Report Scientifico IRP 2016

Re in c gen i erative Med

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Pa log e d iatric Onco

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e Nan n i c o Medi

SCIENTIFIC REPORT 2016

Floor 1

Mass Spectrometry and Metabolomics Lab Diagnosis and Therapy of Lysosomal Disorders Lab P-Care and Critical Care Biology Lab

Floor 2

Cystic Fibrosis Lab Neurodevelopmental Molecular Genetics Lab Clinical Genetics and Epidemiology Lab Neuroscience Lab

Floor 3

Renal lmmunopathology and Molecular Biology Lab Neuroimmunology Lab Solid Tumor Biology Lab

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Floor 4≠5

Pediatric Onco-hematologic Lab Neuroblastoma Lab Bio Nano Characterization Lab

Floor 7

Stem Cells and Regenerative Medicine Lab Nano Inspired Biomedicine Lab

Contents

President introduction

Pag. 5

Scientific Directorate introduction

Pag. 7

The essential about IRP in 2016

Pag. 9

Clinical Genetics and Epidemiology Laboratory

Pag. 11

Group Leaders: Leonardo Salviati – Eva Trevisson

Cystic Fibrosis Laboratory

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Group Leader: Luigi Picci

Diagnosis and Therapy of Lysosomal Disorders Laboratory

Pag. 17

Group Leader: Rosella Tomanin

Mass Spectrometry and Metabolomics Laboratory

Pag. 23

Group Leaders: Eugenio Baraldi - Giuseppe Giordano

Nano Inspired Biomedicine Laboratory

Pag. 29

Group Leader: Marco Agostini

Neurodevelopmental Molecular Genetics Laboratory

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Group Leader: Alessandra Murgia

Neuroimmunology Laboratory

Pag. 17

Group Leader: Bruno Giometto

Neuroscience Laboratory

Pag. 39

Group Leader: Antonio Vallesi

Pediatric Critical Care & Critical Care Biology Laboratory

Pag. 43

Group Leaders: Paola Cogo - Luca Vedovelli

Renal lmmunopathology and Molecular Biology Laboratory

vPag. 47

Group Leader: Luisa Murer

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Stem Cells and Regenerative Medicine Laboratory

Pag. 53

Group Leaders: Maurizio Muraca – Michela Pozzobon

Onco-hematologic Unit - Pediatric Onco-hematology Laboratory

Pag. 67

Group Leader: Giuseppe Basso PI: Benedetta Accordi PI: Lara Mussolin PI: Martina Pigazzi PI: Geertuy Te Kronnie PI: Luca Trentin

Onco-hematologic Unit - Solid Tumor Biology Laboratory

Pag. 99

Group Leader: Giuseppe Basso Referent: Gianni Bisogno PI: Paolo Bonvini PI: Lucia Tombolan PI: Angelica Zin

Onco-hematologic Unit - other groups

Pag. 119

Group Leader: Giuseppe Basso PI: Chiara Frasson PI: Giuseppe Germano PI: Maddalena Paganin PI: Luca Persano PI: Giampietro Viola

Neuroblastoma Laboratory

Pag. 133

Group Leader: Gian Paolo Tonini

Bio Nano Characterization Laboratory

Pag. 141

Group Leader: Filippo Romanato

Pubblications List

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Pag. 155

Andrea Camporese President

This first real scientific report arranged by the Paediatric Research Institute (IRP) – Città della Speranza Foundation (Padova, Italy), testifies the gradual increase of scientific activities and the beginning of this incredible venture. In particular, the constant increase of scientific work is related to GRANT, which can be considered one of the indicators of attractiveness. It is important to underline that to qualitative move forward, regional excellences have to be shared in sectors; the final aim is to become part of a high profile planning system representing the territory entirely, not partially. IRP can and must be an instrument and a catalyst, a partner and a main character; anyone can see it with a different role, but, in any case, it is alive and at the community’s disposal.

Our territory has a huge amount of competences and it must take the lead in order to move from a lowvalue-added manufacturing economy to one based on knowledge. In coming years, the aim is to continue growing by creating solid and lasting agreements with research centres worldwide; being recognised internationally; establishing a large researchers turnover; proceeding with the presentation of projects related with European programs linked with Horizon 2020; opening the doors to private investors, always reminding that all economic results will be re-invested in IRP’s development.

Today investing in biotechnologies and biomedicine is crucial as new products for everyone’s welfare are needed, but it is equally essential to make room for our young minds. For this reason, encourage technology transfer in our territory is more than ever fundamental.

In accordance with our associates and partners, the foregoing aims led to a deep reflection, which focused on the amendment of our governance model, in order to make it more coherent and suitable with the challenges we are facing.

Overall in 2016 IRP had a significant increase of activities performed and organised; for 2016 they can be summarised in these numbers: •

295 Researches in IRP (184 active)

•

16 scientific seminars

•

34 Events

In addiction, about 1500 students visited the Institute.

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I would also like to thank CittĂ della Speranza Foundation, Cariparo Foundation, the University and the polyclinic of Padua, Woman and Child Health Department and all the IRP foundation institutions that supported me with great availability and enthusiasm; I consider this a sign of their desire to take the lead with the construction process of this ambitious, hard and, for this reasons, exciting project. We have the great duty and honour to demonstrate that this region is able to propose a modern, smart and successful model, along with all the stakeholder, i.e. the University, companies, economic groups and citizens.

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Marco Pierotti Scientific directorate

It is my pleasure to introduce to you the first Scientific Report of the Pediatric Research Institute (IRP) “Città della Speranza” Foundation.

Since 2012 the Institute has been actively conducting different research studies and in the last year it highly increased its activities. Currently, it counts more than 180 researchers working in its laboratories.

The idea to create IRP came from the founders of the Città della Speranza Foundation that felt not only the necessity to economically support researchers, research projects and laboratories facilities, but also to create a new Research Institute. In particular, they wanted to have an effectively role in speeding up the process that could lead the ill children to have easily access to all the new research discoveries and medical innovations. Indeed, these could certainly enhance their chances to be properly treated and cured.

The nine floors of IRP Tower host a total of 15 laboratories. Although the majority of them belong to the Department of Women and Children Health, there are also other research Units such as the laboratories of Stem cells and Regenerative Medicine and Nano Inspired Biomedicine.

The research activities carried on by these groups concern topics that go from the paediatric oncology (both oncohaematology and solid tumors) and a broad spectrum of Genetic diseases; to the most important paedriatric pathologies affecting different organs (e.g. brain, lung and kidney).

At the beginning, the presence in the IRP of different research topics have instilled fragmentation problems that did not help to realize a well- structured institute. For this reason, the possibility to have a common point of view was thought to be a good chance to solve these isssues. The common global vision was based on having a context - related research where the starting point is represented by the unsolved clinical problem (mainly related to paediatric pathologies). An accurate analysis of the identified problems could clarified how much and which type of research (basic or applied) it will be necessary to be done. Furthermore, this approach could end up in the concrete realization of diagnostic products, procedures and/or novel effective therapeutic compounds that could help to eventually solve problems of children affected by different kind of diseases. Indeed, it is one of the main mission of this institute to

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be able to make ideas to happen. This goal could be reached sustaining the development of the scientific results through obtaining patents and starting different technology transfer processes.

In this perspective, in IRP it was possible to identified three main scientific areas where researchers, although having heterogeneous knowledge and skills, could converge their experience and create a positive synergy. The research fields are: Paediatric Oncology, Regenerative Medicine and Nano Medicine.

To support the integration of these areas, this scientific directorate has promoted the creation of three technological platforms. The researchers working in these platforms were able to developed methods and protocols specific for several techniques. Specifically, the platform one is involved in the preparation of decellularized extracellular matrix (both from normal and pathological tissues); the second is specialized on the isolation and characterization of exosomes and extracellular vesicles from tumor cells and from differently stimulated stem cells. The third platform is represented by uSTEM (a startup based in the Tower), originally developed at the Department of Industrial Engineering of Padua University. This third platform is highly specialized in the production of iPSC (induced pluripotent stem cells) from disparate sources and their differentiation towards different lineages, all performed with high efficiency and low costs. By using this technique, it will possible to better understand, for instance, neurodegenerative diseases related to genetic defects whose cells (e.g. neurons) are difficult to obtain from adult patient and even more from paediatric ones. Furthermore, the differentiation of iPSC towards a certain cell lineage (e.g. cardiomyocytes, hepatocytes) could be a useful tool to screen new potential therapeutic compounds in a disease and personalized specific manner, in accordance with the “precision medicin� emerging field.

Currently, uSTEM has a tight collaboration with IRP researchers and, together with them, conducts several interesting research projects.

The present report will give you an overview of all the research field, the structures and the accomplishments of the different groups located in the IRP Tower.

My hope and wishes are that the IRP Scientific Report of the next year will witness the success of this approach by reporting the concrete results obtained through the collaboration of the different groups, both in terms of publications in well-known scientific journals and in the development of patents able to develop innovative tools helpful for the needs of the ill children.

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The essentials About IRP in 2016

Scientific Activity

Published papers: Impact factor:

169 821,27

Facts and figures Published papers

Impact Factor

2016 2015 2014 2013 0

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200

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Research Founding € 4.500.000 € 4.000.000 € 3.500.000 € 3.000.000 € 2.500.000 € 2.000.000 € 1.500.000 € 1.000.000 € 500.000 0

2013

2014

2015

2016 9

Clinical Genetics and Epidemiology GROUP LEADERS

Leonardo Salviati Eva Trevisson

GROUP MEMBERS

Post≠doctoral Fellows: Acosta Lopez Manuel Jesus Vazquez Fonseca Luis Rigon Chiara Pinato Claudia Calderan Cristina Cerqua Cristina Desbats Marias Andrea Morbiato Laura

PhD Students: Morbidoni Valeria Rossi Silvia Zordan Roberta

Field of interest

The research of group operating in the Clinical Genetics and Epidemiology laboratory stems directly from its diagnostic activity and throughout these years it has focused on two main fields: the biogenesis of the mitochondrial respiratory chain (RC) and the development of simple models to characterize and validate molecular defects identified in patients with genetic disorders. Thes two lines are strictly related one to the other, Prof Salviati is leading the first line, Dr Trevisson is more directly involved with the second.

For the first line of research we have concentrated in particular on the biogenesis of coenzyme Q and of cytochrome c oxidase, two components of the mitochondrial respiratory chain, in order to identify novel genes involved in these processes, to understand the pathogenesis of disorders affecting these pathways, and to develop novel therapeutic strategies for these disease. In latest years, the interest has expanded to other components such as cytochrome c, mitochondrial DNA-encoded genes, and to disorders affecting mitochondrial dynamics. The techniques developed to study mitochondrial function have been employed for characterizing also other experimental models and we have been able to collaborate with several groups on topics not immediately related to RC dysfunction.

In fact in the second line of reseach we have applied the methodologies originally developed for the study of RC defects to a wide set of disease. We have emploed yeast to provide a functional validation for missense mutations found in a large set of genes, many of which relatd to other types of metabolic or neuromuscular disorders, and sometimes unrelated to metabolism at all. This has allowed us to develop novel tools for patient stratification and to determine phenotype-genotype correlations for many disorders. In parallel we have perfected assays based on hybrind minigenes to characterize defects of mRNA splicing and this approach has be applied to several

11

fig.1

different pathological conditions. Finally we have introduced a novel model organism, c.elegans as a further tool for our studies. These techniques have allowed us to continue a small, but productive field of research focused on defects of intermediate metabolism.

Summary of recent research activities and of main results achieved

Regarding the first line of research, during the period 201 -2016, we have cloned and characterized a series of human genes involved in RC biogenesis (22, ) and developed models with increasing complexity to study their function and dysfunction (yeast, c.elegans, and now genomically edited human cell lines and murine models).

Overall, these studies have allowed us to participate to the discovery of a novel genetic defect of Co biosynthesis (CO 8B) ( ), to identify new patients and to better understand the pathophysiology of these disorders (1,5,9,10,11,15,19,21,25,29, 2) and to evaluate new therapeutic strategies (24).

We have also refined our analysis techniques to study mitochondria respiration, RC assembly and function; this has allowed us to establish several collaborations with national and international groups on the characterization of mitochondrial function in different disorders(4,6,7,17, 1).

Moreover, we have participated to the identification of the genetic defects of other types of disease not directly related to mitochondria or to metabolism ( ,1 ,16,17,26,27,28). Our tools were often crucial to define the pathogenicity of the identified variants. Minigenes were instrumental to validate large sets of mutations in different disorders (12,18) As for disorders of intermediate metabolism, we have focused on genotype phenotype correlations and novel therapeutic approaches for HHH syndrome, and OAT deficiency (8, 0,). Finally we have been able to characterize the molecular defect in several different patients/disorders identifying particular phenotypes (14,2 ,28,) 12

fig.1 Mitochondrial alterations in skeletal muscle of a patient with the severe neonatal form of coenzyme Q deficiency due to COQ2 mutations.

From Desbats et al Eur J Hum Genet.

2015;23:1254-8

fig.2 A yeast system allows to associate MCM5 mutations with Meier-Gorlin syndrome (a) A copy of the mcm5 gene was inactivated in a diploid W303 yeast by homologous recombination with a KANMX4 cassette (which confers resistance to G418). The strains carrying the heterozygous deletion were left to sporulate : only two spores for each tetrad survived and none of them could grow in medium containing G418, confirming that these haploid cells carried the wild-type allele. (b) Diploid yeasts, heterozygous for mcm5 inactivation, were transformed either the wild type or the mutated mcm5 and then left to sporulate. Each colony was then streaked in medium lacking uracil (SM–URA) to confirm the presence of the plasmid and then in (YPD +G418) to confirm the mcm5 genotype.

Wild-type mcm5 restored a normal segregation pattern. In contrast, only 50% of those transformed with mutant mcm5 survived. All the surviving spores carried the wild-type allele. This indicated that the Thr501Ile found in the patient abolishes the function of mcm5.

fig.3 Mitochondrial localization of human COQ2 isoforms Desbats et al. Hum Mol Genet.

2016;25:4256-4265.

fig.2

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fig.3

M. Gigante et al. Clin Genet, (2017).

M. Cassina et al. Eur Hum Genet 25, 71- 75 (2017).

A. etro et al. Eur Hum Genet, (2017).

D. Borgia et al. Hum Mol Genet, (2017).

D. ubero et al. Mitochondrion 0, 51-58 (2016).

H. Salah et al. Sci Transl Med 8, 50ra10 (2016).

E. Panza et al. Brain 1 9, e (2016).

M. Doimo et al. IMD Rep 28, 119-126 (2016).

M. A. Desbats et al. Hum Mol Genet 25, 4256-4265 (2016).

M. Cao et al. Neurogenetics 17, 65-70 (2016).

C. Asencio et al. Eur Hum Genet 24, 67- 72 (2016).

C. Fallerini et al. Clin Genet, (2016). . Pinna et al. Eur Hum Genet 2 , 1068-1071 (2015).

I. Paganini et al. Eur Hum Genet 2 , 96 -968 (2015).

E. Trevisson, M. Clementi, L. Salviati, Genet Med 17, 12- 1 (2015).

L. Southgate et al. Circ Cardiovasc Genet 8, 572-581 (2015).

A. uechler et al. Eur Hum Genet 2 , 75 -760 (2015).

G. Giorgi et al. Clin Chem Lab Med 5 , 1719-172 (2015).

M. A. Desbats et al. Eur Hum Genet 2 , 1254-1258 (2015).

E. Trevisson, M. Forzan, L. Salviati, M. Clementi Clin Genet 85, 86- 89 (2014).

M. Spinazzi, A. Sghirlanzoni, L. Salviati, C. Angelini, Neuropathol Appl Neurobiol 40, 888-898 (2014).

T. P. Nguyen et al. Biochim Biophys Acta 1841, 1628-16 8 (2014).

L. Micale et al. Hum Mutat 5, 841-850 (2014).

M. Doimo et al. Biochim Biophys Acta 1842, 1-6 (2014).

D. De Rocco et al. Biochim Biophys Acta 1842, 269-274 (2014).

M. A. Weterman et al. Brain 1 6, 282-29 (201 ).

S. Sacconi et al. Am Hum Genet 9 , 744-751 (201 ).

L. M. Reis et al. Hum Genet 1 2, 761-770 (201 ).

D. . Fernandez-Ayala et al. BM Open , (201 ).

M. Doimo et al. Hum Mutat 4, 229-2 6 (201 ).

S. Cogliati et al. Cell 155, 160-171 (201 ).

D. Cassandrini et al. Inherit Metab Dis 6, 4 -5 (201 ).

S. Ashraf et al. Clin Invest 12 , 5179-5189 (201 ).

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fig.4

fig.5

National and international collaborations:

fig.4 Deranged apoptosis associated with human cytochrome c mutations associated with thrombocytopenia

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Placido Navas, Universidad Pablo De Olavide, Sevilla, Spain

fig.5 Employing a hybrid minigene system to validate CFTR intronic variants

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Michio Hirano, Columbia University, New ork, USA

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Fabien Pierrel, University of University of Grenoble Alpes, France

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Lars Larsson, arolinska Institute, Sweden

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Rafael Artuch, Hospital Sant oan de D u (IRP-HS D), Barcelona, Spain

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Antoni Barrientos, University of Miami, USA

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Orsetta uffardi, Università di Pavia, Italy

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Alessandra Renieri, Università di Siena, Italy

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Marco Seri, Università di Bologna, Italy

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fig.6 The drug BGP-15 rescues mitochondrial complex III and Complex IV activites in diaphragm with ventilation-induced dysfunction Salah et al. Science Translational Medicine 2016

fig.6

5-10 Selected Publications Salah H, Li M, Cacciani N, Gastaldello S, Ogilvie H, Akkad H, Namuduri AV, Morbidoni V, Artemenko KA, Balogh G, MartinezRedondo V, Jannig P, HedstrĂśm Y, Dworkin B, Bergquist J, Ruas J, Vigh L, Salviati L, Larsson L.

The chaperone co-inducer BGP15 alleviates ventilation-induced diaphragm dysfunction.

Sci Transl Med. 2016 Aug 3;8(350):350ra103. doi: 10.1126/scitranslmed.aaf7099.

Emma F, Montini G, Parikh SM, Salviati L.

Mitochondrial dysfunction in inherited renal disease and acute kidney injury.

Nat Rev Nephrol. 2016 May;12(5):267-80

Spinazzi M, Casarin A, Pertegato V, Salviati L, Angelini C. (co-senior author)

Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells.

Nat Protoc. 2012 May 31;7(6):1235-46

Salviati L, Trevisson E, Rodriguez Hernandez MA, Casarin A, Pertegato V, Doimo M, Cassina M, Haploinsufficiency of COQ4 Agosto C, Desbats MA, Sartori G, causes coenzyme Q10 deficiency.

Sacconi S, Memo L, Zuffardi O, Artuch R, Quinzii C, Dimauro S, Hirano M, Santos-OcaĂąa C, Navas P.

J Med Genet. 2012 Mar;49(3):187-91

Montini G, Malaventura C, Salviati L.

Early coenzyme Q10 supplementation in primary coenzyme Q10 deficiency.

N Engl J Med. 2008 Jun 26;358(26):2849-50.

Cassina M, Cerqua C, Rossi S, Salviati L, Martini A, Clementi M, Trevisson E.

A synonymous splicing mutation in the SF3B4 gene segregates in a family with highly variable Nager syndrome.

Eur J Hum Genet. 2017 Feb;25(3):371-375

Desbats MA, Morbidoni V, SilicBenussi M, Doimo M, Ciminale V, Cassina M, Sacconi S, Hirano M, Basso G, Pierrel F, Navas P, Salviati L, Trevisson E.

The COQ2 genotype predicts the severity of coenzyme Q10 deficiency.

Hum Mol Genet. 2016 Oct 1;25(19):4256-4265

Trevisson E, DiMauro S, Navas P, Salviati L.

Coenzyme Q deficiency in muscle. Curr Opin Neurol. 2011 Oct;24(5):449-56

Trevisson E, Burlina A, Doimo M, Pertegato V, Casarin A, Cesaro L, Navas P, Basso G, Sartori G, Salviati L.

