classified into intrahepatic cholangiocarcinoma (iCCA), perihilar CCA (pCCA), distal CCA (dCCA), and gallbladder cancer (GBC) [1]. They represent 3% of all gastrointestinal neoplasms in adults, the most frequent anatomic subtype

originates from the extrahepatic biliary tract [2], but their incidence is increasing worldwide, mainly due to iCCA [3–5], which is the second most common type of primary liver cancer, responsible for 20% of liver-related deaths [6]. Anatomic subtypes have different risk factors, clinical presentations, molecular alterations, and prognosis [7–9]. Risk factors for iCCA include primary sclerosing cholangitis, Caroli’s disease, lithiasis, liver flukes, viral hepatitis, cirrhosis, obesity, and diabetes [7,10,11]. Given the increasing incidence and prevalence of metabolic syndrome, it is estimated that also the incidence of iCCA will increase in the coming years [12,13]. However, about 50% of cases have no identifiable risk factors and there are no clinically applicable biomarkers for early diagnosis. As a result, BTCs are frequently diagnosed at an advanced stage when treatment options are limited and prognosis remains poor with a 5-year survival of about 5–15% [14,15].

Furthermore, up to 20% of cancers of unknown primary site can be reconducted to a biliary tract origin based on the molecular profile, further highlighting the challenges of an early diagnosis [16]. In Western countries, the incidence of iCCA is higher in older people (≥50 years old) than in younger people, and in Hispanic individuals than in non-Hispanic individuals.

There exist peculiar predisposing factors that may determine an earlier age for CCA onset. The mean age of CCA diagnosis in patients with primary sclerosing cholangitis is the fourth decade of life [17] compared with the seventh decade in the general population [18]. Although various risk factors for cholangiocarcinoma in primary sclerosing cholangitis have been reported, none are sufficient to guide risk stratification for disease surveillance.

Early age at diagnosis is also noted in patients with bile duct cystic disorders, including Caroli's disease [18,19]. Furthermore, in a Japanese cohort, patients <50 years Hepatitis B virus infection was the most prevalent etiologic factor significantly associated with iCCA (10.8% of

patients; ref. 20), which in turn heralded the worst prognosis when comparisons were done with other anatomic sites of the biliary tree.

Recent discoveries of somatic genomic alterations have led to exploration of new potential therapeutic targets [21]. In particular, a comprehensive analysis published by Xxxxxxxx et al. reported a high rate (93/239—38.9%) of potentially targetable somatic genetic alterations in analyzed CCA cases [22]. The potential targets included kinases (FGFR1, XXXX0, XXXX0, XXX0XX, XXX, XXXX, XXXX0, BRAF, and AKT3), oncogenes (IDH1, IDH2, CCND1, CCND3,

and MDM2), and, notably, tumor suppressor genes BRCA1 and BRCA2.

In another study, 75 CCA cases were genotyped for targetable somatic mutations, revealing that 16% and 40% of detected alterations in iCCa and extrahepatic cases, respectively, were affecting genes associated with DNA repair pathways, including XXX0, XXX0, XXX, XXX0, XXX0, and BRCA1 and BRCA2 [23]. The contribution of germline mutations in BRCA1/2 genes to the development of bile duct malignancies has previously been reported. Data from the early 2000s by the Breast Cancer Linkage Consortium (BCLC) reported that the relative

risk (RR) of developing gall bladder or bile duct cancer among BRCA2 carriers is 4.97 (95% confidence interval [CI] 1.50–16.52), whereas other established RR factors for CCA development such as infection with liver parasites, hepatitis C virus, and hepatitis B virus are 4.8, 1.8, and 2.6, respectively. BRCA1 and BRCA2 proteins are involved in the DNA damage response mediated via homologous recombination (HR) [24,25]. BRCA1/2-mutated cells are HR deficient and hence

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accumulate DNA double-strand breaks, resulting in genomic instability and increased predisposition to malignant transformation [26], rendering BRCA1/2 mutation carriers with a distinct clinical phenotype of increased sensitivity to DNA damaging therapies [27-29].

