Search

Background and Aims: Cardiac fibrosis in response to injury leads to myocardial stiffness and heart failure. At the cellular level, fibrosis is triggered by the conversion of cardiac fibroblasts (CF) into extracellular matrix-producing myofibroblasts. miR-24-3p regulates this process in animal models. Here, we investigated whether miR-24-3p plays similar roles in human models., Methods and Results: Gain- and loss-of-function experiments were performed using human induced pluripotent stem cell-derived cardiomyocytes (hCM) and primary hCF under normoxic or ischaemia-simulating conditions. hCM-derived extracellular vesicles (EVs) were added to hCF. Similar experiments were performed using three-dimensional human cardiac microtissues and ex vivo-cultured human cardiac slices.hCF transfection with miR-24-3p mimic prevented TGFβ1-mediated induction of FURIN, CCND1 and SMAD4-miR-24-3p target genes participating in TGFβ1-dependent fibrinogenesis -, regulating hCF-to-myofibroblast conversion. hCM secreted miR-24-3p as EV cargo. hCM-derived EVs modulated hCF activation. Ischaemia-simulating conditions induced miR-24-3p depletion in hCM-EVs and microtissues. Similarly, hypoxia downregulated miR-24-3p in cardiac slices. Analyses of clinical samples revealed decreased miR-24-3p levels in circulating EVs in acute myocardial infarction (AMI) patients, compared with healthy subjects. Post-mortem RNAScope analysis showed miR-24-3p downregulation in myocardium from AMI patients, compared with patients who died from noncardiac diseases. Berberin, a plant-derived agent with miR-24-3p-stimulatory activity, increased miR-24-3p contents in hCM-EVs, downregulated FURIN, CCND1 and SMAD4, and inhibited fibrosis in cardiac microtissues., Conclusions: These findings suggest that hCM may control hCF activation through miR-24-3p secreted as EV cargo. Ischaemia impairs this mechanism, favouring fibrosis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19, or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen, but only a few studies have compared some of these assays., Objectives: The International Society on Thrombosis and Haemostasis Scientific and Standardization Committee Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity, and reproducibility of these assays., Methods: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without lipopolysaccharide stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays., Results: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared with antigen assays. In addition, there was a large intra-assay and interassay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared with assays that isolated EVs by high-speed centrifugation., Conclusion: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody., Competing Interests: Declaration of competing interests F.D.-G. and R.L. filed a patent on microvesicle fibrinolytic activity licensed to Stago and obtained a common grant within the framework of the excellence program innovative tests to customize antiplatelet therapy in chronic kidney disease with acute coronary syndrome. The remaining authors declare no competing financial interests., (Copyright © 2024. Published by Elsevier Inc.)

Background: We previously demonstrated that the human amniotic fluid (hAF) from II trimester of gestation is a feasible source of stromal progenitors (human amniotic fluid stem cells, hAFSC), with significant paracrine potential for regenerative medicine. Extracellular vesicles (EVs) separated and concentrated from hAFSC secretome can deliver pro-survival, proliferative, anti-fibrotic and cardioprotective effects in preclinical models of skeletal and cardiac muscle injury. While hAFSC-EVs isolation can be significantly influenced by in vitro cell culture, here we profiled EVs directly concentrated from hAF as an alternative option and investigated their paracrine potential against oxidative stress., Methods: II trimester hAF samples were obtained as leftover material from prenatal diagnostic amniocentesis following written informed consent. EVs were separated by size exclusion chromatography and concentrated by ultracentrifugation. hAF-EVs were assessed by nanoparticle tracking analysis, transmission electron microscopy, Western Blot, and flow cytometry; their metabolic activity was evaluated by oximetric and luminometric analyses and their cargo profiled by proteomics and RNA sequencing. hAF-EV paracrine potential was tested in preclinical in vitro models of oxidative stress and dysfunction on murine C2C12 cells and on 3D human cardiac microtissue., Results: Our protocol resulted in a yield of 6.31 ± 0.98 × 10 9 EVs particles per hAF milliliter showing round cup-shaped morphology and 209.63 ± 6.10 nm average size, with relevant expression of CD81, CD63 and CD9 tetraspanin markers. hAF-EVs were enriched in CD133/1, CD326, CD24, CD29, and SSEA4 and able to produce ATP by oxygen consumption. While oxidative stress significantly reduced C2C12 survival, hAF-EV priming resulted in significant rescue of cell viability, with notable recovery of ATP synthesis and concomitant reduction of cell damage and lipid peroxidation activity. 