Functional complementation in yeast allows molecular characterization of missense argininosuccinate lyase mutations.

Trevisson E, Salviati L, Baldoin MC, Toldo I, Casarin A, Sacconi S, Cesaro L, Basso G, Burlina AB.

Argininosuccinate lyase deficiency: mutational spectrum in Hum Mutat. 2007 Jul;28(7):694-702 Italian patients and identification of a novel ASL pseudogene.

J Biol Chem. 2009 Oct 16;284(42):28926-34

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CV Leonardo Salviati

CV Eva Trevisson

MD degree 1995 University of Padova Residency in Pediatrics 1999 University of Padova PhD in developmental biology 2004 University of Padova Post-doctoral research fellow, 1999-2002 Dept of Neurology Columbia University, New York USA 2005-2014 Assistant professor of medical genetics University of Padova 2014- Associate Professor of Medical Genetics

MD degree 2004 University of Padova PhD in developmental biology 2009 University of Padova Residency in Medical genetics 2012-2016 University of Siena Post-doctoral research fellow, 2008-2009 Universidad Pablo De Olavide Sevilla 2011-Assistant professor of medical genetics University of Padova

111 peer-reviewed articles, Total IF 612.7 (ISI), Hindex 32 (Scopus)

46 peer-reviewed articles, Total IF 180 (ISI), Hindex 16 (Scopus)

Leonardo Salviati

Eva Trevisson

5 Publications as first/last author 2016 23.7 IF as first/last author 2016 32 H-index (source SCOPUS)

4 Publications as first/last author 2016 16.9 IF as first/last author 2016 16 H-index (source SCOPUS)

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Diagnosis and Therapy of Lysosomal Disorders GROUP LEADERS

Rosella Tomanin

GROUP MEMBERS

PI: Maurizio Scarpa PhD Fellows: Francesca DĂ­ Avanzo Laura Rigon Alessandra Zanetti Elisa Legnini Collaborator: Marika Salvalaio PhD Students: Angelica Rampazzo Stefania Bellesso Master Degree Student: Concetta De Filippis

Field of interest

The Laboratory of Diagnosis and Therapy of Lysosomal Disorders performs both diagnostic and research activities.

For diagnostic purposes, the DTML Lab performs some biochemical and molecular analyses related to Mucopolysaccharidoses. The Lab has recently set-up a targeted sequencing panel for the contemporary molecular analysis of about 50 genes related to Lysosomal Storage Disorders (LSDs).

Most of the Lab work consists of research activity related to LSDs. Main Thematic Areas. Lysosomal Storage Disorders are a group of neurometabolic syndromes, mainly affecting children. All LSDs are monogenic disorders, genetically and biochemically welldefined and, although individually rare, they present an overall incidence, in the general population, of about 1:4000-1:7.000 newborns. They are all transmitted as autosomic recessive disorders, except three, presenting an X-linked inheritance.

Each disorder is due to the deficit of one of the numerous lysosomal hydrolases (almost 70 different ones) deputed to the progressive degradation of macromolecules inside lysosomes.

Such deficit of enzyme activity takes to a progressive storage of undegraded, or partially degraded, molecules in the lysosomes and in the extracellular matrix. Due to housekeeping genes, LSD generally affect all organ-systems, including brain in the severe forms. Among LSDs, we have mainly focused on Mucopolysaccahridoses (MPS), a cluster of disorders due to the deficit of the enzymes deputed to the degradation of mucopolysaccharides or glycosaminoglycans; more than 70% of these patients present an important cognitive impairment and significant behavioral problems. Although still not curable, in the last decade some MPS have taken advantage by the availability of the recombinant functional enzymes, infused weekly or biweekly, as Enzyme Replacement Therapy (ERT).

The treatment, which has shown so far some peripheral efficacy,

17

fig.1

unfortunately does not show any efficacy on the CNS disease, due to the inability of the enzymes to cross the blood-brain barrier (BBB).

In the last 20 years, our group has been mainly working on innovative therapeutic approaches for these disorders and, in particular, we are committed to target the brain disease, by developing and testing alternative therapeutic strategies. Research activity also addresses the understanding of MPS pathophysiology, both systemic and of the neurological involvement and is conducted both in vitro, in primary cells obtained from patients, and in vivo, in the mouse models of the diseases. In addition, the bioinformatics activity represents a relevant interest of our Lab.

Summary of recent research activities and of main results achieved

Main Research Programs Lab work mainly focuses on Mucopolysaccharidoses. MPSs are characterized by the inability to degrade one or more mucopolysaccharides. Due to the deficit of lysosomal enzymes coded by housekeeping genes, MPSs generally affect most, if not all, organ-systems including brain in more than 70 of the cases. Enzyme supplementation, progressively developed and applied in the last 10-15 years to 4 different MPSs (MPS I, MPS II, MPS I A and MPS I) could not be of any help for the diffused brain disease affecting these children, due to the inability of the recombinant enzymes to cross the blood-brain barrier (BBB). This is due to their high molecular weight, poor liposolubility and absence of mechanisms for active entry into the brain. Alternative ways to reach the brain compartment have been tested in recent years, including invasive techniques based on neurosurgery or temporary chemico-physical disruption of the barrier. However, these approaches entail several drawbacks as the invasiveness, the high costs of neurosurgery, the physiological stress or the transient increase of the intracranial pressure, the high risk of infections and damages from toxins, due to the BBB temporary opening. Therefore, to improve drug delivery to the brain, non-invasive techniques have been explored in the last years, a couple of which have been tested by our group. Moreover, since little is known on MPS pathophysiology and, in particular, in their brain pathogenesis, part of our efforts was dedicated to this issue, since we believe that the achievement of more detailed information on this side would be of great help to address new therapeutic targets and to identify innovative therapeutic approaches.

Finally, our group is involved in a multi-dipartimental Strategic Project of the University of Padova, centered on Personal Genomics. Within this project, we have collaboratively conducted a research aimed at understanding the lack of genotype-phenotype correlation commonly evidenced in MPSs. We have also set-up a diagnostic panel for a targeted resequencing approach of Lysosomal Storage Disorders.

Main results obtained 1) Pediatric Neurodegenerative Disorders: targeting brain disease in MPS II by a nanoparticle-mediated drug delivery system. FUNDED, still ongoing

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fig.2

In the last four years, the Laboratory has been largely involved in the evaluation and application, in vitro and in vivo, of a Nanoparticlebased approach, functionalized to reach drug brain-targeting, as a potential non-invasive therapeutic approach for MPS brain disease.

PLGA NPs, engineered with a glycopeptide were first tested for their ability to deliver high molecular weight molecules across the blood-brain barrier. Successful experiments, using Albumin as a model drug [Salvalaio et al, 2016, PLoS One, 26;11(5):e0156452.] were followed by short-medium term experiments conducted by using NPs loaded with the functional therapeutic enzyme. Results obtained as well as further improvements of the delivery systems are under analysis.

fig.1 Primary Mucopolysaccharidosis Type II fibroblasts. ysosomes immunostained with ysotracker (red) and AMP-2 (green). Cell nuclei stained with DAPI (blue). aboratory data, unpublished

fig.2 Correction of enzyme deficit in pathological cells. Tomanin et al, 2012 Acta Pediatrica

2) Understanding brain pathophysiology in the MPS II mouse model through a transcriptomic analysis. Closing We have recently concluded a very complex project, focused on the comprehension of the Mucopolysaccharidosis type II brain pathophysiology. The project consisted in the analysis of 2 different brain areas by the means of whole RNA sequencing. Genes known to be involved in several neurological functions showed a significant differential expression in the animal model for the disease compared to wild type. Application of RNA sequencing to dissect pathogenic alterations of complex syndromes allows to photograph perturbations, both determining and determined by these disorders, which could simultaneously occur in several metabolic and biochemical pathways. Results also emphasize the common, altered pathways between neurodegenerative disorders affecting elderly and those associated with pediatric diseases of genetic origin (Salvalaio et al, 2017 submitted).

3) Global approach to Mucopolysaccharidoses: application of highly specific methods for neonatal diagnosis and evaluation of therapeutic efficacy in MPS patients and in animal models.

FUNDED, closing This was a very extended, multicenter national project (PRIN) in which several biochemical and mass spectrometry techniques were applied to the follow-up of MPS patients under Enzyme Replacement

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fig.3

Therapy or under auto-medication with the phytoestrogen genistein.

Contemporarily, a parallel study was conducted by our group in 2 mouse models, for MPS I and MPS II. Preliminary results so far obtained showed the possibility to set-up a rapid diagnostic testing procedure for glycosaminoglycans detection in urine, to be applied independently of the age of subjects and rapidly applicable to the newborn diagnosis without necessity to have creatinine levels Maccari et al, 2016, Clin Chim Acta; 46 :67-72 . As the project is closing now, most of the data obtained in the course of the project development are still being analyzed.

4) Bioinformatics for Personal Genomics, set-up of new analytical systems for genomic/exomic data and criteria of prioritization.

FUNDED, still ongoing In particular, our research, conducted in close collaboration with the Dept. of Biology and the Biotechnology Centre (CRIBI) of the University of Padova, aimed at identifying potential gene modifiers, possibly to define a genotype-phenotype correlation for MPSs, which is commonly hardly reachable. In particular, we have focused on MPS VI, and the analysis, conducted through the application of several “omics” techniques, was performed in a consanguineous population. Within the same project, we have set-up and validated a gene panel for the contemporary molecular diagnosis of about 50 LSDs and we are at the moment completing its validation (manuscripts related to both issues are in preparation).

National and international collaborations:

The Laboratory of Diagnosis and Therapy of Lysosomal Disorders, in about 25 years’ experience has developed an extended network of collaborations. At the moment, the group has several open collaborations with Institutions, Associations and Companies, including the following:

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Department of Biology and Centre for Biotechnology (CRIBI), University of Padova, Italy (Prof. Giorgio Valle) 20

fig.3 P GA Nanoparticles, about 200 nm diameter. Image taken by using a Scanning Electron Microscope (SEM).

Salvalaio et al, 2016; Plos One 11(5):e0156452

fig.4 Representative confocal images of the g -NPs Alb perinuclear localization in the brain of Idua-ko mice in ected with g NPs Alb. Albumin is shown in green (FITC labeling), (b) NPs in red (rhodamine labeling), (c) nuclei in blue (DAPI staining); (d) represents the merged images. Salvalaio et al, 2016; Plos One 11(5):e0156452

fig.4

10 Selected Publications 2014-2016 Targeted Polymeric Nanoparticles for Brain Delivery of High Molecular Weight Molecules in Lysosomal Storage Disorders

M. Salvalaio, L. Rigon, D. Belletti, F. D'Avanzo, F. Pederzoli, PLoS One, 2016, B. Ruozi, O. Marin, M.A. Vandelli, F. Forni, M. Scarpa. R. 26;11(5):e0156452.

Tomanin, G. Tosi

Glial degeneration with oxidative damage drives neuronal demise in MPS II disease.

Cristina Zalfa, Chiara Verpelli, Francesca D’Avanzo, Rosella Tomanin, Cinzia Vicidomini, Laura Cajola, Renzo Manara, Maurizio Scarpa, Angelo Luigi Vescovi, Lidia De Filippis

Cell Death & Disease, 2016, Dec 1;463:67-72

Neuronopathic lysosomal storage disorders: Approaches to treat the central nervous system.

Scarpa M, Bellettato CM, Lampe C, Begley DJ.

Best Pract Res Clin Endocrinol Metab. 2015 Mar;29(2):159-71

lucocere rosidase deficiency in ze rafis affects primary one ossification t rou increased oxidative stress and reduced nt catenin si nalin .

Zancan I, Bellesso S, Costa R, Salvalaio M, Stroppiano M, Hammond C, Argenton, Filocamo M, Moro E.

Hum Mol Genet. 2015 Mar 1;24(5):1280-94

A Phase 3 Trial of Sebelipase Alfa in Lysosomal Acid Lipase eficiency.

urton , al ani M, eillet , ari I, urro , Camarena Grande C, Coker M, Consuelo-Sánchez A, Deegan P, Di Rocco M, Enns GM, Erbe R, Ezgu F, Ficicioglu C, Furuya KN, Kane J, Laukaitis C, Mengel E, Neilan EG, Nightingale S, Peters H, Scarpa M, Schwab KO, Smolka V, Valayannopoulos V, Wood M, Goodman Z, Yang Y, Eckert S, Rojas-Caro S, Quinn AG.

N Engl J Med. 2015 Sep 10;373(11):1010-20

Clinical efficacy of nzyme Replacement Therapy in paediatric Hunter patients, an independent study of 3.5 years.

Tomanin R, Zanetti A, D Avanzo F, Rampazzo A, Gasparotto N, Parini R, Pascarella A, Concolino D, Procopio E, Fiumara A, Borgo A, Frigo A, Scarpa M.

Orphanet J of Rare Dis. 2014, Sep 18;9(1):129

A Hunter Patient with a Severe Phenotype Reveals Two Large Deletions and Two Duplications Extending 1.2 Mb Distally to IDS Locus.

Zanetti A, Tomanin R, Rampazzo A, Rigon C, Gasparotto N, Journal of Inherited Cassina M, Clementi M, Scarpa M. Metabolic Disease Rep. 2014;17:13-21

Intrathecal delivery of protein therapeutics to the brain: a critical reassessment.

Calias P, Banks WA, Begley D, Scarpa M, Dickson P.

Pharmacol Ther. 2014 Nov;144(2):114-22

Molecular Analysis of Turkish Zanetti A, Onenli-Mungan N, Elcioglu N, Ozbek MN, Kör Maroteaux-Lamy Patients and D, Lenzini E, Scarpa M, Tomanin R. Identification of One o el Mutation in the Arylsulfatase B (ARSB) Gene.

Journal of Inherited Metabolic Disease Rep. 2014;14:1-9

A column-switching HPLC-MS/MS method for mucopolysaccharidosis type I analysis in a multiplex assay for the simultaneous newborn screening of six lysosomal storage disorders.

Biomed Chromatogr.

2014 Aug;28(8):1131-9

Gucciardi A, Legnini E, Di Gangi IM, Corbetta C, Tomanin R, Scarpa M, Giordano G.

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• • • • • • • • • • • • •

Department of Molecular Medicine, University of Padova, Italy (Prof. Enrico Moro) Department of Biomedical Sciences, University of Padova, Italy (Prof. Oriano Marin) PCare Laboratory, Department of Women’s and Children’s Health, University of Padova, Italy (Prof.

Paola Cogo, Dr.ssa Manuela Simonato, Dr. Luca edovelli) Laboratory of Molecular Biology of the idney, Department of Women’s and Children’s Health, University of Padova, Italy (Dr. Susanna Negrisolo) Laboratory of Mass Spectrometry, Department of Women’s and Children’s Health, University of Padova, Italy (Dr. Giuseppe Giordano) Laboratory of Pharmaceutical Technology, Life Science Department, University of Modena and Reggio Emilia, Modena, Italy (Prof. Giovanni Tosi) Laboratory of Molecular Biology, Department of Neurosciences – Meyer Children Hospital, University of Firenze, Italy (Prof. Amelia Morrone) Laboratory of Biochemistry and Glicobiology, Life Science Department, University of Modena and Reggio Emilia, Modena, Italy (Prof. Nicola olpi) Division of Pediatrics, Department of Clinical Sciences, Università Politecnica delle Marche, Ancona (Prof. Orazio Gabrielli) Centro di Diagnostica Genetica e Biochimica delle Malattie Metaboliche, Istituto Giannina Gaslini, Genova, Italy (Dr.ssa Mirella Filocamo).

Brains for Brain” Foundation, Onlus, Padova, Italy BiOasis Technologies Inc., Richmond, BC, Canada BioMarin Pharmaceutical, Novato, CA, USA

CV Rosella Tomanin Dr. Rosella Tomanin has a Master Degree in Biological Sciences, and she received her PhD in Pediatrics in 1997, both at the University of Padova (Italy). She has a specialization in Medical Genetics, received from the University of Ferrara (Italy).

Dr. Tomanin has a laboratory experience of more than 30 years, four of which were spent at McMaster University (Hamilton, Ontario, Canada), as a Post-doc in Molecular Biology and as a Visiting Scientist. Rosella Tomanin is presently Head of the Laboratory of Diagnosis and Therapy of Lysosomal Disorders at the Department of Women’s and Children’s Health of the University of Padova. She published about 50 papers in international journals and she co-authored four book chapters. Her fields of interest are all related to rare diseases, in particular Lysosomal Stora e isorders and Mucopolysacc aridoses, investigating innovative therapeutic strategies, mainly brain-targeted drug delivery systems, and performing studies on LSD pathogenesis, through the application of biochemical, molecular and NGS techniques.

Rossella Tomanin 3 Publications 2016 11.41 IF 2016 18 H-index (source RG)

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Mass Spectrometry and Metabolomics GROUP LEADERS

Field of interest Metabolomics: A New Frontier for Research in Pediatrics

Eugenio Baraldi Giuseppe Giordano

GROUP MEMBERS

PI: Silvia Carraro Post Doctoral Fellows: Antonina Gucciardi Paola Pirillo Elena Donazzolo Bioinformatician: Matteo Stocchero Technician: Mauro Naturale

Metabolomics is the most recent of the “omic� sciences.

Metabolomics can be defined as the quantitative analysis of all the metabolites (small molecules 1 kDa) of a biological sample aiming to the investigation of the multiparametric metabolic response of a living system to pathophysiological stimuli or genetic modifications. A metabolic profile consists of the set of metabolites reflecting enzyme expression and activity, and includes the building blocks and breakdown products of the DNA, RNA, proteins, and cellular components. Also, it is affected by several factors unrelated to the genome, such as interactions with commensal microorganisms, nutritional factors, environmental agents, and any exposure to drugs or toxic substances resulting in discordance between genotype and phenotype.

In many fields of medicine, there is a growing interest in characterizing diseases at molecular level with a view to developing an individually tailored therapeutic approach. Metabolomics is a novel area that promises to contribute significantly to the characterization of various disease phenotypes and to the identification of personal metabolic features that can predict response to therapies.

Based on analytical platforms such as mass spectrometry or NMR spectroscopy, the metabolomics approach enables a comprehensive overview of the metabolites, leading to the characterization of the metabolic fingerprint of a given sample.

These metabolic fingerprints can then be used to distinguish between different disease phenotypes and to predict a drug’s effectiveness and/or toxicity. Several studies published in the last few years applied the metabolomic approach in the field of

23

pediatric medicine. Being a highly informative technique that can be used on samples collected non-invasively (e.g. urine or exhaled breath condensate), metabolomics has appeal for the study of pediatric diseases.

Summary of recent research activities and of main results achieved Analysis of Amniotic Fluid in the Prediction of Preterm Delivery and Bronchopulmonary Dysplasia Preterm delivery (PTD) is a major challenge in the field of obstetrics and neonatology. Since 2006 preterm birth rates have been declining both in the United States and in European countries. Nevertheless, prematurity remains a major cause of morbidity and mortality worldwide, which exceed those of infants born full-term. Preterm neonates are at increased risk of both short- and long-term pathological outcomes and, among these, bronchopulmonary dysplasia (BPD) accounts for the vast majority of cases of chronic lung disease after premature birth. Metabolomics allows simultaneous characterization of low molecular weight compounds and may provide a picture of such a complex condition. The aim of this study was to evaluate whether an unbiased metabolomic analysis of amniotic fluid can be used to investigate the risk of spontaneous PTD and BPD development in the offspring. This study suggests that amniotic fluid metabolic profiling may be promising for identifying spontaneous preterm birth and fetuses at risk for developing BPD. These findings support the hypothesis that some prenatal metabolic dysregulations may play a key role in the pathogenesis of PTD and the development of BPD.