Despite mounting evidences on the genetic bases of CCA, data on early-onset CCA are lacking and for most of all it is unknown if and to what extent the clinical course and therapeutic response of early-onset CCA. To gain insight, we elected to perform a multicenter retrospective study on the genomic characterization of early-onset CCA.

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RATIONALE, HYPOTHESIS, AND SPECIFIC AIMS

Previous studies investigating the genomic alterations in a variety of BTC types [30-34] have found commonalities, such as TP53, KRAS and SMAD4 mutations, with a second tier of less frequently mutated genes including XXXX0X, XXXX0X, XXX0, XXX0 xxx XXX0XX that were not seen in all studies. Molecular differences between the subtypes have tended to be in the frequency of mutations in certain genes, rather than different sets of genes being mutated. Recently, a Chinese cohort study (based on a targeted gene sequencing) that included 26 patients aged ≤45 years with iCCA or extrahepatic CCA [35] found an increased frequency of Additional sex xxxxx like 1 (ASXL1) (p = 0.02) and KMT2C (p = 0.02) mutations compared to their older counterparts.

Here, we hypothesize that a comprehensive approach might indeed elucidate the genetic underpinnings of CCA in younger patients. For molecular analysis, instead of a targeted next generation sequencing platform, which captures selected exons and selected intronic regions, we believe that a more comprehensive analysis using whole-exome sequencing (WES) may identify additional potentially actionable genomic alterations. XXX was recently reported to allow the detection of additional variants in relevant genes in 38% of patients. Mutation detection sensitivity of WES was 95% compared with targeted genome sequencing [36]. Additional information derived from WES (possibly coupled with RNA sequencing) that could influence clinical decision includes: fusion transcripts, expression levels, allele-specific expression, alternate transcripts, RNA-based pathogen diagnostic, tumor mutation load, mutational signatures, expression signatures, HLA genotyping, and neoepitope prediction.

In a cohort of patients aged <45 years, the aims of this study are:

1. To report the mutation frequency and significantly mutated genes in CCA and matched non- tumor liver tissue, and to describe their prognostic impact.

2. To perform a pathway analysis and subtype enriched features according to anatomic location.

3. To detect deleterious germline mutations in cancer-predisposing genes. BTC might be related with Xxxxx syndrome, which has germline mutations of DNA mismatch repair genes, and BTC also occurs in carriers of BRCA mutations. Targeted sequencing allows testing of rare variants, which are more likely to have larger effect size and direct functional consequence, in the genes of interest but leaves out most of the genes, and would miss genes not specified a priori.

4. To establish whether a link exist between germline structural variants in known cancer susceptibility or DNA repair genes to the somatic mutation processes. Of note a previous report [37] identified germline variants of selected cancer-predisposing genes including XXXX0, XXXX0, XXX00X, XXX0, XXX0, XXX0, and TP53, and annotated their significance. As a result, eight patients with BTC were found to have deleterious germline mutations of BRCA1, BRCA2, or RAD51D, and six had deleterious germline mutations of XXX0, XXX0, XXXX0, or POLE. An additional 80 variants of uncertain significance (VUS) in these cancer predisposing genes, some of which may be pathogenic, were found in 75/146 (51.3%) Japanese patients

Universal tumor screening [38] for these cancer-predisposing genes in general BTC cases might be beneficial to patients with BTC and their family members, enabling assessment of their cancer development risk and the effectiveness of new anti-cancer drugs, such as immune checkpoint inhibitors and PARP inhibitors.

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Objectives of the study

Analysis of driver mutation frequency and significantly mutated genes in CCA.

To this end, we will collect in participating Institutions fresh-frozen or formalin-fixed paraffin- embedded (FFPE) tumors and normal tissues from patients with early-onset CCA. WES results will be compared with WES results in non-early onset CCA from publicly available datasets. We will survey somatic mutations, copy number variations (CNVs) and germline mutations to reveal the etiopathologic factors involved in the tumorigenesis of early-onset CCA. Through the comparison of missense mutations between normal liver tissue–tumor pairs, we will examine the association of these molecular alterations with biologic pathways. We will examine the encoding functions of top susceptibility genes with the most frequent germline alterations compared to CCA susceptibility genes as a whole.