3D human cardiac microtissues treated with hAF-EVs and experiencing H 2 O 2 stress and TGFβ stimulation showed improved survival with a remarkable decrease in the onset of fibrosis., Conclusions: Our results suggest that leftover samples of II trimester human amniotic fluid can represent a feasible source of EVs to counteract oxidative damage on target cells, thus offering a novel candidate therapeutic option to counteract skeletal and cardiac muscle injury., Competing Interests: Declaration of competing interest The authors have nothing to disclose nor competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This study compares the impact of two isolation methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), on small extracellular vesicles (sEVs) from primary human cardiac mesenchymal-derived progenitor cells (CPCs). sEV_UC and sEV_SEC exhibit similar size, marker expression, and miRNA cargo, but sEV_UC contains notably higher total protein levels. In vitro assays show that sEV_UC, despite an equal particle count, induces more robust ERK phosphorylation, cytoprotection, and proliferation in iPS-derived cardiomyocytes (iPS-CMs) compared to sEV_SEC. sEV_UC also contains elevated periostin (POSTN) protein levels, resulting in enhanced focal adhesion kinase (FAK) phosphorylation in iPS-CMs. Importantly, this effect persists with treatment with soluble free-sEV protein fraction from SEC (Prote_SEC), indicating that free proteins like POSTN in sEV_UC enhance FAK phosphorylation. In vivo, sEV contamination with soluble proteins doesn't affect cardiac targeting or FAK phosphorylation, underscoring the intrinsic tissue targeting properties of sEV. These findings emphasize the need for standardized sEV isolation methods, as the choice of method can impact experimental outcomes, particularly in vitro., (© 2023 Wiley-VCH GmbH.)

Background: Optimal consolidation for young patilents with relapsed/refractory (R/R) follicular lymphoma (FL) remains uncertain in the rituximab era, with an unclear benefit of autologous stem cell transplantation (ASCT). The multicenter, randomized, phase III FLAZ12 (NCT01827605) trial compared anti-CD20 radioimmunotherapy (RIT) with ASCT as consolidation after chemoimmunotherapy, both followed by rituximab maintenance., Patients and Methods: Patients (age 18-65 years) with R/R FL and without significant comorbidities were enrolled and treated with three courses of conventional, investigator-chosen chemoimmunotherapies. Those experiencing at least a partial response were randomized 1 : 1 to ASCT or RIT before CD34+ collection, and all received postconsolidation rituximab maintenance. Progression-free survival (PFS) was the primary endpoint. The target sample size was 210 (105/group)., Results: Between August 2012 and September 2019, of 164 screened patients, 159 were enrolled [median age 57 (interquartile range 49-62) years, 55% male, 57% stage IV, 20% bulky disease]. The study was closed prematurely because of low accrual. Data were analyzed on 8 June 2023, on an intention-to-treat basis, with a 77-month median follow-up from enrollment. Of the 141 patients (89%), 70 were randomized to ASCT and 71 to RIT. The estimated 3-year PFS in both groups was 62% (hazard ratio 1.11, 95% confidence interval 0.69-1.80, P = 0.6662). The 3-year overall survival also was similar between the two groups. Rates of grade ≥3 hematological toxicity were 94% with ASCT versus 46% with RIT (P < 0.001), and grade ≥3 neutropenia occurred in 94% versus 41%, respectively (P < 0.001). Second cancers occurred in nine patients after ASCT and three after radioimmunotherapy (P = 0.189)., Conclusions: Even if prematurely discontinued, our study did not demonstrate the superiority of ASCT versus RIT. ASCT was more toxic and demanding for patients and health services. Both strategies yielded similar, favorable long-term outcomes, suggesting that consolidation programs milder than ASCT require further investigation in R/R FL., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cardiovascular diseases (CVD) can cause various conditions, including an increase in reactive oxygen species (ROS) levels that can decrease nitric oxide (NO) availability and promote vasoconstriction, leading to arterial hypertension. Physical exercise (PE) has been found to be protective against CVD by helping to