Metabolic perturbations in children with type 1 diabetes Despite a considerable reduction in diabetes-related complications, including end-stage renal disease, over the past 0 years, individuals with type 1 diabetes (T1D) still exhibit a nearly -fold excess mortality as compared with the general population without diabetes, cardiovascular diseases being the leading cause of death in young adults with T1D. While the incidence of T1D is steeply increasing, the age at onset is progressively lowering. An early onset of the disease has been associated to marked morphological alterations of brain morphology and growth. The mechanisms underlying such alterations still remain unexplained. We enrolled children with T1D aged 6-15 years, attending the pediatric diabetes clinic of University of Padova (Italy), and healthy peers to investigate the differences in the urine metabolome and to explore the contribution of HbA1c and clinical features to the observed differences. We performed a liquid chromatography and mass spectrometry analysis (LC-MS) on fasting urinary samples of the 2 groups. We identified 59 urinary metabolites having a higher level in children with T1D, mainly represented by gluco- and mineralcorticoids, phenylalanine and tryptophan catabolites (kynurenine), small peptides, 24

glycerophospholipids, fatty acids, and gut bacterial products. We did not find any association between HbA1c, pubertal status, disease duration, and metabolome profile within the case group.

T1D profoundly disrupts the metabolome of pediatric patients.

The excess of cortisol and aldosterone may contribute to the development of macrovascular complications in adulthood, while the increase of tryptophan derivates may have a role in neuronal damage associated to hyperglycemia. Determinants of such findings, other than HbA1c, should be explored.

Inborn errors of bile acid synthesis are rare genetic disorders of liver metabolism that cause chronic liver diseases Inborn errors of bile acid synthesis are rare genetic disorders of liver metabolism that cause chronic liver diseases, fat malabsorption, and fat-soluble vitamin deficiency during childhood. These defects, due to a defective functioning of enzymes, are characterized by a failure to produce normal bile acids (BAs) and an accumulation of unusual BAs and BAs intermediates. BAs are potent digestive surfactants that promote the absorption of cholesterol, lipids and fat-soluble vitamins acting as emulsifiers.

They provide the primary driving force for the promotion and secretion of bile and are essential for the development of the biliary excretory route for the elimination of endogenous and exogenous toxic substances, including bilirubin, xenobiotics, and drug metabolites. Inborn errors in the BA biosynthetic pathways include cerebrotendinous xanthomatosis (CT , sterol 27-hydroxylase deficiency), -hydroxysteroid- 5-C27-steroid dehydrogenase deficiency (HSD B7), D4- -oxosteroid-5 -reductase deficiency, oxysterol 7 -hydroxylase deficiency, cholesterol 7 -hydroxylase deficiency, -methylacyl-CoA racemase (AMACR), peroxisomal -oxidation, and amino acid N-acyltransferase. The most useful screening test is the analysis of urinary bile acids and bile alcohols by flow injection analysis (FIA) electrospray ionization-tandem mass spectrometry (ESI–MS/MS). The screening procedures 25

indicate that inborn errors of BA metabolism probably account for 1 to 2 of the cases of liver disease in infants, children, and adolescents, making this an important and specific category of metabolic liver disease. Early diagnosis of inborn errors of BAs synthesis is important because if the disorder remains untreated, progressive liver disease, together with neurologic disease, may develop and lead to death or require liver transplantation. The treatment of these defects is based on the oral administration of primary bile acids as cholic acid and chenodeoxycholic acid. However, their use was associated with an increased risk of serious possible adverse events, and treatment needs to be accurately monitored.

5-10 Selected Publications Metabolomics- a new frontier for pediatric research

Carraro S, Giordano G, Reniero F, Perilongo G, Baraldi E

J Pediatr 2009;154: 638-644

Childhood asthma biomarkers: Moschino L, Zanconato S, Bozzetto S, Baraldi E, Carraro S present knowledge and future steps

Paediatr Respir Rev 2015;16(4): 205-12

Untargeted metabolomic analysis of amniotic uid in t ep of preterm delivery and bronchopulmonary dysplasia

Baraldi E, Giordano G, Stocchero M, Moschino L, Zaramella P, Tran MR, Carraro S, Romero R, Gervasi MT

PLoS One 2016 Oct 18;11(10):e0164211

Airway metabolic anomalies in adolescents with bronchopulmonary dysplasia: newi from the metabolomic approach

Carraro S, Giordano G, Pirillo P, Maretti M, Reniero F, Cogo P, Perilongo G, Stocchero M, Baraldi E

J Pediatr 2015 Feb;166(2):234-9.e1

Asthma severity in childhood and meta olomic profilin of reat condensate

Carraro S, Giordano G, Reniero F, Carpi D, Stocchero M, Sterk PJ, Baraldi E

Allergy 2013;68(1):110117

Metabolomics reveals new metabolic perturbations in children with type 1 diabetes

Galderisi A, Pirillo P, Moret V, Stocchero M, Gucciardi A, Perilongo G, Moretti C, Monciotti C, Giordano G, Baraldi E.

Pediatr Diabetes 2017 Apr; doi: 10.1111/ pedi.12524

Analysis and interpretation of acylcarnitine profiles in dried lood spot and plasma of preterm and full-term newborns.

Gucciardi A, Zaramella P, Costa I, Pirillo P, Nardo D, Naturale M, Chiandetti L, Giordano G.

Pediatr Res 2015;77(11):36-47

Metabolomic approach to the clinical diagnosis of cholestasis

Giordano G, Gucciardi A, Di Gangi IM, Pirillo P, Costa I, Donazzolo E, Naturale M, Carraro S, Baraldi E, Reniero F.

Ligand Assay 2012;17 (1): 25-32

Improved synthesis of glycine, taurine and sulfate conjugated bile acids as reference compounds and internal standards for ESI-MS/MS urinary profilin of in orn errors of bile acid synthesis.

Donazzolo E, Gucciardi A, Mazzier D, Peggion C, Pirillo P, Naturale M, Moretto A, Giordano G.

Chem Phys Lipids 2017

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CV Eugenio Baraldi

CV Giuseppe Giordano

Qualifications and or e perience: Director Neonatology-Neonatal Intensive Care Unit(2014-).

Director, School of Pediatrics, University of Padova (2012-2016) Director Master in Neonatology and Neonatal Intensive Care-University of Padova (2015-) President of the “Italian Society of Pediatric Respiratory Diseases” (2010-2014) Professor of Pediatrics (2010) Associate Professor of Pediatrics (2005) “Research Fellow” Harbor UCLA (USA), Department of Pediatrics (1990) Specialization in Allergy and Immunology (1990) Specializations in Pediatrics (1986) M.D. Degree University of Padova (1982) Research activities, editorial boards and reviewer activities: More than 180 full papers published in international ournals H inde , total I 10 0 including NEJM, Lancet, JAMA and AIRCCM Included in the list of “Top Italian Scientist ” (www.topitalianscientists.org) Coordinator of the project “Metabolomics in Pediatrics” Coordinator for the respiratory follow up of a cohort of children with bronchopulmonary dysplasia, since 1990 (JAMA 2009;302:1418-20) ditorial oard mem er: ora , ediatric Pulmonology Reviewer of research programs for: the Italian Ministry for the University and Research (MIUR) and the Guidance Committee for the Assessment of Research (CIVR), Netherlands Asthma Foundation, The Asthma Foundation of Western Australia Teaching activities, conferences Professor at the Pediatric School of the University of Padova since 1990.

Invited Speaker at more than 200 national and international conferences.

Qualifications and or e perience: Head of the Mass Spectrometry and Metabolomics unit at the Women’s and Children’s Health Department, University of Padova rom 00 010 e as Seconded ational pert oint esearc Center of the European Commission; Institute for Health & Consumer rotection IHC ysical C emical posure nit C IS (VA), Italy Head of the Mass Spectrometry Lab at the Women’s and Children’s Health Department he works on: Biochemical diagnosis of Inborn rror of Meta olism acylcarnitines and amino acids profilin of blood spots using ESI-MS/MS. Postmortem diagnosis of fatty acid o idation disorders. ioc emical screenin in urine of disorders of bile acids metabolism by ESI-MS/MS. Measurement of palmitate and linoleate turnover in critically ill infants by monitoring stable isotope labeled tracers in vivo. by GC/MS and GC-(combustion) (pirolysis) -IRMS for the elements 13C, 15N2, 2H2. Measurement of acylcarnitine meta olism in fi ro lasts, y monitorin sta le isotope la eled tracers in itro, y t e used of H LC SI MS MS. O idati e stress, y LC MS MS e aled nitrotyrosine on ast matic c ildren He has been involved in research projects for the application of metabolomics approach by NMR and mass spectrometry: Education Research Doctorate Degree in Developmental Sciences (1990) Doctoral Degree in Biological Sciences, (1984) Teaching activities 2011 Appointed as Professor. Master in methodology and application of mass spectrometry in clinical chemistry. Faculty of Sciences Catania University, Italy 2013 Appointed as Professor. Master in methodology and application of mass spectrometry in clinical chemistry. Faculty of Sciences Catania University, Italy Membership of Research Network 2000-2016 SIMMESN, Italian Society for the Study of Hereditary Metabolic Diseases and Neonatal Screening. (founder member and from 2004 to 2010 Board member) 2006-2016 Member of the Metabolomics Society 2013-2016 Member of the IMaSS Italian Mass Spectrometry Society (founder member and Board member from 2013-2015) Or anization of scientific meetin s 2014 Workshop on “The Metabolomics Approaches: Advanced Analytical Tools for Future, challenges, perspectives and applications . In c ar e of t e Scientific Committee, Italy.

2016 Workshop on “Wandering across pediatric cholestatic liver disease: focus on bile acid synthesis defects”. In charge of the Scientific Committee, Italy

27

fig.5

National and international collaborations:

fig.5 xxxxxx xxx xx xxxxxx xxxxx xxx xxxx xx xxx xx xx x x x x xxxx x

Prof. Roberto Romero, Perinatology Research Branch, NICHD, NIH, DHHS, Wayne State University/Hutzel Women’s Hospital, Detroit, United States of America Dr. Louis Bont, University Medical Center Utrecht, Department of Wilhelmina Childrens Hospital Prof. Hans Bisgaard, Faculty of Health and Medical Science, University of Copenaghen Prof. Piero Rinaldo, Hereditary Metabolic Diseases and Neonatal Screening, Mayo Clinic, Rochester, MN, USA

Eugenio Baraldi 6 Publications as first/last author 2016 15.66 IF as first/last author 2016 45 H-index (source )

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Giuseppe Giordano 5 Publications 2016 1 .11 IF 2016 21 H-index (source )

Nano Inspired Biomedicine

GROUP LEADERS

Marco Agostini GROUP MEMBERS

Field of interest

Dr.Agostini’s research group program focuses on the study of genetic, molecular and metabolic characterization of cancer, genetic pathways involved in the progression and outcome of colon and rectal cancer, and drug delivery system modulation.

In collaboration with researchers of The Methodist Hospital Research Institute (TMHRI, Houston, TX, USA), he is developing nanotechnologic applications to enhance the proteomic profiling for tumor biomarkers discovery and drug delivery system modulation. His aims are to improve cancer detection by identifying specific cancer biomarkers and the timing of their interrelated functional molecular contributions to cancer progression.

Summary of recent research activities and of main results achieved

The research group activities are supported by four major grant funded by CARIPARO and AIRC foundation: Senior Affiliate: Pietro Traldi Post Doctoral Fellows: Chiara Bedin Sara Crotti Sara Dí Aronco PhD Students: Edoardo Dí Angelo Francesca Sensi Students: Andrea Dallí Ora

Innovative tools for therapeutic drug monitoring in cancer patients: toward bed side testing. (CARIPARO Grant Program For Young Investigator on Pediatric Research-2013) We have organized the interactive network: research-clinical groups (Agostini’s group-IRP Proteomics facility-CNR-ISTM, Padova and CRO Aviano, Italy) and Science & Technologyoriented research groups (Agostini’ group-IRP, Padova, Italy, and Prof. Ennio Tasciotti’s team-TMHRI, Houston, TX, USA).

Our working hypothesis is that the availability of new and innovative method for the selection and harvest of low molecular weight compounds such as drugs and their metabolites can expand the potential of LC-MS/MS and MALDI-TOF analytical approaches.

The improvement in term of sensitivity and time analysis using 29

fig.1

small biological samples size offered by this new devices can improve the application of TDM platforms in clinical paediatric setting.

Application of advanced nanotechnology in the development of cancer diagnostics tools. (AIRC Multi-Unit 5X1000) We contribute in this multi-unit project performing the cross-validation of previously developed MALDI-TOF method for the quantification of Irinotecan in human plasma samples in collaboration with the Operative Unit 1 (Prof. Giuseppe Toffoli, CRO-Aviano, Italy) by comparing the their results obtained with the LC-MS/MS. Moreover, we would to evaluate and validate new faster analytical methods based on ambient ionization mass spectrometry for the quantification of analytes of interest (e.g. paclitaxel, sunitinib or irinotecan).

Immunomodulation: its unexplored role in the response prediction for the rectal cancer treatment. (AIRC Investigator Grant-2016) We would like to perform a study in an unique cohort of rectal cancer patients, undergoes to standardized pre-surgery chemoradiotherapy (pCRT) protocol, to go deeper on the process related to the changes in biomarkers levels that we previously detected. Predictive markers will be compared with some know index of immune system activation and inflammation (e.g. white blood cell count, erythrocyte sedimentation rate, C-reactive protein, and soluble fraction of TNF-alpha receptors 1 and 2). The final objective is the prediction, or eventually the monitoring, of patients response to the pCRT through a simple blood sampling.

A proteomic approach for the optimization of personalized bio-mimetic nanoparticles (BNV). (CARIPARO Bando Ricerca Pediatrica 2016-2018) The project aim is the creation of an innovative delivery system for the targeting of inflamed vessels to establish a novel class of therapeutics able to accumulate into target tissues and selectively deliver a wide range of therapeutics. We will riformulate the necessary research to be performed to establish adequate engineering rules to manipulate purified biological materials and properly integrate these findings with synthetic protocols to develop safe, effective, scalable and standardized drug delivery systems.

30

fig.1 Therapeutic Drug Monitoring (TDM) of Imatinib by ambient ionization mass spectrometry.

fig.2 The study of cancer extracellular matrix: tissue engineering applied to oncology.

fig.2

5-10 Selected Publications Diagnostic and prognostic role of cell-free DNA testing for colorectal cancer patients.

Bedin C, Enzo MV, Del Bianco P, Pucciarelli S, Nitti D, Agostini M.

Mass spectrometry in the pharmacokinetic studies of anticancer natural products.

Crotti S, osocco , Maran on , itti , offoli M.

Field-assisted paper spray mass spectrometry for therapeutic drug monitoring: 1.the case of imatinib in plasma.

D'Aronco S, Dall'Armi M, Crotti S, Calandra E, Traldi P, Di Marco , uonadonna , Corona , iodini L, Maran on , osocco , offoli , ostini M.

J Mass Spectrom. 2017 Mar 2. [Epub ahead of print] doi: 10.1002/ jms.3927.

Bottom-Up Synthesis of Carbon Nanoparticles with Higher o oru icin fficacy.

Bayda S, Hadla M, Palazzolo S, Kumar V, Caligiuri I, Ambrosi E, Pontoglio E, Agostini M, Tuccinardi T, Benedetti A, Riello P, Canzonieri , Corona , offoli , izzolio .

J Control Release. 2017 Feb 28;248:144-152.

Extracellular Matrix and Colorectal Crotti S, iccoli M, izzolio , iordano , itti , Cancer: How Surrounding Microen ironment ffects Cancer Cell Behavior?.

,

Int J Cancer. 2017 Apr 15;140(8):1888-1898. ostini

ostini M.

Mass Spectrom Rev. 2017 Mar;36(2):213251.

J Cell Physiol. 2017 May;232(5):967-975.

Altered plasma levels of decanoic acid in colorectal cancer as a new diagnostic biomarker.

Crotti S, noletto , Cancemi , i Marco , raldi , Pucciarelli S, Nitti D, Agostini M.

Anal Bioanal Chem.

2016 Sep;408(23):63218.

Serum miR-125b is a non-invasive predictive biomarker of the preoperative chemoradiotherapy responsiveness in patients with rectal adenocarcinoma.

D'Angelo E, Fassan M, Maretto I, Pucciarelli S, Zanon C, Digito M, Rugge M, Nitti D, Agostini M.

Oncotarget. 2016 May 10;7(19):28647-57.

Enabling cytoplasmic delivery and organelle targeting by surface modification of nanocarriers.

Parodi A, Corbo C, Cevenini A, Molinaro R, Palomba R, andolfi L, ostini M, Sal atore , asciotti .

anomedicine Lond .

2015 Jul;10(12):192340.

A functional biological network centered on XRCC3: a new possible marker of chemoradiotherapy resistance in rectal cancer patients.

Agostini M, Zangrando A, Pastrello C, D’Angelo E, omano , io annoni , iordan M, Maretto I, edin C, Zanon C, i ito M, sposito , Mescoli C, La itrano ML, izzolio , urisica I, iordano , ucciarelli S, itti .

Cancer Biol Ther.

2015;16(8):1160-71.

An integrative approach for the identification of pro nostic and predictive biomarkers in rectal cancer.

ostini M, anssen , im L , n elo , izzini S, Zangrando A, Zanon C, Pastrello C, Maretto I, Digito M, edin C, urisica I, izzolio , iordano , ortoluzzi S, Nitti D, Pucciarelli S.

Oncotarget. 2015 Oct 20;6(32):32561-74.

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CV Marco Agostini, PhD Dr. Agostini earned his B.S. in Biological Sciences (2000) and a Ph.D. in Oncologic and Surgical Sciences (2006) at the University of Padova. He moved to the The Netherlands in 2005 for a postdoctoral fellowship in the Department of Pathology at the Josephine Nefkens Institute in the Erasmus University Medical Center. He accepted an appointment to Assistant Professor in the First Surgery Clinic (Chief Prof. Donato Nitti) Department of Surgery Oncology and astroenterolo ical Sciences at t e ni ersity of ado a in 00 .

Since then, Dr. Agostini has been leading these projects in collaboration with more than 20 other investigators from multiple institutions. He specialized in enetic in 00 at t e ni ersity of adua. He is aut or coaut or of o er pubblications on peer-reviewed journals and he is inventor of the patent: -International Patent WO2014EP58356 20140424 itle: in itro met od for t e dia nosis, pro nosis and treatment of cancer t rou t e analysis in a ody uid sample of the total circulating DNA and the amount of DNA released by non-apoptic cells. (https://worldwide.espacenet.com/publicationDetails/biblio?FT=D&date=20151029&DB=worldwide.espacenet. com&locale=en_EP&CC=WO&NR=2015161880A1&KC=A1&ND=5) -International Patent WO2015145384 itle: Met od for dia nosis of colorectal cancer y determinin t e le el of fatty acids present in a iolo ical uid sample, and apparatus for implementing the method. https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015145384 - National Patent Filing number: 102016000073220 Title: “Strumentazione analitica perfezionata”

National and international collaborations: • • • • • • • •

Department of Pathology at the Josephine Nefkens Institute in the Erasmus University Medical Center, Rotterdam, The Netherland. Prof. Riccardo Fodde (since 2005); Ontario Cancer Institute, IBM Life Sciences Discovery Centre, Toronto, Ontario (CA). Prof. Igor Jurisica e Dr.ssa Chiara Pastrello (since 2013); Department of Nanomedicine, Center for Biomimetic Medicine, The Houston Methodist Research Institute, Houston (TX), USA. Prof. EnnioTasciotti (since 2010); Experimental and Clinical Pharmacology Unit, National Cancer Institute (CRO) Aviano, Italy. Prof.

Giuseppe Toffoli. (since 2006); Department Molecular Science and Nanosystems, University Ca’ Foscari, Venezia, Italy. Dr. Flavio Rizzolio (since 2002); School of Medicine and Surgery, University of Milano Bicocca, Milano, Italy. Dr. Roberto Giovannoni (since 2012); Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padova, Italy.

Prof.ssa Antonella Bertazzo (since 2013); Department of Woman and Child’s Health, University of Padua, Padova, Italy. Dr.ssa Silvia Visentin and Prof. Erich Cosmi (since 2014).