Materials and methods

1. Subjects: We will take advantage of a cohort of patients that underwent surgery at the Humanitas Cancer Center, San Xxxxxxxx Hospital, and other IRCCS Centers possibly interested. A detailed research proposal will be submitted to the Institutional Review Board (IRB) of Humanitas. According to the institutional biobank standard operating procedures, only the part of the cancer- free liver tissue adjacent to the resected tumor and tumor not used for the histopathology diagnosis have been collected. All subjects enrolled in the project are then followed up and clinical data

recorded every 3 months (as per protocol in most Cancer Centers) or at time of any significant event including treatment, disease progression, or death.

2. Sample collection, WES library preparation and sequencing: Tissue samples will be assessed for tumor cellularity by a pathologist at each participating Institution For each patient, two sets of samples will be provided: tumor DNA and germline. If available, Germline DNA will be recovered from normal tissue adjacent to the tumor at each participating site. Genomic DNA (gDNA) will be extracted from surgical (or bioptic) specimens containing at least 20%-40% cancer cells, and from normal adjacent tissue; gDNA extraction will be performed at each participating Institution (Prof. Xxx Xxxx’x Lab for Humanitas University). WES will be centralized and WES libraries will be prepared using Twist Human Comprehensive Exome kit and will be sequenced on Illumina NovaSeq 6000 platform, 2x100 bp.

3. Significantly mutated genes: Genes harboring somatic mutations will be identified by a ‘Tumor vs Normal’ variant calling pipeline. To this end we will implement a bioinformatic strategy using either GATK4-Mutect2 to identify SNV and small indels, or CNVKit to identify copy number aberrations. Mutations will be prioritized by effect prediction (excluding non-coding and silent mutations) and by overlapping to known COSMIC mutations.

4. Germline variant calling content analysis: Germline SNVs and indels will be called using GATK4 Haplotypecaller, following Broad’s GATK best practices. Relevant mutation will be annotated with dbSNP and GnomAD to rule out possible polymorphisms and with ClinVar and OMIM to identify possible pathological variants. The tool LOHLLA will be used to spot regions of

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loss of heterozygosity in tumors. HLA type will be predicted by exploiting normal DNA using the software BWA-KIT.

5. Pathway Analysis for Germline and Somatic Mutations: Identification of known pathways and network enriched by the set of mutated genes will be explored using tools like GSEA, CAMERA and KEGG. LOH regions (detected by somatic analysis) along with mutational data will be used to identify possible markers of cancer susceptibility.

To assess the molecular differences in early-onset CCA and non-early onset individuals, also we will collect publicly available CCA datasets, which contain mutation and clinical information (fromcBioPortal) and analyzed these datasets in a systematic manner. Patients without gender and/or age information will be excluded. Patients will be dichotomized into two groups on the basis of age at diagnosis, and patients ≤45 years will be categorized as early-onset CCA and patients above 45 years as non-early onset CCA.

For the comparison of somatic mutation patterns between these two groups, we first

analyzed somatic mutations for all these patients to identify early-onset CCA or non-early-onset CCA specific genes and then compared the prevalences of the 10 genes most susceptible to somatic mutations in the two cohorts. Disease/progression free survival time will be defined as the time from diagnosis until first occurrence of relapse, progressive disease, secondary cancer or death or, if none occurred, until last contact. Similarly, patients from external cohorts will be dichotomized into early-onset CCA or non-early-onset based on age (<45 years) and survival probability was estimated by Xxxxxx–Xxxxx survival curves. The relative risk of disease/progression free survival will be estimated by univariate Xxx-proportional hazard regression analysis.

Expected Results and Future Developments

We expect to define major characteristics of the genomic landscape in early-onset CCA. We also aim to identify main pathways in CCA that potentially lead to new actionable biomarkers in patients with early-onset CCA.

The role of germline mutations still need to be elucidated in CCA but may provide additional tools for cancer screening in selected patients and families.

Patient Inclusion criteria

c) Patients <45 years old with a diagnosis of cholangiocarcinoma and available FFPE or fresh- frozen tumor tissue will be considered eligible for this retrospective study. If available, non- tumor tissue will be collected for germline DNA analyses. Similarly, matching patients ≥45 years old will also be collected.

d) Availability of clinical and survival data.