maintain redox homeostasis through a decrease in ROS levels, achieved by increased expression of antioxidant enzymes (AOEs) and modulation of heat shock proteins (HSPs). Extracellular vesicles (EVs) circulating in the body are a major source of regulatory signals, including proteins and nucleic acids. Interestingly, the cardioprotective role of EVs released after PE has not been fully described. The aim of this study was to investigate the role of circulating EVs, obtained through Size Exclusion Chromatography (SEC) of plasma samples from healthy young males (age: 26.95 ± 3.07; estimated maximum oxygen consumption rate (VO 2max ): 51.22 ± 4.85 (mL/kg/min)) at basal level (Pre&#95;EVs) and immediately after a single bout of endurance exercise (30' treadmill, 70% heart rate (HR) -Post&#95;EVs). Gene ontology (GO) analysis of proteomic data from isolated EVs, revealed enrichment in proteins endowed with catalytic activity in Post&#95;EVs, compare to Pre&#95;EVs, with MAP2K1 being the most significantly upregulated protein. Enzymatic assays on EVs derived from Pre and Post samples showed increment in Glutathione Reductase (GR) and Catalase (CAT) activity in Post&#95;EVs. At functional level, Post&#95;EVs, but not Pre&#95;EVs, enhanced the activity of antioxidant enzymes (AOEs) and reduced oxidative damage accumulation in treated human iPS-derived cardiomyocytes (hCM) at basal level and under stress conditions (Hydrogen Peroxide (H 2 O 2 ) treatment), resulting in a global cardioprotective effect. In conclusion, our data demonstrated, for the first time, that a single 30-min endurance exercise is able to alter the cargo of circulating EVs, resulting in cardioprotective effect through antioxidant activity., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) constitute a mixed population of ventricular-, atrial-, nodal-like cells, limiting the reliability for studying chamber-specific disease mechanisms. Previous studies characterised CM phenotype based on action potential (AP) morphology, but the classification criteria were still undefined. Our aim was to use in silico models to develop an automated approach for discriminating the electrophysiological differences between hiPSC-CM. We propose the dynamic clamp (DC) technique with the injection of a specific I K1 current as a tool for deriving nine electrical biomarkers and blindly classifying differentiated CM. An unsupervised learning algorithm was applied to discriminate CM phenotypes and principal component analysis was used to visualise cell clustering. Pharmacological validation was performed by specific ion channel blocker and receptor agonist. The proposed approach improves the translational relevance of the hiPSC-CM model for studying mechanisms underlying inherited or acquired atrial arrhythmias in human CM, and for screening anti-arrhythmic agents., (© 2023. The Author(s).)

Background: A long-standing effort is dedicated towards the identification of biomarkers allowing the prediction of graft outcome after kidney transplant. Extracellular vesicles (EVs) circulating in body fluids represent an attractive candidate, as their cargo mirrors the originating cell and its pathophysiological status. The aim of the study was to investigate EV surface antigens as potential predictors of renal outcome after kidney transplant., Methods: We characterized 37 surface antigens by flow cytometry, in serum and urine EVs from 58 patients who were evaluated before, and at 10-14 days, 3 months and 1 year after transplant, for a total of 426 analyzed samples. The outcome was defined according to estimated glomerular filtration rate (eGFR) at 1 year., Results: Endothelial cells and platelets markers (CD31, CD41b, CD42a and CD62P) in serum EVs were higher at baseline in patients with persistent kidney dysfunction at 1 year, and progressively decreased after kidney transplant. Conversely, mesenchymal progenitor cell marker (CD1c, CD105, CD133, SSEEA-4) in urine EVs progressively increased after transplant in patients displaying renal recovery at follow-up. These markers correlated with eGFR, creatinine and proteinuria, associated with patient outcome at univariate analysis and were able to predict patient outcome at receiver operating characteristics curves analysis. A specific EV molecular signature obtained by supervised learning correctly classified patients according to 1-year renal outcome., Conclusions: An EV-based signature, reflecting the cardiovascular profile of the recipient, and the repairing/regenerative features of the graft, could be introduced as a non-invasive tool for a tailored management of follow-up of patients undergoing kidney transplant., (© The Author(s) 2022. Published by Oxford University Press on behalf of the ERA.)