Marco Agostini 5 Publications as first/last author 2016 21 61 IF as first/last author 2016 16 H-index (source )

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Neuroimmunology

GROUP LEADERS

Bruno Giometto GROUP MEMBERS Post≠Doctoral Fellow: De Gaspari Piera Medical Doctors: Rosellini Irene Zoccarato Marco Zuliani Luigi, PhD Scientific secretary: Fleming Joanne

Field of interest

The key clinical and research interest of my team and I is Neuroimmunology with a particular focus on paraneoplastic and autoimmune neurological diseases. Neuroimmunology is the study of the interaction between immunology and the nervous system. A group of encephalopathies has been demonstrated to be mediated by an autoimmune mechanism. These diseases could affect children and adult patients. The recent discovery of autoantibodies associated with these diseases has changed the diagnostic approach.

In patients affected by autoimmune encephalopathies, the body, for partly known reasons, produces antibodies directed against itself, and in particular against the brain, leading to specific neurological syndromes. To date, various antibodies have been described and classified according to their targets as a) nuclear and cytoplasmic antigens; b) intracellular synaptic antigens and c) surface neuronal antigens.

The first category are antibodies directed against the nucleus or cytoplasm of the neuronal cells. These antibodies are also defined as “onconeural antibodies�, because they are associated with paraneoplastic syndromes. In this situation, an extracerebral neoplasm leads to an inflammatory response of the central nervous system (CNS). The most common onconeural antibodies are anti-Hu and anti-Ma2. Anti-Hu antibodies are IgG immunoglobulins that recognize RNA binding proteins, which are expressed in the nucleus of the neurons and in the cells of tumors. The tumor most frequently associated with anti-Hu is small cell lung cancer. Anti-Ma2 antibodies are directed against an antigen belonging to the Ma protein family, involved in mRNA biogenesis. Affected patients are usually males under 40 years of age and the associated tumor is usually testicular or lung cancer. Different studies have demonstrated that onconeural antibodies should be considered specific biomarkers, whereas

33

fig.1

the pathogenic mechanism of disease is due to the activation of cytotoxic T lymphocytes. The same pathogenic mechanism is used by the second category of autoantibodies: the intracellular synaptic antigens. An example of this type of antigen are antibodies directed against glutamate decarboxylase (GAD). Patients with anti-GAD antibodies develop stiff person syndrome, ataxia, encephalitis and epilepsy, usually in a non-paraneoplastic context.

The third category of antibodies, directed against surface antigens (e.g. NMDA, AMPA, and GABAB receptors), were identified more recently. They are called Neuronal Surface Antibodies (NsAbs) and are not always associated with tumors. In this case, the pathogenic mechanism is caused by the antibodies themselves which act directly on neuronal cells. The tumors associated with these disorders are teratoma and thymoma in the majority of the cases.

The main difference between paraneoplastic neurologic syndromes associated with onconeural antibodies and syndromes associated with NsAbs is the ability to respond to therapy. The former, associated with onconeurals, arise subacutely, progress rapidly and respond to oncologic but not to immunological therapy.

Differently, those associated with NsAbs arise acutely or subacutely and respond to immunological therapies (corticosteroids, intravenous immunoglobulin, plasma exchange or immunosuppressor drugs); indeed, antibody removal or suppression by pharmacological therapy positively influences disease progression.

Although different antibodies related to neurological autoimmune syndromes have been identified to date, some patients with clinical features of autoimmune encephalopathies still test negative for onconeural, GAD and NbAbs antibodies (“seronegative patients�). Negativity does not imply the absence of autoantibodies, but it could simply be that the specific antibody causing the syndrome has not yet been identified.

Summary of recent research activities and of main results achieved

My team and I actively work at both diagnostic and research levels in the field of paraneoplastic and autoimmune neurological syndromes. We have acquired specific skills in paraneoplastic and autoimmune neurological syndromes at both national and international level, becoming one of the national reference centers for the diagnosis of this type of neurological disease. Our research has given rise to international collaboration opportunities and to the coordination of two European projects funded by the European Commission. Said projects have led to the collection of a large series of cases with paraneoplastic neurological diseases, autoimmune encephalopathies and to the characterization of autoantibodies directed against neuronal antigens. Young researchers awarded European neurological society grants have in the past taken part in our research activities, enabling them to gain experience and grow as researchers, while paving the way to the assignment of permanent positions at departments of Neurology. The skills

34

fig.2

acquired and the role built at the national level now enable our center to collect sufficient case sets of these diseases, which are rare but can be appropriately studied in our laboratory.

Medical Diagnostic Activities My group is widely recognized at the national level as a reference center for the analysis of anti-neuronal antibodies.

Each year, we receive more than 500 patient samples (both CSF and serum) from all over Italy. The great expertise of my collaborators, acquired over the years, and their collective experience in international laboratories, enable us to accurately diagnose autoimmune neurological syndromes based on the detection of autoantibodies. We actively work in collaboration with the Treviso Hospital Laboratory of Autoimmune Analysis (Dr G. Barberio and. L. Caberlotto) and monthly we guarantee the analysis and diagnosis of samples from roughly 0-40 patients suspected of having autoimmune neurological syndromes. The easiest method to establish the autoimmune etiology of the encephalitis is to check the presence of antibodies in both the serum and cerebrospinal fluid (CSF) of patients.

We can guarantee the accurate detection of onconeural, anti-GAD and NsAbs in serum and CSF by using available commercial kits. Specifically, immunoblotting kits are used to check the presence of onconeural antibodies (e.g. Hu, Ma2, C 2/CRMP5, Ri, amphiphysin), ELISA-immunoenzymatic kits are used for GAD detection, whereas surface antigens are analyzed by indirect immunofluorescence kits.

fig.1 An example of Immuno BLOT used to detect anti-YO antibodies.

The diagnostic test usually performed in the case of onconeural antibodies is immunoblot.

This method is designed to identify antibodies directed against neural recombinant proteins detected in special strips. The antibodies present in the positive samples will bind to the relative antigens, after which an enzymatic reaction will develop a visible color band. In the picture, the presence of a color band is a clear example of patient serum testing positive for anti Yo antibodies.

fig.2 An example of immunofluorescence staining in cells transfected with different NsAbs (e.g. NMDAR, CSPR2, AQP4).

Conversely, NsAbs are detected through indirect immuno uorescence techni ues, in this case using Euroimmun commercial kits.

Cell lines transfected with vectors expressing the antigens involved, are incubated with patient serum and or CSF; in the case of antibody positivity the cell will be uorescent with clear, recognizable patterns. The panel picture above gives an example of the presence of NMDAR, CSPR2 and AQP4 antibodies (in green) in di erent patient sera.

Research Activities: Despite the increasing spectrum of antibody reactivity, some subjects affected by suspected autoimmune encephalopathies (AE) still test negative for onconeural, GAD and NsAbs (i.e. “seronegative� autoimmune encephalitis patients). This negativity does not imply the absence of antibody markers, 35

fig.3

fig.3 IHC optimization of anti-YO, NMDAR, AQP4, CSPR2 antibodies detection.

Fixed rat brain cryosections were incubated with the serum of patients with antibodies directed against Yo, NMDAR, AQP4, CSPR2 antigens.

Immunohistochemistry was performed to detect the antigenantibody reaction. Biotin-labeled IgG were incubated with streptavidin con ugated with HRP. The staining was developed with a brown colorimetric enzymatic reaction using diaminobenzidine (DAB substrate).

Yo antibodies are directed against Purkin e cells present in the cerebellum; NMDAR and CSPR2 antibodies are directed against receptors localized in the hippocampal region of the brain; and AQP4 antibodies are directed against the cell membrane of astrocytes.

36

rather it could simply be that the protein involved has not yet been characterized. Further studies are thus warranted to find new antigens related to the immunological response in this kind of neurological patient. Based on this hypothesis, the current aim of our laboratory is to identify novel antineural biomarkers in these “seronegative” patients with suspected AE. Thanks to our diagnostic activity, dating back to 2005, our laboratory has been collecting sera and/or CSF from all over Italy, belonging to patients defined by the literature as having possible autoimmune encephalopathies. Currently, we are preliminarily screening these samples by performing indirect immunohistochemistry on fixed rat brain cryosections.

This first step will enable us to: a) ensure the presence of autoantibody response in these samples; b) identify recognizable staining patterns; c) define the localization of the possible antibody response in the brain. Although this is the commonly used approach, it does have some limitations.

The sensitivity and positivity of the results rely on laboratory expertise. False positive or doubtful results can often occur, which is why they should be confirmed by other more specific techniques. Accordingly, we are drawing on the expertise of our team to optimize this technique with a view to defining an accurate, standardized protocol to ensure reliable, reproducible results. Our idea is to identify new proteins responsible for the autoimmune response in “seronegative” patients and to seek to investigate and understand the pathway and mechanism by which the antibody activates the immunological response. Furthermore, the clinical information collected in our database represents an important tool not only to characterize the clinical pattern associated with these novel autoantibodies, but also to correlate associated tumors in the case of paraneoplastic encephalitis.

5-10 Selected Publications Godani M, Zoccarato M, Beronio A, Zuliani L, Benedetti L, Giometto B, Del Sette M, Raggio E, Baldi R, Vincent A.

Voltage-Gated Potassium Channel Antibodies in SlowProgression Motor Neuron Disease.

Neurodegener Dis. 2017;17(1):59-62

Waters P, Reindl M, Saiz A, Schanda K, Tuller F, Kral V, Nytrova P, Sobek O, Nielsen HH, Barington T, Lillevang ST, Illes Z, Rentzsch K, Berthele A, Berki T, Granieri L, Bertolotto A, Giometto B, Zuliani L, et al.

Multicentre comparison of a diagnostic assay: aquaporin-4 antibodies in neuromyelitis optica.

J Neurol Neurosurg Psychiatry. 2016 Sep;87(9):1005-15. doi: 10.1136/jnnp-2015312601

Suppiej A, Nosadini M, Zuliani L, Plasma exchange in pediatric anti-NMDAR encephalitis: A Pelizza MF, Toldo I, Bertossi C, Tison systematic review.

T, Zoccarato M, Marson P, Giometto B, Dale RC, Sartori S.

Brain Dev. 2016 Aug;38(7):613-22.

Sabater L, Gaig C, Gelpi E, Bataller L, Lewerenz J, TorresVega E, Contreras A, Giometto B, Compta Y, Embid C, Vilaseca I, Iranzo A, SantamarĂ­a J, Dalmau J, Graus F.

A novel non-rapid-eye movement and rapid-eye-movement parasomnia with sleep breathing disorder associated with antibodies to IgLON5: a case series, characterisation of the antigen, and post-mortem study

Lancet Neurol. 2014, Jun;13(6):575-86. Epub 2014 Apr 3. 32 IF 23.468

Zuliani L, Graus F, Giometto B, Bien C, Vincent A.

Central nervous system neuronal surface antibody associated syndromes: review and guidelines for recognition

J Neurol Neurosurg Psychiatry. 2012 83:638-45. Epub 2012 Mar 24 134

Giometto B, Vitaliani R, LindeckPozza E, Grisold W, Vedeler C.

Treatment for paraneoplastic neuropathies

Cochrane Database Syst Rev 2012 Dec 12.

17

Briani C, Vitaliani R, Grisold W, Honnorat J, Graus F, Antoine JC, Bertolini G, Giometto B;

PNS Euronetwork. Spectrum paraneoplastic disease associated with lymphoma.

Neurology 76:705-10, 2011

37

CV Bruno Giometto, MD Current position: Head, Department of Neurology, Azienda ULSS 6 Euganea, Padova, Italy Previous position: Head, Department of Neurology, Azienda ULSS 9 Treviso, Italy (2003-2014) Academic qualifications: Degree in Medicine, awarded 1981, University Padova Residency in Neurology, awarded 1985, University Padova Residency in Neuropathology, awarded 1992, University Verona Personal Statement: I a e o er 0 years e perience in t e field of neuroimmunolo y, focusin particularly on paraneoplastic neurolo ical syndromes. I coordinated the PNS Euronetwork between 2002 and 2011, with funding from two European grants. I am currently director of the Department of Neurology of Azienda ULSS 6 Euganea of Padova, which is also a national reference center in Italy for PNS and other neuroimmunological disorders. I was Vice President of the Italian Society for Neurology from 2013-2015 Memberships: 1989-present Member of Italian Society of Neuropathology (AINP) 1993-present Formerly Vice President, currently member of Board of Arbitrators 1998-present Member of Italian Neuroimmunology Association (AINI) 2002-present Member of Paraneoplastic European Network 01 present Mem er of Su speciality Scientific anel for euroimmunolo y of uropean ssociation of Neurology (EAN)

National and international collaborations: Our group has an active collaboration, stabilized over the years, with different excellent neuroimmunological research centers present in Italy, such as Fondazione Istituto Neurologico “C. Mondino” of Pavia (Dr. Diego Franciotta) and the Unit of Neurology - University Policlinic “A. Gemelli” (Prof. A. Evoli). At the international level, our group is connected to the Department of Neurology of the Hospital Clinic of Barcellona (Prof. F. Graus) and the Nuffield Department of Clinical Neurosciences of Oxford (Prof. A. incent), both groups focus on autoimmune encephalitis research.

Bruno Giometto 2 Publications as last author 2016 5,882 IF 2016 35 H-index (source RG)

38

Neuroscience GROUP LEADERS

Antonino Vallesi

GROUP MEMBERS

Field of interest

In the Executive Function Lab, we perform empirical research mainly dealing with brain regions and networks involved in selective attention and executive control of behavior in normal conditions. Additionally, we are interested in understanding how brain functioning changes with adverse events such as normal aging, tumors and lesions, or psychiatric disorders.

Finally, we are also investigating how significant life experiences such as bilingualism or cognitive reserve shape our mind-brain relationship. The methods we use range from experimental psychology and neuropsychology, to EEG and fMRI.

Summary of recent research activities and of main results achieved Theme 1. Hemispheric asymmetries in executive functions: evidence in healthy and lesioned brains Post≠doctoral Fellows: Ambrosini Ettore Capizzi Mariagrazia Furlan Michele Tarantino Vincenza PhD Students: Arbula Sandra Mazzonetto Ilaria Tafuro Alessandra Visalli Antonino

We studied whether and why key high-level cognitive functions show hemispheric lateralization independently of the task context. Some ERP studies showed that flexibly switching between different (spatial, verbal) task rules relies on mostly domain-general criterion-setting mechanisms. Two fMRI studies additionally showed that criterion-setting (studied with task-switching and inductive reasoning) activates left frontoparietal regions independently of the task context, although the latter determines whether the co-activation of right frontal regions occurs or not. On the other hand, another set of ERP and fMRI studies demonstrated that monitoring for the occurrence of critical events is mediated by right-lateralized ERP components and underlying prefrontal sources. In two additional electrophysiological studies, we demonstrated that even intrinsic, resting-state asymmetric electro-cortical activity

39

fig.1

in dorsolateral prefrontal cortex predicts individual differences in phasic (criterion-setting, conflict resolution) and sustained (monitoring) executive functions, regardless of the specific task. In neuropsychological studies with brain tumor patients, we also found that the left (but not right) frontal cortex is “critically� involved in adopting strict response criteria when switching from a harsh to an accurate response strategy. The same left frontal region is critically involved when criterion-setting requires withholding a response in verbal and spatial Go/No-go tasks.

Theme 2. Can life experience boost executive functions?

We first focused on simultaneous interpretation, which requires the highest level of language management, with possible cognitive benefits beyond the language-specific ones. To study language-specific control, simultaneous interpretation students were tested on a 3-language switching task, which affords a measure of inhibition of abandoned task sets (n-2 repetition costs). The results revealed that different languages could be flexibly controlled through various mechanisms (inhibitory and not) at various stages of training and practice. Regarding domain-general cognitive skills, studies with professional interpreters and matched multilinguals revealed an advantage for the interpreters on most short-term and working memory measures, and on sustained control (mixing costs) during task-switching, suggesting benefits in maintaining multiple task sets beyond language domain. We also studied the impact of Air Traffic Control (ATC) training on higher cognitive processes. With a control-matched longitudinal approach, we found that training boosted reactive control processes in ATC students that already relied more on proactive control strategies before training.

EEG data are still under analysis. Our results provide novel findings on how ecological, real-life training, focused on creating skills necessary for cognitively demanding jobs, enhances specific cognitive control processes and contributes in explaining inter-individual differences in executive functioning.

40

fig.1 The figure reports the decoding performance, using a multivariate pattern analysis (MVPA) approach, calculated as a function of di erent sets of voxels, separately for the left and the right inferior frontal gyrus (IFG) and for two sessions during inductive reasoning with spatial and verbal rules. The black line depicts the 50% chance level accuracy, while the red line depicts the 5th percentile of the null distribution derived by permutation testing. We were able to reliably decode the representation of rule domain only in the left IFG. We suggest that sophisticated neural systems in the left IFG are able to carry out abstract operations, such as extracting patterns and high-order relations starting from the available information independently of the task-context (verbal, spatial).

fig.2 The figure shows the performance of neuro-oncological patients with lesions in left frontal, right frontal and non-frontal brain areas on two versions of a Go No-Go task. The discriminability measure (left panel) is an estimate of the ability to distinguish Go and No-Go stimuli while controlling for possible di erences in response bias, with 0 representing chance performance. The response bias measure (right panel) re ects a tendency in initiating or withholding the response without the impact of stimulus discriminability, with high values indicating more conservative response bias. The upper parts of the panels show the results from a voxel-based lesion-symptom mapping analysis: areas significantly associated with low discriminability (left) and more conservative response bias (right) confirm the behavioral results. Color bars indicate -scores. In this study we show that lesions in left ventrolateral and dorsolateral prefrontal areas cause more decisional errors, while lesions in right ventrolateral and medial prefrontal cause more attentional errors, without any specific inhibitory impairment observed in neither left nor right prefrontal patients.

fig.2

Theme 3. Can experience modulate age≠ related changes in executive functions?

We showed that dual-tasking abilities in cognitive aging are predicted by the cognitive reserve and in particular by the level of formal instruction. Other investigations on how it is possible to actively intervene to improve or at least maintain older adults’ cognitive and neural functioning through structured training activities are in progress.

5-10 Selected Publications Asymmetry in Prefrontal RestingAmbrosini E., Vallesi A. state EEG Spectral Power Underlies Indi idual ifferences in asic and Sustained Cognitive Control.

Neuroimage, 2016, Vol. 124 (1), pp. 843-857.

Ho air traffic control trainin s apes co niti e control strate ies

Arbula S., Capizzi, M.G., Lombardo, N., Vallesi A.

Plos ONE, 2016, Vol. 11(6), pp. e0157731.

estin t e domain eneral nature of monitoring in spatial and verbal cognitive domains.

Capizzi M., Ambrosini E., Arbula S., Mazzonetto I., Vallesi A.

europsyc olo ia, 2016 Vol. 89, pp. 83-95

lectrop ysiolo ical idence for Domain-general Processes in as s itc in

Capizzi M., Ambrosini E., Arbula S., Mazzonetto I., Vallesi A.

Front Hum Neurosci, 2016, Vol 10, pp. 124

lectrop ysiolo ical correlates of t e co niti e control processes underpinning mixing and s itc in costs.

Tarantino V., Mazzonetto I., Vallesi A.

Brain Res, 2016, Vol 1646, pp. 160–173

Domain-general Stroop performance Ambrosini E., Vallesi A. and emisp eric asymmetries: a resting-state EEG study.

J Cogn Neurosci. (in press , OI:10.11 jocn_a_01076

ite matter inte rity and tas s itc in performance: a I study.

Vallesi A., Mastrorilli E., Causin F., D’Avella D., Bertoldo A.

Neuroscience, 2016, Vol. 329, pp. 349–362

Speed-accuracy strategy regulations in prefrontal tumor patients.

Campanella F., Skrap M., Vallesi A.

europsyc olo ia, 2016, Vol. 82, pp. 1-10.

Mental time line distortion in ri t Marin ., itteri M., ella uppa ., Mene rain dama ed patients: e idence Biasutti E., Priftis K., Vallesi A. from a dynamic spatio-temporal task.