Study Design

This is a retrospective study and no formal hypothesis regarding the sample size has been postulated a priori. Univariate survival was performed using Xxx regression and the log rank test.

FEASIBILITY AND RESOURCES

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We know this is ambitious project, however the possibility of failure associated to the overall project is low. Further, the possibility of identifying pathways that can be targeted to modulate inflammation and cancer progression in iCCA makes the risk worth taking. Furthermore, this risk does not compromise the success of the project that have high feasibility for the following reasons: first, the experimental design is based on solid preliminary data obtained by the team members; second, the team members are familiar with all the techniques described in the project; third, the network is solid, has strong experience in Translational research, and extensive access to human liver samples.

Description of facilities and research environment:

Humanitas University is an international higher education institution, established in 2014 and recognized by the Italian Ministry of Education, University and Research (MIUR). Humanitas is dedicated to basic and translational research in life sciences. Translational studies are carried out in tight collaboration with the Humanitas Research Hospital (recognized by Joint Commission International xxxxx://xxx.xxxxxxxxxxxxxxx.xxx/), a 750 bed hospital devoted to clinical research mainly in the context of immune-related, neurological, cardiovascular and neoplastic diseases. Research activities are carried out by 350 researchers and physicians, divided into more than 24 research groups including several prestigious, top-level groups covering many research areas.

Our researchers and physicians have access to up to 20,000 sqm laboratory space devoted to research and training activities. The Scientific Coordinator is Xxxx Xxxxxxx Xxxxxxxxx. An International Advisory Board assesses and evaluates Humanitas pre-clinical research. According to the “Excellence rank” of Scimago Research Group, Humanitas ranks among the top 10% worldwide. Humanitas's Laboratories are fully equipped with instruments for molecular and cellular biology research and all conform to Biosafety Level 2 (BSL2). Support to the researchers is given by the Grant Office which assists researchers in the management of funded research projects and by the Innovation Office which assists researchers in Technology Transfer issues.

IMPACT ON CANCER

iCCA is characterized by a grim prognosis with a 5-year survival of less than 20%. Indeed, response to chemotherapy is very low and diagnosis if commonly performed in advanced stages of disease. We herein propose to perform a complete genomic characterization of early-onset CCA by identifying driver genes and predisposing mutations. Universal tumor screening [36] for these cancer- predisposing genes in general BTC cases might be beneficial to patients with BTC and their family members, enabling assessment of their cancer development risk and the effectiveness of new anti- cancer drugs, such as immune checkpoint inhibitors and PARP inhibitors.

Ethical considerations Patient protection

The responsible investigator will ensure that this study is conducted in agreement with either the Declaration of Helsinki (Tokyo, Venice, Hong Kong and Somerset West amendments) or the laws and regulations of the country.

The protocol has been written, and the study will be conducted according to the ICH Guideline for Good Clinical Practice

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The protocol and its annexes are subject to review and approval by the competent Independent Ethics Committee(s) (“IEC”).

Subject identification – Personal Data protection

The data will be anonymously collected by using an electronic case report form in accordance with the current international recommendations and strict rules of confidentiality. Access to eCFR is guaranteed by personal identification code sent after activation of the investigative center. Each patient will be assigned with an anonymous code of identification. Samples will be labeled with the same identity code of the patient generated by e-CRF and stored in each participating center until the end of the project.

As in any routine care clinical research study, the patients will have to indicate their no-objection once they have been duly informed on the nature of the study. The no-objection form will be dated and signed by the investigator (original filed by the investigator).

Informed consent

All patients have already received and signed an Informed consent in the frame of Humanitas Biobank repository.

It is emphasized that the participation is voluntary and that the patient is allowed to refuse further participation in the protocol whenever he/she wants. This will not prejudice the patient’s subsequent care. Documented informed consent must be obtained for all patients included in the study before they are registered at the Data Center. This must be done in accordance with the national and local regulatory requirements.