The organ-specific metastatization of breast cancer to bone is driven by specific interactions between the host microenvironment and cancer cells (CCs). However, it is still unclear the role that circulating immune cells, including neutrophils, play during bone colonization (i.e. pro-tumoral vs. anti-tumoral). Here, we aimed at analyzing the migratory behavior of neutrophils when exposed to breast CCs colonizing the bone and their contribution to the growth of breast cancer micrometastases. Based on our previous bone metastasis models, we designed a microfluidic system that allows to independently introduce human vascularized breast cancer metastatic seeds within a bone-mimicking microenvironment containing osteo-differentiated mesenchymal stromal cells and endothelial cells (ECs). ECs self-assembled into microvascular networks and connected the bone-mimicking microenvironment with the metastatic seed. Compared to controls without CCs, metastatic seeds compromised the architecture of microvascular networks resulting in a lower number of junctions (5.7 ​± ​1.2 vs. 18.8 ​± ​4.5, p ​= ​0.025) and shorter network length (10.5 ​± ​1.0 vs. 13.4 ​± ​0.8 [mm], p ​= ​0.042). Further, vascular permeability was significantly higher with CCs (2.60 ​× ​10 -8  ​± ​3.59 ​× ​10 -8  ​vs. 0.53 ​× ​10 -8  ​± ​0.44 ​× ​10 -8 [cm/s], p ​= ​0.05). Following metastatic seed maturation, neutrophils were injected into microvascular networks resulting in a higher extravasation rate when CCs were present (27.9 ​± ​13.7 vs. 14.7 ​± ​12.4 [%], p ​= ​0.01). Strikingly, the percentage of dying CCs increased in presence of neutrophils, as confirmed by confocal imaging and flow cytometry on isolated cells from the metastatic seeds. The biofabricated metastatic niche represents a powerful tool to analyze the mechanisms of interaction between circulating immune cells and organ-specific micrometastases and to test novel drug combinations targeting the metastatic microenvironment., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Inflammatory response following SARS-CoV-2 infection results in substantial increase of amounts of intravascular pro-coagulant extracellular vesicles (EVs) expressing tissue factor (CD142) on their surface. CD142-EV turned out to be useful as diagnostic biomarker in COVID-19 patients. Here we aimed at studying the prognostic capacity of CD142-EV in SARS-CoV-2 infection. Expression of CD142-EV was evaluated in 261 subjects admitted to hospital for pneumonia and with a positive molecular test for SARS-CoV-2. The study population consisted of a discovery cohort of selected patients (n = 60) and an independent validation cohort including unselected consecutive enrolled patients (n = 201). CD142-EV levels were correlated with post-hospitalization course of the disease and compared to the clinically available 4C Mortality Score as referral. CD142-EV showed a reliable performance to predict patient prognosis in the discovery cohort (AUC = 0.906) with an accuracy of 81.7%, that was confirmed in the validation cohort (AUC = 0.736). Kaplan-Meier curves highlighted a high discrimination power in unselected subjects with CD142-EV being able to stratify the majority of patients according to their prognosis. We obtained a comparable accuracy, being not inferior in terms of prediction of patients' prognosis and risk of mortality, with 4C Mortality Score. The expression of surface vesicular CD142 and its reliability as prognostic marker was technically validated using different immunocapture strategies and assays. The detection of CD142 on EV surface gains considerable interest as risk stratification tool to support clinical decision making in COVID-19., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Rationale: Aging in the heart is a gradual process, involving continuous changes in cardiovascular cells, including cardiomyocytes (CMs), namely cellular senescence. These changes finally lead to adverse organ remodeling and resulting in heart failure. This study exploits CMs from human induced pluripotent stem cells (iCMs) as