Are simultaneous interpreters expert bilinguals, unique bilinguals, or ot

Babcock L., Vallesi A.

ello .,

europsyc olo y, 2016, Vol. 30(3), pp.

338-45. ilin ualism: Lan ua e and Cognition (in press , oi: 10.101 S1366728915000735

41

fig.3

CV Antonino Vallesi, PhD ntonino allesi is an ssociate rofessor in syc o iolo y ysiolo ical syc olo y at t e ni ersity of adua since 014. He recei ed is master de ree in syc olo y from ni ersity of adua in 00 cum laude and t en is in euroscience cum laude at SISS , rieste in 00 . He as a post doctoral fello at t e otman esearc Institute at aycrest, oronto 00 00 and an ssistant rofessor at SISS 00 01 and t en at ni ersity of adua 01 014 . In 011 e recei ed t e ertelson ard from t e uropean Society of Co niti e syc olo y. In 01 e as a arded it an C startin rant it a pro ect on Life perience Modulation of ecuti e unction symmetries. e met ods e uses include fM I, and neuropsyc olo y. He as super ised more t an students and post doctoral fello s, se eral trainees and students. He as aut ored more t an peer re ie ed articles in international scientific ournals, oo c apters, and se eral contri utions to national and international conferences. He teac es e ery year euroima in , Co niti e neuroscience, Introduction to syc olo y and Met odolo y at ni ersity of adua. He as ser ed as ssociate ditor for rontiers in syc olo y Co nition section and is in t e editorial oard of ournal of Co niti e n ancement Sprin er 01 ioMed esearc International ction ditor, since 01 etc. He as ser ed as an ad oc re ie er for se eral ournals includin los iolo y rain eurosci io e a Cere Corte euro iol in euroima e Hum rain Mapp Co n eurosci. He as een a rant re ie er for Horizon 0 0 MSC I 01 014 MI SI 014 H Call 01 S C Canada 011 1 Romanian NCDI 2011-13.

Antonino Vallesi 13 Publications as first/last author 2016 2 IF as first/last author 2016 22 H-index (source esearch ate)

42

fig.3 The scatterplots show the correlations between the participants hemispheric asymmetry in resting-state electrophysiological brain activity (as assessed by a asymmetry measure, x axis) in di erent regions of interest and a behavioral measure of their ability to resist to interfering information (as assessed by the -transformed Stroop e ect, y axis) during a verbal and a spatial task (blue and orange points, respectively). The blue and orange regression lines re ect the skipped Pearson correlation (r) for the verbal and spatial Stroop e ects, respectively. Positive values of asymmetry index indicate a stronger right-lateralized brain activity at rest. These results indicate that participants with a stronger resting-state-related left-lateralized activity in the prefrontal cortex were more able to inhibit irrelevant information in both cognitive domains.

Pediatric Critical Care & Critical Care Biology GROUP LEADERS

Paola Cogo Luca Vedovelli

GROUP MEMBERS

Field of interest

Our research team has 30 years of experience in translational medicine of acute lung diseases (including animal models, newborns, and adults) and in genetic mutations of lung surfactant-specific proteins.

We conducted several clinical nutrition studies in newborns and infants.

Our focus is now on markers of neurologic and pulmonary injuries in pediatric cardiac-surgery, neonatal and adults intensive care.

Our studies are conducted with an analytical chemistry approach involving quantitative measurements of the molecules and the use of stable isotopes to follow metabolic pathways in vivo in humans.

Summary of recent research activities and of main results achieved Post≠doctoral Fellows: Manuela Simonato Medical Doctors: Aldo Baritussio Virgilio P. Carnielli Lorenza Dalla Massara Giovanna Verlato Students: Sonia Giambelluca

In recent years, we focused on the neurological and pulmonary injuries occurring during pediatric cardiac surgery for congenital heart defects. Congenital heart defects are the most common congenital disease affecting about 1 of all births. Improved surgical techniques have reduced operative mortality to and major concerns are now focused on the long-term outcome, especially on neurological and neurodevelopmental disorders along with lung injuries. We are trying to define one or more easily assayed biomarker that correlates with the neurological and pulmonary outcome of children undergoing open-heart surgery for congenital heart diseases. We recently described how the minimum temperature reached during surgery is the most important factor that influences the rise of a brain injury neuromarker and how the type of cardiac diseases is linked to a specific pulmonary surfactant status.

43

fig.1

Studies on preterm and newborn infants’ nutrition (focused on parental nutrition) are also ongoing with important results obtained describing the metabolism of lipids components and their relationship with the diseases. Lipidomics studies are also ongoing regarding the metabolism of lipids in the fetus and during pregnancy.

Pulmonary surfactant status in critical diseases is also a key topic of the laboratory. We used stable isotope tracers (that are safe to use in humans) and mass spectrometry to describe lipids and proteins kinetics in different disease both in children and adults.

We are also studying a protective ventilation protocol to be used in the intensive care in adult patients. We will measure inflammation mediators and surfactant components expression comparing the results with the ones obtained in standard-ventilated patients.

Along with clinical studies, cellular and molecular studies are ongoing to describe the cellular activity of drugs (i.e. amiodarone) on various types of cells and diseases. Moreover, the healing effect of the amniotic membrane is under investigation with different human primary cells.

44

fig.2

fig.1 fig2 Plasma GFAP (Glial Fibrillary Acidic Protein) in patients with univentricular heart physiology.

We showed that GFAP, a brain in ury biomarker, increases during cardiopulmonary bypass only in the first stage surgery of univentricular hearts palliation (Norwood Stage I) and peaks at the end of rewarming. From Vedovelli et al.

CJC 2015.

5-10 Selected Publications Vedovelli L, Padalino M, D'Aronco S, Stellin G, Ori C, Carnielli VP, Simonato M, Cogo P. M. . ella am a, . Carmenati, . scenzo, M. Malatesta, C. Spa noli, C. ia etti, I. urattini, and . . Carnielli . . Carnielli, C. ior etti, M. Simonato, L. Vedovelli, and P. Cogo

lial fi rillary acidic protein plasma le els are correlated it de ree of ypot ermia durin cardiopulmonary ypass in con enital eart disease sur ery.

One tra ram of rotein to reterm Infants rom irt to 1 00 : Sin le linded andomized Clinical rial

eonatal espiratory iseases in t e e orn Infant: o el Insi ts from Sta le Isotope racer Studies

D'Aronco S, Simonato M, Vedovelli L, aritussio , erlato , o ile S, ior etti C, especa M, Carnielli V and Cogo P

Surfactant protein and neonatal pneumonia

S. Carraro, . iordano, . irillo, M. Maretti, . eniero, P. Cogo, . erilon o, M. Stocc ero, and . araldi

ir ay meta olic anomalies in adolescents it ronc opulmonary dysplasia: ne insi ts from t e meta olomic approac

. . udo , I. a dee, . C eun , H. Liu, L. Vedovelli, . inelli, . enyon, S. arapuram, and C. M. Hutni

ffects of amniotic mem rane e tract on primary uman corneal epit elial and lim al cells

S. Lanini, . Zumla, . . . Ioannidis, . i Caro, S. ris na, L. ostin, . irardi, M. letsc ette, . Strada, . aritussio, . ortella, . polone, S. Ca uto, . Satolli, . remsner, . airo, and . Ippolito

Interact Cardio asc orac Sur . 01 ec 1. pii: i . doi: 10.10 ic ts i . ediatr. astroenterol. utr., ol. , no. , pp.

4, un. 01 . eonatolo y, ol. 10 , no. 4, pp. , 01 .

concentrations are increased in

re adapti e randomised trials or non randomised studies t e est ay to address t e ola out rea in est frica

ediatric esearc .

01 Oct 4 :401 .

. ediatr., ol. 1 , no. , pp. 4 .e1, e .

01 Clin. p. Op t almol., ol. 4 , no. , pp.

44 44 , 01 Lancet. Infect.

1 , no. , pp. un. 01 .

. etto, M. Mondini, C. ezzella, arenteral utrition Is One of t e Most Si nificant is L. omani, . Luci nano, L. ansani, actors for osocomial Infections in a ediatric Cardiac . ar enio, and P. Cogo Intensi e Care nit . eca, . oldrini, . o annson, . Clinical and ultrastructural spectrum of diffuse lun . S ie , . Citti, S. etrini, . Salerno, disease associated it surfactant protein C mutations S. Cazzato, . esta, . Messina, . Onofri, . Cenacc i, . estermar , . llman, P. Cogo, . Cutrera, and O. an ai e assler , la a , S in ell S, Hallman M, arreau H, Carnielli , an den n er , Meisner C, n el C, Sc a M, Halliday HL, oets C OSIS rial roup

arly In aled udesonide for t e re ention of ronc opulmonary ysplasia.

is., ol.

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. . arenter. nteral utr., o . 01 ur. . Hum. enet., ol. , no. , pp.

10 1041, u . 01

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45

fig.3

fig.4

CV Paola Cogo

CV Luca Vedovelli

rof. Co o, M , as e tensi e clinical and la oratory e pertise in Italy and a road from almost 0 years.

S e s associate professor of pediatrics and c air of t e di ision of pediatrics ni ersity of dine . S e s c air of t e researc section on Con enital Heart disease, S IC Society. S e as t e scientific Coordinator of t e pediatric Italian neuro intensi e care committee SI I Society . S e s aut or of 1 1 Scopus inde ed documents 1 citations it an factor of 0. rof. Co o researc team is specialized in analytical tec ni ues includin ad anced isotope ratio mass spectrometry focused on t e study of t e diseases t at re ards t e critical care settin s of ot c ildren and adults.

r. edo elli, as e tensi e la oratory e pertise in analytical c emistry, ac ie ed in Italy and a road Har ard Medical Sc ool, oston . He is t e leader of t e Critical Care iolo y la oratory since 014, role o tained after winning the Grant Program for oun In esti ators on ediatric esearc ondazione Cariparo .

He is aut or of 1 Scopus inde ed documents citations it an factor of 4. r. edo elli researc focused on neurolo ical outcome in con enital eart diseases it insi ts in pediatric nutrition and re enerati e medicine.

fig.3 fig.4 Plasma GFAP (Glial Fibrillary Acidic Protein) in patients with biventricular heart physiology.

We showed that GFAP, a brain in ury biomarker, increases during cardiopulmonary bypass and it is directly correlated with hypothermia degree in patients with biventricular physiology.

From Vedovelli et al. Interact CardioVasc Thorac Surg 2016.

National and international collaborations:

The laboratories actively collaborate with national and international universities, institutions and companies for clinical studies, know-how exchange and grant applications: Neonatology, Ospedali Riuniti di Ancona (prof. irgilio Carnielli) Newborn Intensive Care Unit (NICU), St. Louis Children’s Hospital, USA (Dr. Aaron Hamvas) Intensive Care Cardiosurgery Unit, Bambino Ges Pediatric Hospital, Roma Chiesi Farmaceutici, Parma Department of Pediatrics, University of Siena (prof. Giuseppe Buonocore) Anaesthesia and Resuscitation, San Gerardo Hospital, Monza (prof. Antonio Pesenti) Pediatric and Neonatal Intensive Care Unit, Padova University Hospital Pediatric Cardiosurgery, Padova University Hospital (prof. Giovanni Stellin) Department of Medicine, Anesthesia and Resuscitation, Padova University Hospital (prof. Carlo Ori) Paola Cogo

Luca Vedovelli

30.54 IF as first/last author 2016 13 Publications as first/last author 2016 20 H-index (source )

3.71 IF as first/last author 2016 1 Publications as first/last author 2016 4 H-index (source )

46

Immunopathology and Molecular Biology of kidney GROUP LEADERS

Luisa Murer

GROUP MEMBERS

Post≠ doctoral Fellows: Negrisolo Susanna PhD Student: Carraro Andrea Technician: Fregonese Giulia Medical Doctors: Benetti Elisa, PhD Vidal Enrico, PhD Longo Germana Meneghesso Davide Parolin Mattia

Field of interest

The Laboratory of Immunopathology and Molecular Biology of the Kidney is part of the Pediatric Nephrology Dialysis and Transplant unit of the Dept. of Women’s and children’s Health of Hospital/University of Padua. The Unit is a center of excellence and reference for Pediatric Nephrology and Rare kidney disease. It is also part of the international registries and networks (i.e. ERK-Net) for the diagnosis and treatment of kidney rare diseases. The structure offers the most advanced knowledge for the diagnosis, treatment and follow-up of acute and chronic kidney and urologic diseases. Furthermore, it works in close collaboration with various specialists to follow children patients from prenatal diagnosis to kidney transplantation, using a multidisciplinary and comprehensive approach.

The Laboratory of Immunopathology and Molecular Biology of the Kidney provides an analysis pattern for the immune-histological classification of primary and secondary pediatric renal diseases and for the follow-up of pediatric kidney transplantation recipients. It also coordinates the management of molecular tests for genetic kidney disease.

Furthermore, the laboratory has a remarkable biobank of paraffin and frozen renal tissues of transplanted pediatric patients.

The Pediatric Nephrology Dialysis and Transplant unit together with the laboratory conduct and coordinate important scientific studies, from the clinical trials to the translational research, in particular concerning kidney transplantation, congenital abnormalities of the kidney and urinary tract (CAKUT), nephrotic syndrome, acute kidney injury (AKI) and dialysis in childhood.

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fig.1

Summary of recent research activities and of main results achieved Congenital anomalies of the kidney and urinary tract (CAKUT) account for more than 30% of all developmental anomalies. One of the most severe CAKUT phenotype is renal hypodysplasia (RHD: OMIM #610805), which is a defect in the number and/or normal differentiation of nephronic units with a subsequent impairment of kidney function. RHD accounts for almost 25% of all causes of end stage renal failure in children. Mutations of at least 17 genes involved in the early stages of kidney development and some copy number variations (CNV) have been associated with RHD. Both point mutation and CNV disorders can explain about 25% of RHD cases but nevertheless, the majority of patients remains without a genetic diagnosis, that would be helpful for prognostic evaluation. Our laboratory studied the genes involved in early nephrogenesis (e.g. PAX2, SIX1, GATA3) and their epigenetic regulation by miRNAs, by mutational analysis.

In the last three years, we have been participating in the University of Padua Strategic project “Bioinfogen� that involves four research units with the aim to create new bioinformatics tools to facilitate NGS data analysis in Mendelian diseases. Our unit is investigating on RHD genetic causes in 20 patients and their healthy relatives, with whole exome sequencing. To date we identified a compound heterozygous mutation in IN S gene and we now we are continuing the validation studies. Our findings suggest that Inversin (INVS) mutations could be associated to a wider spectrum of kidney phenotypes of NPHP including RHD or that INVS could be a candidate gene for 48

fig.1 Output of NGS data analysis tool developed during Bioinfogen Pro ect in collaboration with bioinformatic unit

fig.2

isolated bilateral RHD. These data were communicated at 48th Annual Scientific Meeting of the European Society

fig.2 Exovesicles isolated in patients urine (photo obtained by nanosight NS300)

of Paediatric Nephrology (ESPN 2015). Furthermore, we highlighted a variant in a candidate gene in 25% of the analyzed cases. In 4/5 cases the variant was a

ariant

with Uncertain Significance in known CAKUT genes often associated to syndromic forms. In remains 15/20 patients WES data analysis will be continued performing splicing regions analysis and considering oligogenic inheritance patterns. These data were communicated at Italian Society of Nephrology congress (SIN 2016) and in the “International Conference: The kidney in genetic and rare disease�, where they were awarded as best poster.

These results will permit to lay the basis for setting up a perspective RHD diagnostic panel, allowing the screening of genes involved in the kidney developmental abnormalities.

Genetic diagnosis of RHD allows us to offer timely patient related treatment strategies and to give genetic counseling to the family.

Another milestone of our laboratory is the study of factors affecting the survival of kidney transplantation in the pediatric population.

Kidney transplantation is the treatment of choice for endstage kidney disease. Although the immunosuppressive therapy have improved the survival graft rate; at 20 years after transplantation it remains still negligible (65%), data unacceptable for pediatric patients. In the last decade the unit conducted studies related to viral infections acquired by the organ transplanted and their impact on pediatric recipient and association studies between genetic polymorphisms involved in cardio vascular risk and their correlation with the transplant survival.

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A new field of interest we are currently developing towards the identification of early kidney transplant rejection markers. To date the gold standard in the diagnosis of renal allograft failure (main cause of kidney rejection), is the biopsy. However, this invasive method does not avoid risks for patients. Emerging data support the presence in biological fluids of noninvasive biomarkers predictive of rejection. Urinary and blood extra cellular vesicles (EVs) could represent a source of mediator of rejection.

In our new project we defined a specific and precise time-line model to be able to monitor both acute and chronic kidney rejections. This model will help us to explain how the development of humoral acute and chronic kidney rejection are two different phenomena belonging to the same “ad continuum� process (i.e. immunological profile as initial event and histological/clinical manifestations latter events).

In this perspective, the identification of biomarkers in the early phase, that can be predictive of the possibly chronic outcome, it acquires an extreme importance. In this study we expect to be able to uniquely identify predictive immunological, genetic biomarkers of post-transplant kidney damage (e.g. EVs and their miRNA content) using multi-disciplinary skills and the application of innovative technologies. Our results will contribute to elucidates the role of the humoral immune mechanisms in the development of allograft rejection and the identification of noninvasive biomarkers. Indeed, predictive biomarkers allow sensitive/accurate monitoring of kidney graft function, early and specific diagnosis of rejection, assessment of long term outcome. Their identification will help to define a specific patient oriented immunosuppressive therapy ,extremely important in children, to develop a noninvasive diagnostic tool and to create an health cost-effective strategy.

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10 Selected Publications Dello Strologo L, Murer L, Guzzo I, Morolli F, Pipicelli Am, Benetti E, Longo G, Testa S, Ricci A, Ginevri F, Ghio L, Cardillo M, Piazza A, Nanni Costa A.

Renal transplantation in sensitized children and young adults: a nationwide approach.

Nephrol Dial Transplant.

2016 oct 14. Pii: gfw369. [epub ahead of print] IF 4.085, cit -

Mele C, Lemaire M, Iatropoulos P, Characterization of a new DGKE intronic mutation in Piras R, Bresin E, Bettoni S, Bick genetically unsolved cases of familial atypical hemolytic D, Helbling D, Veith R, Valoti E, uremic syndrome.

Donadelli R, Murer L, Neunhäuserer M, Breno M, Frémeaux- Bacchi V, Lifton R, Remuzzi G, Noris M.

Clin J Am Soc Nephrol.

2015 jun; 10(6):1011-9. epub 2015 apr 8.

IF 4.657, cit. 5

Giglio S, Provenzano A, Mazzinghi B, Becherucci F, Giunti L, Sansavini G, Ravaglia F, Roperto Rm, Farsetti S, Benetti E, Rotondi M, Murer L, Lazzeri E, Lasagni L, Materassi M, Romagnani P.

Heterogeneous genetic alterations in sporadic nephrotic syndrome associate with resistance to immunosuppression.

Journal of The American Society of Nephrology, (2015) vol. 26; p. 230-236, IF 8.491, cit 17

Vidal E, Amigoni A, Brugnolaro V, Ghirardo G, Gamba P, Pettenazzo A, Zanon G, Cosma C, Plebani M, Murer L.

Near-infrared spectroscopy as continuous real-time monitoring for kidney graft perfusion.

Pediatric Nephrology, 2014. vol. 29; p. 909914, IF 2.338, cit. -

Bower M, Salomon R, Allanson J, Antignac C, Benedicenti F, Benetti E, [...], Murer L, Nguyen Ht, […], Eccles M, Schimmenti La, Heidet L.

Update of PAX2 mutations in Renal Coloboma Syndrome and esta lis ment of a locus specific data ase.

Human Mutation, (2012). VOL. 33, P. 457466, IF 5.089 cit.29

Benetti E, Caridi G, Malaventura C, Dagnino M, Leonardi E, Artifoni L, Ghiggeri GM, Tosatto SC, Murer L

A novel WT1 gene mutation in a three-generation family with progressive isolated focal segmental glomerulosclerosis.