For European Union member states, the informed consent procedure must conform to the ICH guidelines on Good Clinical Practice. This implies that “the written informed consent form should be signed and personally dated by the patient or by the patient’s legally acceptable representative”.

Conflict of Interest

Any investigator and/or research staff member who has a conflict of interest with this study (such as patent ownership, royalties, or financial gain greater than the minimum allowable by their institution) must fully disclose the nature of the conflict of interest.

Data ownership

According to the ICH Guidelines on Good Clinical Practice the sponsor of a study (the Institution, should the investigator or study coordinator act as sponsor in the performance of her/his institutional duties under the employment or collaboration agreement with Humanitas) is the owner of the data resulting therefrom. All centers and investigators participating in the study should be made aware of such circumstance and invited not to disseminate information or data without the Institution’s prior express consent.

Publication Policy

After completion of the study, the project coordinator will prepare a draft manuscript containing final results of the study on the basis of the statistical analysis. The manuscript will be derived to the co- authors for comments and after revision will be sent to a major scientific journal.

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All publications, abstracts, presentations, manuscripts and slides including data from the present study will be submitted to and reviewed by the Study Coordinator for coordination and homogeneity purposes: specific advance periods for submission and review may be specified in the protocol. The timing of publications (in the event several Centers should be participating in the Study) may be coordinated, and publication delayed if patentable inventions should be involved (for the time required in order to file the relevant patent applications); otherwise, according to the MoH’s Decree of May 12, 2006, investigators cannot be precluded from or limited in publishing the results of their studies (IECs must verify that no excessive restriction is contained in the protocols submitted to their review and approval).

Study time table

Project starting date: February 2021

Project completion of data collection: March 2021 Project data analysis: May 2021

Project presentation of scientific report: September 2021

REFERENCES

1. Rimassa L, Xxxxxxxxx N, Xxxxxx A, Xxxx A. The immune milieu of cholangiocarcinoma: From molecular pathogenesis to precision medicine. J. Autoimmun. 2019, 100, 17–26.

2. Xxxxx T, Xxxxxxxx G, Xxxxxx D, Xxxxxx D, Xx Xxxxxxx X, Xxxxx JW. Current Status on Cholangiocarcinoma and Gallbladder Cancer. Liver Cancer 2017, 6, 59–65.

3. Xxxxx N, Xxxxxxx, B. Incidence of Cholangiocarcinoma in the USA from 2001 to 2015: A US Cancer Statistics Analysis of 50 States. Cureus 2019, 11, e3962.

4. Xxxx, S.A.; Xxxxxxxx, S.; Xxxxxx, X. Xxxxxxxxxxxxxxxxxx: Epidemiology and risk factors. Liver Int. 2019, 39, 19–31.

5. Xxxxxxxxx, P.; Xxxxxxxx, M.; Xxxxxxx, G.; Xxxxxx, D.; Xxxxxx, P.; Xx-Xxxxx, H.B.; Xx Xxxxxxx, C.; Xxxxx, E. Global trends in mortality from intrahepatic and extrahepatic cholangiocarcinoma. X. Xxxxxxx. 2019, 71, 104–114.

6. Global Burden of Disease Liver Cancer Collaboration; Akinyemiju T, Xxxxx X, Xxxxx X.; Xxxx X, Xxxxxxxxx X.X, Xxxxx X, Xx-Xxxxxxx X, Xxxxx-Xxxxxx, X, Xxxxxx, X.; et al. The Burden of Primary Liver Cancer and Underlying Etiologies From 1990 to 2015 at the Global, Regional, and National Level. JAMA Oncol. 2017, 3, 1683–1691.

7. Xxxxxxxxxx, N.; Xxxxx, G.J. Cholangiocarcinoma. Lancet 2014, 383, 2168–2179.

8. Xxxxxxxx, H.; Xxxx, Y.; Xxxxxx, Y.; Xxxxxxx, T, El Xxxxxxx, A.; Xxxx, M.; Xxxx, N.; Xxxxxx, F.; Xxxxxxxxxx, T, Xxxxxx S, et al. Genomic spectra of biliary tract cancer. Nat. Xxxxx. 2015, 47, 1003–1010.