a tool to model and characterize mechanisms involved in aging. Methods and Results: Human somatic cells were reprogrammed into human induced pluripotent stem cells and subsequently differentiated in iCMs. A senescent-like phenotype (SenCMs) was induced by short exposure (3 hours) to doxorubicin (Dox) at the sub-lethal concentration of 0.2 µM. Dox treatment induced expression of cyclin-dependent kinase inhibitors p21 and p16, and increased positivity to senescence-associated beta-galactosidase when compared to untreated iCMs. SenCMs showed increased oxidative stress, alteration in mitochondrial morphology and depolarized mitochondrial membrane potential, which resulted in decreased ATP production. Functionally, when compared to iCMs, SenCMs showed, prolonged multicellular QTc and single cell APD, with increased APD variability and delayed afterdepolarizations (DADs) incidence, two well-known arrhythmogenic indexes. These effects were largely ascribable to augmented late sodium current (I NaL ) and reduced delayed rectifier potassium current (Ikr). Moreover sarcoplasmic reticulum (SR) Ca 2+ content was reduced because of downregulated SERCA2 and increased RyR2-mediated Ca 2+ leak. Electrical and intracellular Ca 2+ alterations were mostly justified by increased CaMKII activity in SenCMs. Finally, SenCMs phenotype was furtherly confirmed by analyzing physiological aging in CMs isolated from old mice in comparison to young ones. Conclusions: Overall, we showed that SenCMs recapitulate the phenotype of aged primary CMs in terms of senescence markers, electrical and Ca 2+ handling properties and metabolic features. Thus, Dox-induced SenCMs can be considered a novel in vitro platform to study aging mechanisms and to envision cardiac specific anti-aging approach in humans., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Cardiovascular (CV) disease represents the most common cause of death in developed countries. Risk assessment is highly relevant to intervene at individual level and implement prevention strategies. Circulating extracellular vesicles (EVs) are involved in the development and progression of CV diseases and are considered promising biomarkers. We aimed at identifying an EV signature to improve the stratification of patients according to CV risk and likelihood to develop fatal CV events. EVs were characterized by nanoparticle tracking analysis and flow cytometry for a standardized panel of 37 surface antigens in a cross-sectional multicenter cohort (n = 486). CV profile was defined by presence of different indicators (age, sex, body mass index, hypertension, hyperlipidemia, diabetes, coronary artery disease, cardiac heart failure, chronic kidney disease, smoking habit, organ damage) and according to the 10-year risk of fatal CV events estimated using SCORE charts of European Society of Cardiology. By combining expression levels of EV antigens using unsupervised learning, patients were classified into 3 clusters: Cluster-I (n = 288), Cluster-II (n = 83), Cluster-III (n = 30). A separate analysis was conducted on patients displaying acute CV events (n = 82). Prevalence of hypertension, diabetes, chronic heart failure, and organ damage (defined as left ventricular hypertrophy and/or microalbuminuria) increased progressively from Cluster-I to Cluster-III. Several EV antigens, including markers for platelets (CD41b-CD42a-CD62P), leukocytes (CD1c-CD2-CD3-CD4-CD8-CD14-CD19-CD20-CD25-CD40-CD45-CD69-CD86), and endothelium (CD31-CD105) were independently associated with CV risk indicators and correlated to age, blood pressure, glucometabolic profile, renal function, and SCORE risk. EV profiling, obtained from minimally invasive blood sampling, allows accurate patient stratification according to CV risk profile., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: We compared 2 years of rituximab maintenance (RM) with a response-adapted postinduction approach in patients with follicular lymphoma who responded to induction immunochemotherapy., Methods: We randomly assigned treatment-naïve, advanced-stage, high-tumor