Clinical Journal of The American Society of Nephrology, (2010) VOL. 5, P. 698-702.

IF 8.491, cit.9

Barzon L*- Murer L*, Pacenti M, Biasolo Ma, Della Vella M, Benetti E, Zanon Gf, Palu G.

Investigation of intrarenal viral infections in kidney transplant recipients unveils an association between parvovirus B19 and chronic allograft injury.

The Journal of Infectious Diseases, (2009). VOL. 199, P. 372-380, IF 6.344, cit 17

Dello Strologo L, Guzzo I, Laurenzi Use of Rituximab in focal glomerulosclerosis relapses C, Vivarelli M, Parodi A, Barbano after renal transplantation.

G, Camilla R,Scozzola F, Amore A, Ginevri F, Murer L.

Transplantation, (2009).

VOL. 88, P. 417-420, IF 3.690, cit 36

Benetti E, Artifoni L, Salviati L Pinello L, errotta S, Zuffardi O, Zacc ello , Murer L.

Renal hypoplasia without optic Coloboma associated with Nephrology Dialysis PAX2 gene deletion.

Transplantation, (2007). vol. 22, p. 2076-2078.

If 4.085, cit 147

Murer L, Addabbo F, Carmosino M, Procino G, Tamma G, Montini G, Rigamonti W, Zucchetta P, DellaVella M, Venturini A, Zacchello G, Svelto M, Valenti G.

Selective decrease in urinary AQUAPORIN 2 and increase in PROSTAGLANDIN E2 excretion is associated with postobstructive polyuria in human congenital hydronephrosis.

Journal Of The American Society Of Nephrology, (2004).

Vol. 15, P. 2705-2712, IF 8.491, Cit 32

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CV Luisa Murer, MD Dr. Luisa Murer is a pediatric nephrologist and she is the chief of the Pediatric Nephrology, Dialysis and Transplant Unit. She is also the Head of Immunopathology and Molecular Biology of the Kidney Laboratory of “Women’s and Children’s Health” Department of University/Hospital of Padova. Since October 2016 she is President of Pediatric Nephrology Italian Society (SINePe). She studied at the University of Padua. She obtained the Medical Doctor degree in 1986, the Residency in Paediatrics School in 1990, the Ph.D. in Developmental Sciences in 1994 and the Residency Pediatric Nephrology in 1995. She moved to the UK in 1987 granted by a fellowship in the Paediatric Research Unit Guy’s Hospital, London.Since 2001 she is Adjunct Professor of many Specialization Schools of Women’s and Children’s Health Department of University of Padova.She is author/co-author of over 100 publications on peer-reviewed journals with a cumulative impact factor score of 359.772 and total of 2376 citations.

National and international collaborations: • • • • • • • • • • • • • • • • •

Pediatric Nephrology, Centre for Child and Adolescent Medicine, Universitätsklinikum Heidelberg. Germany Division of Nephrology, Columbia University, College of Physicians and Surgeons, New York, USA Dipartimento di Biologia e CRIBI Biotech Center, Università di Padova, Padova, Italy IRCCS “Mario Negri”, Centro di Ricerche Cliniche per le Malattie Rare “Aldo e Cele Daccò”, Ranica, Bergamo, Italy UOC Nefrologia, Dialisi e Trapianto - IRCCS Giannina Gaslini, Genova, Italy UO Nefrologia e Dialisi e Trapianto-Ospedale Pediatrico Bambino Gesù IRCCS, Roma, Italy SOC Nefrologia Pediatrica e Struttura di Genetica-AO Universitaria Meyer, Firenze, Italy Dipartimento di Nefrologia e IRRI Ospedale San Bortolo, icenza, Italy UOSD Nefrologia Clinica e UOC Nefrologia 2, AO Padova, Padova, Italy UOC Nefrologia e Dialisi Pediatrica-Fond. IRCCS Cà Granda Ospedale Maggiore Policlinico Milano, Italy Istituto Ricerca Scientifica Ospedale San Raffaele di Milano, Milan, Italy UOC Nefrologia Pediatrica-AO Universitaria-Città della Salute e della Scienza, Torino, Italy IRCCS Fondazione Casa Sollievo della Sofferenza, San Giovanni Rotondo, Foggia, Italy Centro di Medicina Molecolare, Ospedali Riuniti di Foggia UOC Nefrologia, Dialisi e Trapianto e Servizio di Nefrologia Pediatrica, AO-U di Bari, Italy Divisione di Nefrologia dell’Adulto e del Bambino, AU Policlinico, Napoli, Italy Divisione di Nefrologia dell’Adulto e del Bambino, Azienda Universitaria Policlinico Seconda Università di Napoli

Luisa Murer 6 Publications as author, co-author 2016 19.102 IF as author, co-author 2016 27 H-index (source RG)

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Stem Cells and Regenerative Medicine GROUP LEADERS

Michela Pozzobon Maurizio Muraca GROUP MEMBERS

Post≠ Doctoral Fellows: Chiara Franzin Martina Piccoli PhD Students: Caterina Trevisan Michele Grasso Mattia Saggioro Giovanna Spiro Annamaria Tolomeo Students: Stefania Dí Agostino Edoardo Maghin

Field of interest

The group has been focused in the past 12 years on developing strategies for tissue regeneration for the paediatric patients.

Following the interest of the group founder, the paediatric surgeon professor Paolo De Coppi that since 11 years works at the great Ormond street Hospital, London, the group has acquired different skills in the field of stem cell biology, molecular biology and murine animal model creating a strong expertise on regenerative medicine field.

Dr. Michela Pozzobon has become the coordinator of the laboratory since 2010 and during these years doctor Pozzobon demonstrate to be able to conduct the research for the lab coordinating first of all the people of the group, secondly the research experiments, the results and their publication, the writing of the projects and the funds administration.

Maurizio Muraca joined the group in 2015, bringing his benchto-bedside experience in Regenerative Medicine developed through his clinical background (Internal Medicine and Transplantation Medicine), his work in basic science, also as Responsible for Research in Regenerative Medicine at the Bambino Gesù Hospital, his experience in regulatory issues developed at the Agenzia Italiana del Farmaco and at the European Medicines Agency, his organization and accreditation experience as Head of Clinical Chemistry and Microbiology at the Bambino Gesù Hospital and as Responsible for the development of the Cell Factory at the same Institution.

Maurizio Muraca brought his experience in the study of extracellular vesicles, developing novel perspectives in the use of these biological nanoparticles as therapeutic tools The group has been the first in Padova to develop stem cell research both on fetal and adult stem cells. In particular, the research activity is focused on:

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1. amniotic fluid stem cells (discovered by the professor De Coppi) and on satellite cells, the skeletal muscle stem cell population. Both in vitro and in vivo approaches are used to study and understand the advantages and limits of stem cells in tissue engineering applications. Murine models (for muscle and endothelial regeneration) are used to prove the in vitro attained results.

2. iPS on murine and human amniotic fluid stem cells. The basic idea is to reprogram fetal stem cells to sequentially differentiate them toward muscle and seed with cells the decellularized tissues in order to develop a new tissue construct ready to be implanted 3. extracellular matrix as tissue bulk and cell recipient. Decellularization methods of skeletal muscle tissue (of murine and human source) is a specific toll used to muscle rebuilding (in congenital malformations such as diaphragmatic hernia, in muscle defects such as volume muscle loss due to trauma, tumor resection). The decellularization process is used for both healthy and diseased tissues.

4. since prof Muraca joined the group (2015), the research field on shedding vesicles has been developed. Details of the research will be specified below.

Dr. Michela Pozzobon Summary of recent research activities and of main results achieved

The main research activities are focused on (1) human and murine amniotic fluid stem cells, (2) muscle precursor cells, (3) muscle extracellular matrix. Here below the main results are summarized.

1.1 We focused on the ability of human amniotic fluid stem cells to form new endothelium. Abstract of the recent published results is following.

Abstract. Introduction: Endothelial dysfunction is found in different pathologies such as diabetes and renal and heart diseases, representing one of the major health problems. The reduced vasodilation of impaired endothelium starts a prothrombotic state associated with irregular blood flow. We aimed to explore the potential of amniotic fluid stem (AFS) cells as a source for regenerative medicine in this field; for the first time, we focused on third-trimester amniotic fluid AFS cells and compared them with the already-described AFS cells from the second trimester. Methods: Cells from the two trimesters were cultured, selected and expanded in normoxia (20 % oxygen) and hypoxia (5 % oxygen). Cells were analysed to compare markers, proliferation rate and differentiation abilities. Endothelial potential was assessed not only in vitro—Matrigel tube formation assay, acetylated human low-density lipoprotein (AcLDL) uptake but also in vivo (Matrigel plug with cell injection and two animal models). Specifically, 54

fig.1

for the latter, we used established protocols to assess the involvement of AFS cells in two different mouse models of endothelial dysfunction: (1) a chronic ischemia model with local injection of cells and (2) an electric carotid damage where cells were systemically injected. Results: We isolated and expanded AFS cells from third-trimester amniotic fluid samples by using CD117 as a selection marker. Hypoxia enhanced the proliferation rate, the surface protein pattern was conserved between the trimesters and comparable differentiation was achieved after culture in both normoxia and hypoxia. Notably, the expression of early endothelial transcription factors and AngiomiRs was detected before and after induction. When in vivo, AFS cells from both trimesters expanded in hypoxia were able to rescue the surface blood flow when locally injected in mice after chronic ischemia damage, and importantly AFS cells at term of gestation possessed enhanced ability to fix carotid artery electric damage compared with AFS cells from the second trimester. Conclusions: To the best of our knowledge, this is the first research work that fully characterizes AFS cells from the third trimester for regenerative medicine purposes. The results highlight how AFS cells, in particular at term of gestation and cultured in hypoxia, can be considered a promising source of stem cells possessing significant endothelial regenerative potential.

(1.2) We reprogrammed murine AFS cells with a nonviral method, using Piggybac (PB). PB transposition is hostfactor independent, and has recently been demonstrated to be functional in various human and mouse cell lines. The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events.

PB-mediated delivery allows the option of xenofree production of iPS cells contrary to current viral production protocols that use xenobiotic conditions and for these reasons it could became

fig.1 Characterization of diaphragmatic acellular sca old angiogenetic potential. (A) Coryon Allantoid Membrane (CAM) assay appearance after days of incubation with negative control, positive control and decellularized diaphragm; (B) Number of vessels converging towards each sample at baseline (0d) and after days; vessels were counted in a blinded fashion; (C) Immunoblotted membrane array of fresh and decellularized tissue homogenate.

53 angiogenetic factors were detected; (D) Pixel intensity of some of the principal angiogenetic factors; Intensity was calculated using UVITEC Cambridge software (mean SD); . (E) Quantification of VEGF from fresh and decellularized tissue homogenate (mean SD); (F) Quantification of SDF-1a from fresh and decellularized tissue homogenate (mean SD); (G) Quantification of HGF from fresh and decellularized tissue homogenate (mean SD); (H) Quantification of EGF from fresh and decellularized tissue homogenate (mean SD).

55

fig.2

a clinically acceptable method of deriving reprogrammed cells.

We used the validated sets of primers (i) and (ii) we characterized AFS cells-derived iPS cells. (Franzin et al, BOC 2015; Bertin et al, Stem Cell Research 2016).

(2) Using human muscle precursor cells, we set a published protocol of muscle cell extraction from more than 10 different muscle of origin both from fresh and frozen samples.

Abstract: One of the major issues concerning human skeletal muscle progenitor cells is represented by the efficient isolation and in vitro expansion of cells retaining the ability to proliferate, migrate and differentiate once transplanted. Here we describe a method (1) effective in obtaining human muscle precursor cells both from fresh and frozen biopsies coming from different muscles, (2) selective to yield cells uniformly positive for CD56 and negative for CD34 without FACS sorting, (3) reliable in maintaining proliferative and in vitro differentiative capacity up to passage 10. (Franzin et al, Mol Methods 2016) (3) We developed the project: “A tissue engineering approach for diaphragm repair in a congenital diaphragmatic hernia mouse model�

Abstract: Congenital diaphragmatic hernia (CDH) is a phenotypically and genetically heterogeneous disorder. The missing diaphragm can be repaired using prosthetic materials which are limited because they do not grow with the child, can get infected and they do not replace the myogenic function promoting recurrence of the hernia. In this scenario, we propose an after-birth therapy through a tissue engineering approach as therapy for CDH. In a mouse model of CDH, we will use a supporting three-dimensional scaffolds and stem cells as new therapeutic tool. This surgical mouse model will be the first created for this pathology. The great advantage of using the biological scaffold is based on the fact that decellularized 56

fig.2 Sca old subcutaneous implantation and vessel after DET treatment. (A) Schematic representation of experimental plan (B) Macroscopic appearance of the implanted DD at day 0 and of DD explanted after and 15 days of implantation; (C) Quantification of haemoglobin over total protein content in control (skin) and DD after and 15 days of implantation (mean SD); (D) Immuno uorescence staining against SMA and GFP performed in explanted DD after and 15 days from implantation; nuclei were counterstained with DAPI; (E) Immuno uorescence staining against SMA and aminin performed in fresh diaphragm and DD; nuclei were counterstained with DAPI; (F) Immuno uorescence staining against SMA and aminin performed in DD after 48h of incubation with HUVECs; nuclei were counterstained with DAPI; Scale bar 100microm.

fig.3 Comparison of angiogenetic response after orthotropic implantation of DD vs ePTFE. (A) Schematic representation of experimental plan (B) Immuno uorescence staining against CD31 and aminin performed in explanted DD and ePTFE after and 15 days from implantation; nuclei were counterstained with DAPI; (C) Quantification of CD31+ cells over total cells content in explanted DD and ePTFE after and 15 days from implantation (mean SD); (D) Immuno uorescence staining against vWF and SMA performed in explanted DD and ePTFE after and 15 days from implantation; nuclei were counterstained with DAPI; (E) Quantification of CXCR4 mRNA extracted from DD and ePTFE after and 15 days from implantation (mean SD); (F) SMA+ vWF+ vessels dimension distribution calculated in fresh tissue, DD and ePTFE after and 15 days from implantation; Scale bar 100microm.

fig.3

matrices closely resemble the native tissue and offers the best environment to the seeded cells that will survive and proliferate because of the tridimensional shape. The scaffold will be seeded with amniotic fluid stem (AFS) cells resuspended alternatively in two type of biocompatible and absorbable gel to ameliorating the cells survival and proliferation, obtaining a 3D homogeneous distribution of cells within the matrix domain and, finally, to enhance AFS cells myogenic potential through application of mechanical-biochemical stimulations. In this perspective, AFS cells seem to be a good source for muscle differentiation on the basis of our previous work in a mouse model of muscle disease. AFS re-cellularized scaffold will be engrafted in congenital diaphragmatic hernia mouse model and animals will be sacrificed after 1, 2 and 4 weeks. In conclusion, we proposed an innovative tissue engineering approach in a new animal model of CDH; the attained results will be analyzed through RT-PCR, force measurement and immunocitochemistry techniques. In this study, we aimed to characterize a decellularized patch and find a method to re-cellularize it for future clinical application.

We characterized in vitro the decellularized scaffold in order to assess whether it will be in vivo a comfortable environment for the stem cells. We published paper: Piccoli M et al, Biomaterials 2015.

“Improvement of diaphragmatic performance through orthotopic application of decellularized extracellular matrix patch”. We demonstrated that the decellularized matrix structure will enable the cells to survive and growth because of the tridimensional shape and the pores.

The following paper on the same topic is in submission: Decellularized diaphragmatic muscle drives a constructive angiogenetic response in vivo. Alvarèz Fallas ME, Piccoli M, Franzin C, Sgrò A, Dedja A, Urbani L, Bertin E, Trevisan C, Gamba PG, De Coppi P, Pozzobon M. Abstract. Skeletal muscle tissue engineering aims at providing a suitable graft to support and repair large congenital and acquired defects. To this end, the best material is the one that better mimics the native tissue, avoids rejection, sustains cell viability, proliferation and the overall regeneration promoting angiogenesis. In the past decade, biological acellular scaffolds have been described as a good tool for tissue engineering purposes since after decellularization they are mainly composed of extracellular matrix (ECM) that represents a unique cytokine reservoir and protein combination, both absent in synthetic scaffolds. Our group already developed a diaphragm-derived scaffold, which after implantation led to a pro-regenerative immunological response. In this work, we focused mainly on the ability of our skeletal muscle acellular scaffold to promote vascularization both ex-vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array evaluation confirmed the presence of pro-angiogenic molecules in the decellularized tissue. When transplanted in vivo, the naturally derived ECM after 7 and 15 days was remodelled and functional vessel formation was detected; on the contrary, the synthetic matrix made of polytetrafluoroethylene promoted the foreign body reaction. Therefore, we demonstrated the high angiogenic potential and the absence of immunological activity of a diaphragm-derived acellular 57

matrix that can be suitable as a scaffold for skeletal muscle tissue engineering purposes.

Results are summarized: We recently studied the engraftment of recellularized patch as substitution in a diaphragmatic defect.

In particular, two systems were analyzed: (i) decellularized scaffold seeded with muscle presursor cells resuspended in a rich-plasma platelet gel (Rebulla et al, 2010), (ii) decellularized scaffold seeded with muscle precursor cells resuspended in hyaluronic acid-based hydrogel (Rossi et al, 2010). (i) To develop this task we completed a thesis project using human muscle precursor cells in decellularized diaphragm. Also a paper titled “Generation of a functioning skeletal muscle xenograft with mouse decellularized diaphragmatic extracellular matrix and human muscle precursor cells” will be submitted and here below the results are summarized.

Abstract. Naturally derived acellular matrices are biocompatible materials obtained by tissue decellularization, being considered promising tools for tissue engineering purposes. However, the in vivo application of acellular matrices has only partially proven to be effective when facing large defects, highlighting the need of more complex constructs for tissue substitution. Up to now, generation of functional grafts from acellular ex-vivo tissues has been problematic largely due to two main reasons: (i) poor likeness of the scaffolds with the tissue to be regenerated, and (ii) difficulty in primary cell expansion maintaining the proliferation and differentiation properties especially when cells are joined with the biological support. In the present work we demonstrated that paediatric human muscle precursor cells are an attractive cell source, being able not only to proliferate and differentiate in vitro, but also to give rise to a functioning D skeletal muscle tissue. We also proved that the seeded decellularized scaffold allowed cell proliferation and differentiation, confirming the potential of this type of material for tissue engineering approaches. Finally, the obtained xenograft was able to activate a regeneration response upon damage, endorsing this combination as a promising step towards the generation of a functional graft for future clinical applications. (ii) For this task at first in a mouse model of volume muscle loss we used ialuronic acid and platelet gel with human muscle cells to replenish the muscle. The synthetic polymer and the gel kept cells alive and in proliferation as shown below.

Secondly, we followed decellularization of quadriceps and characterisation.

Patent approved August 2016: “Matrice acellulare per ricostruzione in vivo di muscolo scheletrico”.

Inventors: Franzin Chiara, Piccoli Martina, Pozzobon Michela, Urbani Luca.

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fig.4

5-10 Selected Publications Michela Pozzobon irst steps to define murine amniotic uid stem cell microenvironment

ertin , iccoli M, ranzin C, Spiro , on S, ed a , Sc ia i , asc in , onaldo , ra etta , e Coppi , Pozzobon M

Isolation and pansion of Muscle ranzin C, iccoli M, r ani L, iz C, recursor Cells from Human Pozzobon M. S eletal Muscle iopsies.

am a , e Coppi ,

Reprogramming of mouse amniotic uid cells usin a i y ac transposon system

ertin , iccoli M, ranzin C, Coppi P, Pozzobon M.

Endothelial properties of thirdtrimester amniotic uid stem cells cultured in ypo ia. Stem

Sc ia o , ranzin C, l iero M, iccoli M, Spiro , ertin , r ani L, isentin S, Cosmi , adini , e Coppi , Pozzobon M.

unctional Human odocytes enerated in Or anoids from mniotic luid Stem Cells.

Sci ep. 01 1 : 0 0

a y , Milei o s y M,

e

o

Methods Mol Biol.