9. Xxxxxxx A, Xxxx, P, Xxxxx, XXX, Xxxxxx, R, XxXxxxxx, M.G.; Xxxxx, A.; Xxxxxxxxx, P.; Xxxxxx D, Xxxxxxxxxxx J, Xxxxx JW. Advanced intrahepatic cholangiocarcinoma: Post-hoc analysis of the ABC-01, -02 and -03 clinical trials. X. Xxxx. Cancer Inst. 2019, 112, 200–210.

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10. Xxxxx S, Xxxxx, G.J. Pathogenesis, Diagnosis, and Management of Cholangiocarcinoma. Gastroenterology 2013, 145, 1215–1229.

11. Xxxxx S, Xxxx SA, Xxxxxxxxxx XX, Xxxxxx RK, Xxxxx GJ. Cholangiocarcinoma—evolving concepts and therapeutic strategies. Nat. Rev. Clin. Oncol. 2018, 15, 95–111.

12. Xxxxxxx JL, Xxxxxxx JE, Xxxxxxxxx-Xxxxxxxxx A, Xxxxx X, Xxxxxxxxx-Xxxxx, J.; Xxx Xxxx AL, Xxxxxxxx, M.J.; Xxxxx, R.; Xxxxx, H.D.; Xxxxxxxx, C.; et al. Body Mass Index, Diabetes and Intrahepatic Cholangiocarcinoma Risk: The Liver Cancer Pooling Project and Meta- analysis. Xx. X. Xxxxxxxxxxxxx. 2018, 113, 1494–1505.

00. Xxxxxxx X, Xxxxxxxx X, Xxxx X, Xxxxxxx X, Xxxxxxxx, X.; Xxxxxxxx, J.B. Molecular perturbations in cholangiocarcinoma: Is it time for precision medicine? Liver Int. 2019, 39, 32–42.

14. Xxxxxx, R.K.; Xxxxxxxxxxx J, Xxxxx, GJ, Xxx, A.X. Systemic therapies for intrahepatic cholangiocarcinoma. X. Xxxxxxx. 2020, 72, 353–363.

15. Xxxxxxx A, Xxxxxxxx J, XxXxxxxx MG, Xxxxx, J.W. Molecular targeted therapies: Ready for “prime time” in biliary tract cancer. X. Xxxxxxx. 2020, 73, 170–185.

16. Xxxxxxxxxx XX, Xxxxx MS, Xxxxxx DR, Xxxxxx RV, Xxxx S, Xxxxx R.; Xxxxx, F.A. Molecular Gene Expression Profiling to Predict the Tissue of Origin and Direct Site-Specific Therapy in Patients With Carcinoma of Unknown Primary Site: A Prospective Trial of the Xxxxx Xxxxxx Research Institute. X. Xxxx. Oncol. 2013, 31, 217–223.

17. Xxxxxxx MH, Xxxxxxx GJ, Xxxxxx S, Xxxxxxx GJ, Xxxxxxxx J, Xxxxxxx SP. Cholangiocarcinoma and dominant strictures in patients with primary sclerosing cholangitis: a 25-year single-centre experience. Eur J Gastroenterol Hepatol. 2012; 24:1051–58.

18. Xxxxx GL, El-Serag HB. Risk factors for cholangiocarcinoma. Hepatology. 2011; 54:173–84.

00. Xxxxxxx X, Xxxxxx X, Xxxxxx X, Xxxxxxx XX. Bile duct cysts in adults. Br J Surg. 2004; 91:1538–48.

20. Xxxxxx X, Xxxx M, Xxxxxxx H, Xxxx S, Xxxxxxxxx A, Xxxxxx H, Xxxxxxxx M. Risk factors and characteristics of young patients with the biliary tract carcinoma: results of a project study for biliary surgery by the Japanese Society of Hepato-Biliary-Pancreatic Surgery. J Hepatobiliary Pancreat Sci. 2020 Sep;27(9):571-580. doi: 10.1002/jhbp.776.

21. Xxxx JS,Xxxx K, Xxx L et al. New routes to targeted therapy of intrahepatic cholangiocarcinomas revealed by next-generation sequencing. The Oncologist 2014;19:235– 242.