burden follicular lymphoma patients to receive standard RM or a response-adapted postinduction approach on the basis of metabolic response and molecular assessment of minimal residual disease (MRD). The experimental arm used three types of postinduction therapies: for complete metabolic response (CMR) and MRD-negative patients, observation; for CMR and MRD-positive (end of induction or follow-up) patients, four doses of rituximab (one per week, maximum three courses) until MRD-negative; and for non-CMR patients, one dose of ibritumomab tiuxetan followed by standard RM. The study was designed as noninferiority trial with progression-free survival (PFS) as the primary end point., Results: Overall, 807 patients were randomly assigned. After a median follow-up of 53 months (range, 1-92 months), patients in the standard arm had a significantly better PFS than those in the experimental arm (3-year PFS 86% v 72%; P < .001). The better PFS of the standard versus experimental arm was confirmed in all the study subgroups except non-CMR patients (n = 65; P = .274). The 3-year overall survival was 98% (95% CI, 96 to 99) and 97% (95% CI, 95 to 99) in the reference and experimental arms, respectively ( P = .238)., Conclusion: A metabolic and molecular response-adapted therapy as assessed in the FOLL12 study was associated with significantly inferior PFS compared with 2-year RM. The better efficacy of standard RM was confirmed in the subgroup analysis and particularly for patients achieving both CMR and MRD-negative., Competing Interests: Stefano LuminariConsulting or Advisory Role: Roche, Gilead Sciences, Celgene, Genmab, Regeneron, IncyteTravel, Accommodations, Expenses: Janssen, Celgene Sara GalimbertiSpeakers' Bureau: Novartis Italy, Jazz Pharmaceuticals, Incyte, AbbVieTravel, Accommodations, Expenses: Jazz Pharmaceuticals, Janssen Oncology, Incyte, Novartis Italy Annibale VersariConsulting or Advisory Role: NovartisTravel, Accommodations, Expenses: Novartis Alessandra TucciConsulting or Advisory Role: Janssen, TakedaTravel, Accommodations, Expenses: Sandoz Luigi MarcheselliUncompensated Relationships: Sandoz SpA Simone FerreroConsulting or Advisory Role: Janssen-Cilag, EUSA Pharma, Clinigen Group, IncyteSpeakers' Bureau: Janssen-Cilag, Servier, EUSA Pharma, GentiliResearch Funding: Gilead Sciences (Inst), MorphoSys (Inst), Janssen (Inst)Travel, Accommodations, Expenses: Roche, Servier, Sanofi, Janssen-Cilag, EUSA Pharma, Gentili Luca ArcainiConsulting or Advisory Role: Roche, Janssen-Cilag, Verastem, Incyte, EUSA Pharma, Celgene/Bristol Myers SquibbSpeakers' Bureau: EUSA PharmaResearch Funding: Gilead SciencesTravel, Accommodations, Expenses: Roche, Celgene, Janssen-Cilag, EUSA Pharma Federica CavalloHonoraria: ServierConsulting or Advisory Role: Gilead Sciences, RocheTravel, Accommodations, Expenses: Celgene Tetiana SkrypetsHonoraria: Takeda Ilaria del GiudiceConsulting or Advisory Role: Tolero Pharmaceuticals, AstraZeneca Stephane ChauvieStock and Other Ownership Interests: DixitHonoraria: Sirtex MedicalSpeakers' Bureau: TERUMO, Sirtex MedicalResearch Funding: Roche Antonello PintoHonoraria: Roche/Genentech, Merck Sharp & Dohme, Bristol Myers Squibb, Celgene, Servier, IncyteConsulting or Advisory Role: Servier, Roche/Genentech, Merck, TakedaSpeakers' Bureau: Roche/Genentech Marco LadettoHonoraria: AbbVie, Amgen, ADC Therapeutics, BeiGene, Celgene, Gentili, Kite/Gilead, Novartis, Incyte, Janssen, Jazz Pharmaceuticals, Regeneron, RocheConsulting or Advisory Role: Jazz Pharmaceuticals, Roche, Janssen, Regeneron, Gilead Sciences, Novartis, IncyteSpeakers' Bureau: IncyteResearch Funding: ADC Therapeutics (Inst), Janssen (Inst) Annalisa ChiarenzaConsulting or Advisory Role: Roche/Genentech Annalisa ArcariConsulting or Advisory Role: Janssen-CilagTravel, Accommodations, Expenses: Janssen-Cilag, Takeda Massimo FedericoConsulting or Advisory Role: Takeda, Menarini, Erytech Pharma, MedivationTravel, Accommodations, Expenses: Takeda Pharmaceutical Taiwan, LTDNo other potential conflicts of interest were reported.