01 1 1 :1 04 Stem Cell esearc , 01 , 1 : 10 Cell es 1 : 0 .

er. 01 Oct

inaris C, enedetti , o elli , ate M, izzo , Conti S, omasoni S, Corna , ozzo on M, Ca allotti , o oo , Mori i M, eni ni , emuzzi .

m Soc 01 Oct

Improvement of diaphragmatic performance through orthotopic application of decellularized e tracellular matri patc .

iccoli M, r ani L, l arez allas M , ranzin C, ed a , ertin , Zuccolotto , osato , a an , l assore , e Coppi P, Pozzobon M.

iomaterials. 01 Oct 4: 4 .

Immune Regulatory Properties of C 11 pos mniotic luid Stem Cells ary ccordin to estational e.

i rapani M, assi , ontana , iacomello L, ozzo on M, uillot , e Coppi , rampera M.

ep rol. .

Stem Cells e . 01 an 1 4 1 :1 4

CV Michela Pozzobon, PhD Michela Pozzobon obtained her degree in Chemistry and Pharmaceutical Technology in 1999 and her PhD in Biology and Regenerative Medicine in 2008, at University of Padua. From 001 to 004 s e or ed in lymp oma field at O ford ni ersity, o n adcliffe Hospital, since 010 s e as een Stem Cells and Regenerative Medicine laboratory operative responsible and since une 01 senior researc er of t e ni ersity of adua. S e is author/coauthos of over 50 publications in peer-reviewed journals.

S e is co in entor of t e patent: Matrice acellulare per ricostruzione in vivo di muscolo scheletrico.�

fig.4 Storage and characterization of diaphragmatic decellularized matrix in order to evaluate the best condition for a long lasting sca old preservation. Trevisan C et al, Paper in submission

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National and international collaborations Michela Pozzobon • • • • • • • • • • • • • • • • • •

Prof Bisogno, Dipartimento Salute Donna e Bambino, Padova. Field of collaboration: Tumor microenvironment of pediatric sarcoma Prof Zavan, Dipartimento di Scienze Biomediche. Field of collaboration: extracellular matrix Prof. Pavan, Dipartimento di Ingegneria Industriale, Padova. Field of collaboration: mechanical properties and devices Prof. Balkwill, Barts Cancer Institute, Centre of Cancer and Inflammation. London. Field of collaboration: macrophages and tumor microenvironment.

Dr Agostini, Dipartimento di Scienze Chirurgiche e Gastroenterologiche, Padova. Field of collaboration: proteomics and colon tumor.

Prof Elvassore , Dipartimento di Impianti di Ingegneria industriale, Padova. Field of collaboration: Tissue engineering Dr. Vitiello, Dipartimento di Biologia Vallisneri, Padova.Field of collaboration: animal model and muscle biology Prof Vettor, Dipartimento di Medicina e Scienze Chirurgiche, Padova, Field of collaboration: muscle stem cells and adipocytes.

Prof. Angelini , Dipartimento Medical Diagn Sci.Special Ther. Padova, Field of collaboration: animal model of disease for stem cell therapy.

Dott. Tomanin, Dipartimento della Salute della Donna e del Bambino, Padova, Field of collaboration: animal model of disease for stem cell therapy.

Prof Rosato , Istituto Oncologico Veneto (IOV), Padova Field of collaboration: Imaging analysis Prof Bonaldo, Dipartimento di Biologia Vallisneri, Padova. Field of collaboration: animal model for muscle therapy with amniocytes.

Prof Remuzzi, Mario Negri, Bergamo Field of collaboration: animal model for stem cell therapy.

Dr Bollini, Dipartimento Medicina Sperimentale Genova, Field of collaboration: exosomes form AFS cells and heart repair Prof Nagy, Mount of Sinai Joseph and Wolf. Lunenfeld research Institute, Toronto Canada.

Field of collaboration: study of the development of mouse fetus and murine model Prof Cavazzana-Calvo, Department of Biotherapy, Paris France. Field of collaboration: ematopoiesis for stem cells Dr Judith Melki, UMR 788 Inserm-Université Le Kremlin- Bicetre Cedex france. Field of collaboration: animal model

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• •

Prof Atala, director of Insitute for regenerative Medicine, Richard H. Dean Biomedical Building, WinstonSalem, NC . Field of collaboration: amniocytes.

Dr Adjiaye, Max Plank Institue for Molecular Genetics Molecular Embryology and Aging Group, Berlin Germany. Field of collaboration: stem cell biology technique

Michela Pozzobon 3 Publications as first/last author 2016 15.0 IF as first/last author 2016 24 H-index (source )

Prof Maurizio Muraca Summary of research activities Extracellular vesicles derived from mesenchymal stem cells as immune modulatory tools Extracellular vesicles (EVs) are nanometer-size particles secreted by different types of cells in vivo and in vitro. EVs contain proteins, growth factors, mRNA and other molecules encapsulated in a lipid sphere and are best classified according to their size and intracellular origin, although the heterogeneity of these nanoparticles is very complex and only partially understood. Exosomes are around 0,1 micron in size, are derived from multivesicular bodies, a late endosomal compartment, and are secreted via fusion of multivesicular bodies with the plasma membrane. Shedding vesicles (also kown as “microvesicles”) are a heterogeneous population of membrane vesicles up to 1 micron in size directly released from the cell membrane through the disruption of cortical cytoskeleton. Such tiny vesicles are complex biological machines which can convey signals by interacting at the cell surface, by internalization into endocytic compartments, or by fusion with plasma membranes. In parallel with the recognition of their biological importance, EVs have become the object of increasing interest for possible clinical applications, and many Authorities now believe they will replace their cells of origin as therapeutic tools, because of several reasons.

Regarding safety, tumorigenic risk and ectopic colonization are clearly less of concern with EVs

61

fig.5

than with living cells. Results in animal models suggest that local EV administration is feasible e.g. for lung diseases (inhalant or intratracheal), to reach the CNS via the intranasal route and topical to eyes. Since the EVs are not living cells reacting to different environments, but they rather convey a definite set of signals, their effect should be both more predictable and reproducible.

The immunomodulatory effect of MSCs seems to be largely mediated by E s (Budoni et al. 201 ; Del Fattore et al. 2015), and a case of successful treatment with MSC-EVs in a patient with severe gastrointestinal steroid-resistant GVHD was reported (Kordelas et al. 2014). Administration of allogenic vs. autologous EVs should not be a major issue, since EVs do not express MHC and thus should not evoke an immune rejection, at variance with MSCs, whose MHC expression is influenced by the recipient environment. These nanoparticles can thus be produced by “waste” human tissue such as Wharton jelly from the umbilical cord and can be readily administered “offthe-shelf”. Specific cell targeting by EVs can increase the therapeutic index of EV-carried drugs, both by increasing drug concentration in the affected tissue and reducing unwanted distribution to other sensitive tissues and organs (Tian et al. 201 ; Wiklander et al. 2015). Finally, E s are much easier to be produced and cryopreserved under GMP requirements than living cells, with significant cost reduction, thus eliminating a major barrier to the diffusion of advanced therapies.

Clinical-grade EVs have been produced and tested in preliminary clinical trials on cancer immune therapy (Morse et al. 2005). We have investigated the immunomodulatory properties of EVs derived from MSCs in several in vitro and in vivo models. The inhibitory effects of MSCs on B-cell proliferation and differentiation in a CpG-stimulated peripheral blood mononuclear cell co-culture system could be fully reproduced by EVs isolated from MSC culture supernatants in a dosedependent fashion(Budoni et al. 2013). A dose-dependent inhibitory activity of MSC-EVs was also observed for IgM, IgG and IgA production. Moreover, in the same coculture system 7-AAnegative and Annexin-positive MSC-EVs isolated from mesenchymal stromal cells were labelled with the membrane red fluorescent dye PKH26 and were shown to be internalized in a subset of CD86/CD19 positive cells corresponding to activated B lymphocytes. We then studied the effects of MSC-EVs on T cells (Del Fattore et al. 2014) in human PBMC cultures treated with human T cell activator CD3/CD28 beads. Stimulation increased the number of proliferating CD3+ cells as well as of T regulatory cells (Treg). Co-culture with MSCs inhibited the proliferation of CD3+ cells, with no significant changes in apoptosis. Addition of MSC-EVs to PBMCs did not affect proliferation of CD3+ cells, but induced the apoptosis of CD3+ cells and of the CD4+ sub-population and increased the proliferation and the apoptosis of Treg. Moreover, MSC-EV treatment increased 62

fig.6

the Treg/Teff ratio and the immunosuppressive cytokine IL10 concentration in culture medium. The above results were

fig. 5 Preparation of sca old for recellularization with human muscle precursor cells (A-D).

Analysis of the recelluarized sca old after 4, and 15 days of culture (E-J).

confirmed by other investigators, who also showed that MSCEVs suppress NK cells and shift macrophage polarization to an immune suppressive M2 phenotype, inducing the differentiation of CD4+ T cells to Treg and the production of IL-10 (Di Trapani

fig. 6 Hyaluronic acid-based hydrogel recellularized with human muscle precursor cells. In vivo experiments and analysis of cell and construct engraftment after skeletal muscle damage.

et al. 2016).Thus, MSC-EVs exert their immune modulatory activity both on innate and on adaptive immunity.

Extracellular vesicles derived from mesenchymal stem cells for the treatment of inflammatory bowel disease: a bench≠ to≠ bedside approach.

In order to address more specifically the treatment of Inflammatory Bowel Disease, we studied an animal model of inflammatory bowel disease induced by Dextran Sulfate Sodium (DSS) (Del Fattore A, Luciano R, Fierabracci A, Muraca M 2014). Mice injected daily with human MSC-EVs showed less weight loss, improved disease activity index and a less severe reduction in colon length when compared to DSS/vehicle-treated controls. Real time RT-PCR analysis performed on RNA extracted from colon tissue revealed a strong inhibition of the induction of inflammatory cytokines with respect to untreated animals.

The above results prompted a collaboration with the Company Esperite, which acquired from the Bambino Gesù Hospital the patent by Muraca and Fierabracci on the use of MSCEVs as immune modulatory tools. Esperite has developed the GMP production of clinical grade EVs at the Cell Factory in Niel (Belgium) and has signed a collaboration agreement with the Department of Women’s and Children’s Health of the University of Padova to implement a first-in-man study on the use of MSC-EVs for the treatment of fistulas in Crohn’s 63

disease resistant to conventional therapy. A dossier has been submitted to the Italian Regulatory Authorities asking permission for this pioneering clinical application.

Extracellular vesicles derived from the osteoblastic lineage as therapeutic and diagnostic tools (In collaboration with A. Teti and A. Cappariello, University of LĂ­ Aquila) We have accumulated evidence that EVs are involved in bone biology. We observed that osteoblasts seeded in transwells (pore size 1 m) exchanged fluorescent probes overtime, probably by E s.

EVs were isolated from osteoblast culture media by ultracentrifugation followed by sorting by FACS. Transmission electron microscopy showed that they were intact, irregularly shaped, sized 0.15-1 m and well preserved. FACS analysis showed that 50% of EVs presented on their surface the most potent pro-ostoclastogenic cytokine, RANKL. This EV population labelled with cytoplasmic and membrane fluorescent probes fully integrated with target osteoblasts, suggesting that both membrane-bound and intravesicular components were transferred to target cells. The RNA-specific linker Syto RNA Select revealed the presence of RNA inside the EVs that was transferred to target cells. RANKL-positive EVs injected intraperitoneally in newborn WT CD1 mice delivered fluorescent lipophylic probe preferentially to the bone suggesting potential use for systemic targeted therapy as well as for imaging techniques.

RANKL-positive EVs also dose-dependently triggered osteoclastogenesis in RANKL KO mice suggesting in vivo biological activity. Osteogenic sarcoma cell line, U2OS and SAOS2, communicated by EVs with osteoblasts, monocytes and endothelial cells transferring fluorescent probes. Osteoblast E s loaded with the chemotherapeutics doxorubicin (DXR) incubated in vitro with tumor cells induced tumor cell death similar to standard treatment with 1uM of free DXR. HPLC preliminarily revealed that 100.000 D R-loaded E s efficaciously shuttled about

ng (10 nM) of D R to target cells, suggesting an strongly

enhanced antitumoural potency of DXR (two orders of magnitude) when encapsulated in EVs.

Extracellular vesicles derived from mesenchymal stem cells as anti≠angiogenic tools (In collaboration with A. Viola, University of Padova) The group of Antonella

iola demonstrated that when primed by inflammatory cytokines, both in

vitro and in vivo, MSCs change their functional properties in favor of a strong anti-angiogenic activity (Zanotti et al. 2016). Working in collaboration with Viola’s group, we showed that the anti-angiogenic actions of MSCs are exerted by the E s released upon stimulation by pro-inflammatory cytokines using both in vitro and in vivo models. We are thus further studying anti-angiogenic EVs, derived from primed MSCs, as a possible therapeutic tool for a variety of disorders.

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5-10 Selected Publications Maurizio Muraca ut micro iota deri ed outer mem rane esicles: under recognized major players in health and disease?

Muraca M, uti nani L, iera racci , eti , erilon o

Immunore ulatory effects of Mesenc ymal Stem Cell deri ed tracellular esicles on lymphocytes.

el attore , Luciano , ascucci L, offredo M, , Scapaticci M, iera racci , Muraca M.

Biotechnological approach for systemic delivery of membrane eceptor cti ator of Li and L acti e domain into t e circulation.

Cappariello , aone , Maurizi , Capulli M, ucci Muraca M, eti .

Ho far are e from t e clinical use of placental-derived mesenchymal stem cells?

.

iorda

,

iera racci , Lazzari L, Muraca M, arolini O.

isco Med 1 : 4 8,2015.

Cell Transplantation 24, 1 , 01 .

Biomaterials. 2015 pr 4 : .

pert Opin iol er. : 1 , 01 . 114.

ifferential effects of e tracellular esicles secreted by mesenchymal stem cells from different sources on lio lastoma cells.

el attore , Luciano , Saracino , attafarano , izzo C, ascucci L, lessandri , essina , errotta , iera racci , Muraca M.

pert Opin iol er.

1 4 :4 04, 014.

e immunosuppressi e effect of mesenchymal stromal cells on B lymphocytes is mediated by membrane vesicles

udoni M, iera racci , Luciano , etrini S, i Ciommo V, Muraca M.

Cell Transplant. : , 01

Bone marrow derived liver stem cells LSC en raft more efficiently li ers under oin re ection t an epatocytes: a potential novel therapeutic approach for the treatment of liver rejection.

ital I, eraresso C, o i , Hui , Ome ara , oz a , emetriou , Muraca M.

Hepatocyte transplantation as a treatment for glycogen storage disease type I

Muraca M, erunda , eri , ilei M , eltracco , Meroni M, iron , urlina

Intraportal hepatocyte transplantation in the pi . emodynamic and histopathological study.

Muraca M, eri , arenti , eltracco , ranato , ilei M , erraresso C, allarin , Zanusso , iron , oz a , erunda .

ranato , .

Sur ery 1 90,2002.

:

Lancet 18,2002.

: 1

4

Transplantation : 0 , 00 . Stem Cells Dev. 2015 Jan 1 4 1 :1 4

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CV Maurizio Muraca, MD, PhD resent position: ssociate rofessor of Medicine, ept. of omen s and C ildren s Healt , ni ersity of ado a ni ersity of ado a: M. . 1 Specialist in Internal Medicine 1 astroenterolo y 1 , ni ersity of Leu en, el ium: in ioc emistry 1 Head of Clinical C emistry and Micro iolo y, esponsi le for t e esearc rea in e enerati e Medicine Cell actory, am ino es esearc Hospital, ome 004 014 Qualified erson for t e production of d anced erapy Medical roducts Italian Medicines ency I , 00 pert in d anced erapies clinical at t e uropean Medicines ency ut or and co aut or of 1 4 ori inal articles in peer re ie ed international ournals, international oo s in n lis , oo s in Italian, 1 c apters in international oo s, 1 ori inal articles and oo c apters in Italian. He participated in t e first international clinical trial on t e treatment of li er failure it a ioartificial li er de ice nn Sur . 004 : 0 and coordinated t e first clinical epatocyte transplantation in urope Lancet 00 : 1 . He is in entor of four patents in cell t erapy. ards: 10 11 00 : Internazionale ard iuseppe Sciacca , section Medicine 1 0 011: ard entlemen d Italia for scientific acti ity

Maurizio Muraca 1 Publications as first/last author 2016 3.09 IF as first/last author 2016 29 H-index (source )

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Onco≠ hematology Unit

The IRP hosts most of the laboratories of the pediatric Onco-hematology Unit (uoc) directed by Prof. Giuseppe Basso. The laboratories include: 1) Laboratory of pediatric Onco-hematology Group Leader and Referent: Giuseppe Basso • PI: Benedetta Accordi Reserch Area: Phosphoproteomics • PI: Lara Mussolin Reserch Area: Non Hodgkin Lymphoma - Molecular Diagnostics • PI: Martina Pigazzi Reserch area: Molecular Genetics • PI: Geertuy te Kronnie Reserch area: Gene Expression NGS • PI: Luca Trentin Reserch Area: MLL Leukemia 2) Laboratory of Solid Tumor Biology Group Leader: Giuseppe Basso Referent: Gianni Bisogno • PI: Paolo Bonvini Reserch Area: Basic Signal Trasduction • PI: Lucia Tombolan Reserch Area: Solid Tumor Genomics • PI: Angelica Zin Reserch Area: Soft Tissue Sarcomas Molecular Diagnosis-Biobank 3) Other groups of the Unit Group Leader and Referent: Giuseppe Basso • Group Leader: Chiara Frasson Reserch area: Cytometry / Sorting • Group Leader: Giuseppe Germano Reserch area: ebrafish and Leukemia • Group Leader: Maddalena Paganin Reserch area: Gene Sequencing ALL • Group Leader: Luca Persano Reserch Area: Brain Tumors • Group Leader: Giam Pietro Viola Reserch area: Experimantal Pharmacology 4) Laboratory of Neuroblastoma Group Leader and Referent: Gian Paolo Tonini

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Photo of Onco≠hematology Unit

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Laboratory of pediatric Onco≠ hematology GROUP LEADER AND REFERENT

Field of interest • •

Giuseppe Basso

Research Laboratory Oncological Diagnosis and Molecular Therapy Laboratory

The Laboratory of Pediatric Oncology (LPO) is an organizational structure of the Pediatric Oncohematology Clinic.

The LPO, even with specific objectives, contributes to the achievement of the mission and vision of the Division of Pediatric Oncology Clinic.

The processes in which is divided the laboratory are as follows:

1. Provision of laboratory services for diagnosis in pediatric oncology and hematology 2. Design and research in pediatric oncology and hematology 3. Collection, handling and storage of biological material for research purposes.

The Pediatric Oncohematolology Clinic is national reference center for the diagnosis of acute leukemias and has an active role in the drafting, evaluation, monitoring and coordination of national and European protocols for diagnosis and treatment of lymphomas, sarcomas, brain tumors. It also plays the role of Biological Bank of pediatric samples of leukemias, lymphomas, sarcomas.

The objectives of the Pediatric Oncohematology Laboratory are aimed at provide the pediatric patient with malignant hematological diseases:

• •

the best diagnostic techniques and monitoring of cancer disease, using the most advanced laboratory techniques; deepening in research, development and application of new biomedical knowledge.

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The laboratory plays a leading role in the study of minimal residual disease, both in the molecular and flow cytometry part, by participating in international monitoring of standardization and quality control.

Collaboration with most important world institutions.

Clinical Research Leukemia / Myelodysplasia / Myeloproliferative Syndromes: • development and application of national and international diagnostic and therapy protocols; • centralization of morphological diagnosis and haematological counseling of acute lymphoblastic and myeloblastic leukemia, myelodysplasia and Bone marrow insufficiency , for Italian Pediatric Oncohematology centers.