22. Xxxxxxxx H, Xxxx Y, Xxxxxx Y et al. Genomic spectra of biliary tract cancer. Xxx Xxxxx 2015;47:1003–1010.

23. Xxxxx CR, Xxxxxx R, Xxxx Y et al. Mutation profiling in cholangiocarcinoma: Prognostic and therapeutic implications. PLoS One 2014;9:e115383.

24. Breast Cancer Linkage Consortium, Easton D, Xxxxxxxx D et al. Cancer risks in BRCA2 mutation carriers. J Natl Cancer Inst 1999;91:1310–1316.

25. Xxxx HR, Xx JK, Xxxxxxx E et al. Epidemiology of cholangiocarcinoma: An update focusing on risk factors. Cancer Sci 2010;101:579–585.

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26. Venkitaraman AR. Cancer susceptibility and the functions of BRCA1 and BRCA2. Cell 2002;108:171–182.

27. Venkitaraman AR. Functions of BRCA1 and BRCA2 in the biological response to DNA damage. J Cell Sci 2001;114:3591–3598.

28. Xxxx A, Xxxxxxxx X. The relationship between the roles of BRCA genes in DNA repair and cancer predisposition. Trends Mol Med 2002;8:571–576.

29. Xxxxxx H, XxXxxx N, Xxxx XX et al. Targeting the DNA repair defect in BRCAmutant cells as a therapeutic strategy. Nature 2005;434:917–921.

30. Simbolo M, Xxxxxx M, Ruzzenente A, Xxxxxxxxx A, Xxxx LD, Xxxxx V, et al. Multigene mutational profiling of cholangiocarcinomas identifies actionable molecular subgroups. Oncotarget 2014;5:2839–2852.

31. Xx M, Xxxxx Z, Xx X, Xx J, Xx X, Xxx Z, et al. Whole-exome and targeted gene sequencing of gallbladder carcinoma identifies recurrent mutations in the ErbB pathway. Xxx Xxxxx 2014;46:1–7.

32. Xxx S, Xx J, Xxxx H, Xxxxx C, Xxxxx X, Xxx JS, et al. Mutational landscape of intrahepatic cholangiocarcinoma. Nat Commun 2014;5:5696.

33. Xxxxxxxx H, Xxxx Y, Xxxxxx Y, Xxxxxxx T, Xxxxxxxxx A, Xxxx M, et al. Genomic spectra of biliary tract cancer. Xxx Xxxxx 2015;47:1003–1010.

00. Xxxxxxxxxx X, Xxxxx X, Xxxxxxx XX, Xxxxxx X, Xxxx J, et al. Integrative genomic analysis of cholangiocarcinoma identifies distinct IDH-mutant molecular profiles. Cell Rep 2017;18:2780–2794.

35. Xxxx H, Xxxx H, Xxx J, Xx M, Xxxx W, Xxxx X. Xxxxxxx Features and Clinical Characteristics of Adolescents and Young Adults With Cholangiocarcinoma. Front Oncol. 2020 Xxx 14;9:1439.

00. Xxxxxxx X, Xxxxxx X, Xxxxxxxx X, Xxxxxxx M, Xxxxxxx M, Xxxxxxx Y, Xxxxxx C, Xxxx-Xxx- Xxx X, Xxxxxx X, Meurice G, Xxxxxxx E, Xxxxxxxx S, Xxxxxxx C, Xxxxxxx JY, Xxxxxx E, Xxxxx JC, Xxxxx F, Xxxxxxx L. Added Value of Whole-Exome and Transcriptome Sequencing for Clinical Molecular Screenings of Advanced Cancer Patients With Solid Tumors. Cancer J. 2018 Jul/Aug;24(4):153-162.

37. Xxxxxxx CP, Xxxxxx M, Xxxxxx T, Xxxxxxx M, Xxxxxx M, Xxxxxx R, et al. Genomic characterization of biliary tract cancers identifies driver genes and predisposing mutations. J Hepatol. 2018 May;68(5):959-969.

38. Xxxxxxx L, Xxxxxxxx F, Xxxxxx N, de la Chapelle A, Hampel H, Xxxxxxxx LA, et al. Identification of Xxxxx syndrome among patients with colorectal cancer. JAMA 2012;308:1555–1565.

Manuale di laboratorio del 08/03/2022 (v1.0)

Protocollo clinico “Genomic Landscape of Early-Onset Cholangiocarcinoma and Its Prognostic Implications” Controllo qualità del gDNA (a cura del Centro collaboratore)

Il gDNA estratto deve essere quantificato mediante metodo fluorimetrico (es. Qubit), e l’integrità deve essere valutata possibilmente mediante la piattaforma TapeStation (Agilent Technologies). Il DIN fornito da quest’ultima deve essere ≥2, affinché il campione sia considerato idoneo al processamento mediante NGS.

Linee guida per la spedizione dei campioni di gDNA al Centro coordinatore dell’NGS (Center for Omic Sciences, COSR)

• Il collaboratore deve fornire un’aliquota di 500 ng di gDNA (normale e tumorale), in un volume minimo di 10

μl e massimo di 100 μl.

• Il collaboratore deve compilare il modulo* Template_Content_DNA_ColottaF_1443_UNIC_colangiocarcinomi con le informazioni sui campioni di DNA forniti, ed inviarlo, unitamente ai file pdf del controllo qualità su TapeStation, agli indirizzi mail xxxxxxxxx.xxxxx@xxx.xx , xxxxx.xxxxxxxx@xxx.xx , xxxxxxxxx.xxxxxx@xxx.xx , xxxxx.xxxxxxxx@xxx.xx prima della spedizione dei campioni stessi, per approvazione da parte di COSR.

* le istruzioni per la compilazione del modulo sono incluse nel file excel

• I campioni devono essere spediti in tubi Eppendorf da 1,5 ml, ben chiusi con parafilm e opportunamente etichettati.

• L’inserimento in ACC LIMS delle informazioni legate a "patients" e "samples", con la generazione dei relativi barcode, sarà effettuato a cura del collaboratore. Il progetto di riferimento in ACC LIMS è UCOL. Per eventuali problematiche relative all’accesso ad ACC LIMS e/o al suo utilizzo, è necessario rivolgersi ad Xxxxxx Xxxx (XxxxxxXxxxxxXxxxxx.Xxxx@xxx.xx)

• Le informazioni relative alle librerie ("sample sequencing") saranno inserite in ACC LIMS da COSR, generando i relativi barcode.

• E’ richiesta al collaboratore la comunicazione tramite e-mail a xxxxxxxxx.xxxxx@xxx.xx , xxxxx.xxxxxxxx@xxx.xx, xxxxxxxxx.xxxxxx@xxx.xx, xxxxx.xxxxxxxx@xxx.xx della data prevista di spedizione dei campioni ed il tracking number della stessa.

• I campioni devono essere spediti con siberini (non è necessario ghiacco secco), all’attenzione di:

Xxxx. Xxxxx Xxxxxxxxx Center for Omics Sciences (COSR)

IRCCS Xxxxxxxx Xxx Xxxxxxxx, Xxxxxxxx 0X0

Xxx Xxxxxxxxx 00

00000 Xxxxx, Xxxxx

x00 00 00000000

Ricevimento campioni, gestione campioni e librerie (a cura di XXXX)

• I campioni in ingresso saranno sottoposti ad ulteriore controllo qualità (Qubit e TapeStation) da parte di COSR, e in caso di eventuali problematiche, sarà cura di COSR contattare il collaboratore.

• Eventuali campioni residui non saranno rispediti al collaboratore, ma saranno conservati presso il COSR per tutta la durata dello studio. Al termine di tale periodo saranno distrutti.

• Le librerie preparate dal COSR non saranno spedite al collaboratore, ma saranno conservate per tutta la durata dello studio presso COSR. Al termine di tale periodo saranno distrutte.

Per eventuali dubbi o chiarimenti, contattare: xxxxxxxxx.xxxxx@xxx.xx

xxxxx.xxxxxxxx@xxx.xx xxxxxxxxx.xxxxxx@xxx.xx xxxxx.xxxxxxxx@xxx.xx

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