Lymphomas and histiocytosis: • development and coordination of national protocols for the diagnosis and therapy of non-Hodgkin lymphomas; • centralization of diagnosis and counseling Italian Pediatric Oncohematology Centers; • monitoring of protocols application and toxicity ; collecting data for evaluating therapy results; • coordination of international activities related to diagnosis and treatment of non-Hodgkin lymphoma in children and adolescents; • development and application of national and international protocols for Hodgkin lymphoma and Langerhans Histiocytosis and Lymphohistiocytosis.

Solid Tumors: • development and coordination of national and international protocols for soft tissue sarcomas in the framework of the ‘ European Pediatrics Sarcoma Study Group - (EpSSG): RMS2005 and NRSTS2005; for liver tumors: SIOPEL ; for rare cancers: Project TREP; • centralization of diagnosis, centralized molecular diagnostics and counseling for Italian Pediatric Oncohematology Centers; • monitoring of protocols application and toxicity ; collecting data for evaluating therapy results; • development and participation in national and international protocols for other oncological diseases such as bone tumors, kidney tumors, retinoblastoma; • study of new drugs and treatment outcome in childhood solid tumors.

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Brain Tumors: • development and coordination of the European Protocol SIOP - LGG 2004 for low-grade gliomas of the Central Nervous System (CNS ); • development and participation in national and international protocols for diagnosis and therapy for CNS neoplasms.

Hematopoietic stem cell transplantation: • implementation and evaluation of the reduced conditioning regimens toxicity; new therapies for graft host disease; • participation in the drafting and implementation of national and International protocols conditioning and treatment of graft versus host disease; • new Indications for allogeneic hematopoietic stem cell transplantation (HSCT).

Infectious diseases and supportive care: • early Detection and pre - emptive therapy of fungal and viral infections; • use Of growth factors in childhood; • vaccinations in the oncohematologic patient; • epidemiology of sepsis in pediatric oncohematology. mmunodeficiencies: • development and participation in national protocols for diagnosis and treatment of primary immunodeficiencies.

Non Oncologic Hematology: • development and participation in national protocols for diagnosis and treatment of neutropenia, constitutional and acquired bone marrow aplasia, constitutional marrow failure; • participation to developmental surveillance protocols of complications of thalassemic patients; • development and participation in national and international epidemiological studies, diagnostic and clinical monitoring of sickle cell disease in non-endemic areas; ( Scates - Transcranial Doppler; ENERCA III; CASIRE Renal Study; CASIRE Twin - Sib Study); • participation in the drafting of national guidelines for the diagnosis and treatment of sickle cell disease; • development and coordination with Thrombocytopenia Regional Register in children of diagnostic and treatment protocol of thrombocytopenia.

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Psycho-oncology: neurocognitive rehabilitation of patients treated for brain tumors; Observational, multicenter, multidisciplinary, multi-method, international study in partnership with the National Institute for Child Health and Development, NIH, USA and the Neuropsychiatric Institute, UCLA, USA; • identification of psychosocial family factors that determine a better adaptation and a positive quality of life in children treated for oncological disease; • study for the identification of “ post - traumatic stress symptoms predictors in children’s parents; • assessment of the state of isolation and anxiety in patients in hospital for a bone marrow transplant; • multicenter observational study on cognitive neuro complications of sickle cell disease.

• •

New drugs: search for new drugs and therapeutic approaches to patients not treatable with standard treatment (phase II protocols ); • participation in national and international clinical trials on new drugs; • research on “off label” drugs in children.

•

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10-20 Selected Publications Buitenkamp TD, Izraeli S, Zimmermann M, Forestier E, Heerema NA, van den HeuvelEibrink MM, Pieters R, Korbijn CM, Silverman LB, Schmiegelow K, Liang DC, Horibe K, Arico M, Biondi A, Basso G, Rabin KR, Schrappe M, Cario G, Mann G, Morak M, Panzer-Grümayer R, Mondelaers V, Lammens T, Cavé H, Stark B, Ganmore I, Moorman AV, Vora A, Hunger SP, Pui CH, Mullighan CG, Manabe A, Escherich G, Kowalczyk JR, Whitlock JA, Zwaan CM.

Acute lymphoblastic leukemia in children with Down syndrome: a retrospective analysis from the Ponte di Legno study group.

Blood. 2014, 123(1):70-7.

Meissner B, Bartram T, Eckert C, Trka J, Panzer-Grümayer R, Hermanova I, Ellinghaus E, Franke A, Möricke A, Schrauder A, Teigler-Schlegel A, Dörge P, von Stackelberg A, Basso G, Bartram CR, Kirschner-Schwabe R, Bornhäuser B, Bourquin JP, Cazzaniga G, Hauer J, Attarbaschi A, Izraeli S, Zaliova M, Cario G, Zimmermann M, Avigad S, Sokalska-Duhme M, Metzler M, Schrappe M, Koehler R, Te Kronnie G, Stanulla M.

Frequent and sex-biased deletion of SLX4IP by illegitimate V(D)J-mediated recombination in childhood acute lymphoblastic leukemia.

Hum Mol Genet. 2014, 23(3):590-601.

Pillozzi S, Accordi B, Rebora P, Serafin , alsecc i M , asso , Arcangeli A.

ifferential e pression of 1 and 1 enes in pediatric acute lymp o lastic leu emia identifies different prognostic subgroups.

Leukemia. 2014, 28(6):1352-5.

Azzolin L, Panciera T, Soligo S, Enzo E, Bicciato S, Dupont S, Bresolin S, Frasson C, Basso G, Guzzardo V, Fassina A, Cordenonsi M, Piccolo S.

Z incorporation in t e catenin destruction comple orchestrates the Wnt response.

Cell. 2014, 158(1):157-70.

Nikolaev SI, Garieri M, Santoni F, Falconnet E, Ribaux P, Guipponi M, Murray A, Groet J, Giarin E, Basso G, Nizetic D, Antonarakis SE.

Frequent cases of RAS-mutated Down syndrome acute lymphoblastic leukaemia lack JAK2 mutations.

Nat Commun. 2014, 5:4654

Paganin M, Fabbri G, Conter V, Barisone E, Polato K, Cazzaniga G, Giraldi E, Fagioli F, Aricò M, Valsecchi MG, Basso G.

Postinduction minimal residual disease monitoring by polymerase chain reaction in children with acute lymphoblastic leukemia.

J Clin Oncol. 2014, 32(31):3553-8.

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Conter V, Valsecchi MG, Parasole R, Putti MC, Locatelli F, Barisone E, Lo Nigro L, Santoro N, Aricò M, Ziino O, Pession A, Testi AM, Micalizzi C, Casale F, Zecca M, Casazza G, Tamaro P, La Barba G, Notarangelo LD, Silvestri D, Colombini A, Rizzari C, Biondi A, Masera G, Basso G.

C ild ood i ris acute lymp o lastic leu emia in first remission: results after chemotherapy or transplant from the AIEOP ALL 2000 study.

Blood. 2014, 123(10):1470-8.

Bertacchini J, Guida M, Accordi B, Mediani L, Martelli AM, Barozzi P, Petricoin E 3rd, Liotta L, Milani G, Giordan M, Luppi M, Forghieri F, De Pol A, Cocco L, Basso G, Marmiroli S.

Feedbacks and adaptive capabilities of the PI3K/Akt/ mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis.

Leukemia. 2014, 28(11):2197-205.

Manara E, Baron E, Tregnago C, Aveic S, Bisio V, Bresolin S, Masetti R, Locatelli F, Basso G, Pigazzi M.

MLL-AF6 fusion oncogene sequesters AF6 into the nucleus to trigger RAS activation in myeloid leukemia.

Blood. 2014, 124(2):263-72.

Fischer U, Forster M, Rinaldi A, enomics and dru profilin of fatal C HL positi e Risch T, Sungalee S, Warnatz acute lymp o lastic leu emia identifies recurrent HJ, Bornhauser B, Gombert M, mutation patterns and therapeutic options.

Kratsch C, Stütz AM, Sultan M, Tchinda J, Worth CL, Amstislavskiy V, Badarinarayan N, Baruchel A, Bartram T, Basso G, Canpolat C, Cario G, Cavé H, Dakaj D, Delorenzi M, Dobay MP, Eckert C, Ellinghaus E, Eugster S, Frismantas V, Ginzel S, Haas OA, Heidenreich O, Hemmrich-Stanisak G, Hezaveh K, Höll JI, Hornhardt S, Husemann P, Kachroo P, Kratz CP, Kronnie GT, Marovca B, Niggli F, McHardy AC, Moorman AV, Panzer-Grümayer R, Petersen BS, Raeder B, Ralser M, Rosenstiel P, Schäfer D, Schrappe M, Schreiber S, Schütte M, Stade B, Thiele R, Weid Nv, Vora A, Zaliova M, Zhang L, Zichner T, Zimmermann M, Lehrach H, Borkhardt A, Bourquin JP, Franke A, Korbel JO, Stanulla M, Yaspo ML.

Nat Genet. 2015, 47(9):1020-9.

Lana T, de Lorenzo P, Bresolin S, Bronzini I, den Boer ML, Cavé H, ro o , Stanulla M, Zalio a M, Harrison CJ, de Groot H, Grazia Valsecchi M, Biondi A, Basso G, Cazzaniga G, Te Kronnie G

efinement of I Z 1 status in pediatric p iladelp ia positive acute lymphoblastic leukemia.

Leukemia. 2015, 29(10):2107-10.

Gambacorti-Passerini C, Mussolin L, Brugieres L.

Abrupt Relapse of ALK-Positive Lymphoma after Discontinuation of Crizotinib.

N Engl J Med. 2016, 374(1): 95-6.

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Conter V, Valsecchi MG, Buldini B, Early T-cell precursor acute lymphoblastic leukaemia Parasole R, Locatelli F, Colombini in children treated in AIEOP centres with AIEOP-BFM A, Rizzari C, Putti MC, Barisone protocols: a retrospective analysis.

E, Lo Nigro L, Santoro N, Ziino O, Pession A, Testi AM, Micalizzi C, Casale F, Pierani P, Cesaro S, Cellini M, Silvestri D, Cazzaniga G, Biondi A, Basso G.

Lancet Haematol. 2016, 3(2):e80-6.

Palanichamy JK, Tran TM, Howard RNA-binding protein IGF2BP3 targeting of oncogenic JM, Contreras JR, Fernando transcripts promotes hematopoietic progenitor TR, Sterne-Weiler T, Katzman S, proliferation.

Toloue M, Yan W, Basso G, Pigazzi M, Sanford JR, Rao DS.

J Clin Invest. 2016, 126(4):1495-511.

Tosello V, Bordin F, Yu J, Agnusdei V, Indraccolo S, Basso G, Amadori A, Piovan E.

Leukemia. 2016, 30(4):812-22.

Calcineurin and GSK-3 inhibition sensitizes T-cell acute lymphoblastic leukemia cells to apoptosis through X-linked inhibitor of apoptosis protein degradation.

Helsmoortel HH, Bresolin S, LI o ere pression defines a no el fetal li e Lammens T, CavĂŠ H, Noellke P, subgroup of juvenile myelomonocytic leukemia.

Caye A, Ghazavi F, de Vries A, Hasle H, Labarque V, Masetti R, Stary J, An den Heuvel-Eibrink MM, PhilippĂŠ J, Van Roy N, Benoit Y, Speleman F, Niemeyer C, Flotho C, Basso G, Te Kronnie G, Van Vlierberghe P, De Moerloose B.

Blood. 2016, 127(9):1163-72.

Tregnago C, Manara E, Zampini M, Bisio V, Borga C, Bresolin S, Aveic S, Germano G, Basso G, Pigazzi M.

C

Leukemia. 2016, 30(9):1887-96.

Desbats MA, Morbidoni V, SilicBenussi M, Doimo M, Ciminale V, Cassina M, Sacconi S, Hirano M, Basso G, Pierrel F, Navas P, Salviati L, Trevisson E.

The COQ2 genotype predicts the severity of coenzyme Q10 deficiency.

Hum Mol Genet. 2016, 25(19):4256-4265.

Panciera T, Azzolin L, Fujimura A, Di Biagio D, Frasson C, Bresolin S, Soligo S, Basso G, Bicciato S, Rosato A, Cordenonsi M, Piccolo S.

Induction of panda le issue Specific Stem ro enitor Cells through Transient Expression of YAP/TAZ.

Cell Stem Cell. 2016, 19(6):725-737.

en a es C

to initiate leu emo enesis.

Oshima K, Khiabanian H, da Silva- Mutational landscape, clonal evolution patterns, and Almeida AC, Tzoneva G, Abate F, role of RAS mutations in relapsed acute lymphoblastic Ambesi-Impiombato A, Sanchezleukemia.

Martin M, Carpenter Z, Penson A, Perez-Garcia A, Eckert C, Nicolas C, Balbin M, Sulis ML, Kato M, Koh K, Paganin M, Basso G, GastierFoster JM, Devidas M, Loh ML, Kirschner-Schwabe R, Palomero T, Rabadan R, Ferrando AA.

Proc Natl Acad Sci U S A. 2016, 113(40):11306-11311.

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CV Giuseppe Basso, MD - Internal student at Pediatrics Clinic, Padua University since November 1972.

- Degree in Medicine in 1974.

- Specialization in Pediatrics in 1977.

- Specialization in Hematology in 1980.

- University intern physician at Pediatrics Clinic of Padua University since 1.12.74 to 31.07.80.

- University fellow since 01.10.76 to 1981 at Pediatrics Clinic Institute.

Confirmed researc er on 1.10. 0.

Research Activity Since 1994 he has been teaching various courses in General and Pediatric hematology at the School of Pediatrics, at the School of Pediatrics, at the School of Laboratory Medicine and at the School of Hematology at Padua University.

His research interest has always dealt with haematological neoplasms of childhood, in particular acute lymphoblastics and acute myeloid leu emias. His field of or includes all steps of la oratory or up for dia nosis of leu emias, particularly o cytometry for immunop anotypin , ploidy and functional studies. He also oe ed on mocular iolo y both in his laboratory and in collaboration with other collegues. Since 1990 he is in charge of the Biological Bank for acute leukemias of the AIEOP (Italian Association for Pediaric Hematology and Oncology) and he is responsible for centralized diagnosis of all cases entering the Italian therapeutic protocols. He is also member of many international committees on pediatric leukemias, solid tumor and hepatoblastomas biology.

Membership Italian Society of Pediatrics, Italian Society of hematology, AIEOP (Italian association for Pediatric Hematology and Oncology), National Coordinator of Acute Leukemias Phenotype AIEOP Study Group, Committee for Diagnosis and Biology of International BFM Family, Founder member of EWOG – European Working Group for Myelodisplastic Syndromes in Childhood, Solid Tumors FONOP (Operative task force on pediatric oncology) study goup, Leukemias Biology FONOP study group, International Society for Laboratory of Hematology, Inernational Society of Analytical Cytology, American Association for the Advancement of Science, American Society of Heamatology, Cytometry Italian Group, International Society of Pediatric Oncology (SIOP), Liver Tumor Study Group: head of the Liver Tissue Bank SIOP, International Task Force on Standards of Practice for Diagnostic Hematologic Cytometry. President AIEOP years 2000-2001. President I O Italian ediaric Hematolo y Oncolo y oundation . Mem er of Scientific aluation e pert roup (National Agency of evaluation of the University research).

Research Projects Member of National Health Commission of Research.

Member of national Commission of Research Health. Città della Speranza Foundation, CNR (National Research Council) Head of researc units financed y M S Ministry of ni ersity and for Scientific and ec nolo ic esearc , I C (Italian association for cancer research), Città della Speranza Foundation, CNR (national Research Council), Network of excellence.european Leukemia net. (VI program). FP7-NPM-2008-SMALL-2 MONAD Program. Partner of MONAD Program-FP7 EU, Partner of ENCCA Program-FP7-EU, Partner of TRANSCAN Program-FP7 EU. Currently PI for sponsored clinical trials.Italian Ministry of Health Research Program 2013: Tool promoting empowerment in pediatric hematology oncology – to evaluate the quality of pediatric hematology oncology care.

Publications He is re ie er of International scientific papers as ournal of Clinical Oncolo y, lood, Leu emia, roteomics. He is author of more than 450 peer reviewer publications. IF = 3.590,96 – H index 74

Giuseppe Basso 4 Publications as first/last author 2016 17.73 IF as first/last author 2016 6 H-index (source )

76

RESERCH AREA: Phosphoproteomics

PI:

Summary of recent research activities and of main results achieved os

Benedetta Accordi

o oteomic

ofilin

t ou

e e se

ase

Protein Arrays of Pediatric Acute Leukemias The principal aim of our research is the identification of new

GROUP MEMBER

disease biomarkers and new therapeutic targets for acute

Post≠Doctoral Fellow

pediatric leukemias through phosphoproteomic profiling

Valentina Serafin

by Reverse Phase Protein Arrays (RPPA). We believe that monitoring the activation status of signal transduction pathways will be key to identify patient subgroups that can benefit from the use of specific kinase inhibitors and to point out proteins suitable for patients risk stratification and targeted therapy.

RPPA are ideally designed to quantify the activation levels of a large amount of proteins in up to 320 primary samples simultaneously, without experimental variability and from a very low amount of cells. In recent years through RPPA we identified and then validated new potential biomarkers and therapeutic targets for B-ALL, T-ALL and AML pediatric patients. e e se

ase

otein A a s a

lied to ot e diseases

The Phosphoproteomics group is in charge of the RPPA facility management and is involved in several national and international collaborative studies focused on the phosphoproteomic profiling of different neoplastic and non neoplastic diseases. This cutting-edge technology has already been applied with success to profile the activation status of

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fig.1

cellular signalling pathways in adult cancer patients affected

by AML, T-ALL, B-CLL and Primary Effusion Lymphoma, or in patients affected by immune disorders such as Psoriatic Arthritis and acute or chronic Graft versus Host Disease.

Moreover, RPPA were also used to describe the effects of treatment with specific kinase inhibitors in human Leukemia, Medulloblastoma, Breast and Prostate cancer cell lines.

Ongoing/Future projects: Combining metabolic and signaling inhibition in pediatric T-ALL In contrast to its extensive genetic characterization, T-ALL remains poorly characterized from a bioenergetical point of view. Recent exciting evidence in mouse models established that metabolism of T-ALL cells with mutated Notch shifts towards glutaminolysis, but if cells carry both Notch1 and PTEN mutation/deletion, Notch inhibition shifts metabolism towards glycolysis most likely due to concomitant hyperactivation of PI3K pathway. We thus aim to better define the metabolic program of human pediatric T-ALL genetic phenotypes, along with the signaling pathways regulating this program (through RPPA), and to correlate them to the cytotoxic effects induced by inhibition of aerobic glycolysis/glutaminolysis alone or in combination with signaling pathway-specific targeting and conventional chemotherapy.

78

fig.1 Example of an RPPA experiment. (A) A representative image of an RPPA slide, with highlighted in red the group of spots representing one sample. (B) The RPPA screening identifies di erentially activated kinases between patients groups. (C) Di erentially activated kinases are targeted with specific inhibitors in in vitro models and e ects are evaluated through MTT experiments and or other functional assays. (D) Selected inhibitors are tested in in vivo mice models.

Validation of new therapeutic targets in pediatric B- and T-ALL We previously identified through RPPA analyses new promising drug targets in Prednisone Poor Responder T-ALL patients and in ETP-ALL patients. We are currently working on in vitro and in vivo models to validate these targets and identify the most suitable kinase inhibitors to induce cancer cells death, with the final aim to give insights into molecular mechanisms determining resistance and to point out new drugs to support conventional therapies. Moreover, we identified in a specific subgroup of pediatric B-ALL the hyperactivation of a non-receptor cytoplasmic tyrosine kinase at diagnosis in patients who will encounter relapse. Now we plan to screen the activation of this kinase through RPPA in a wider and more representative cohort of patients at diagnosis and relapse to investigate its activation also at relapse occurrence. We will then proceed with in vitro and in vivo validation to evaluate its potential in new therapies for relapsed patients.

National and international collaborations: •

Dr. S. Indraccolo, Immunology and Molecular Oncology Unit, Istituto Oncologico Veneto (Italy).

•

Dr. U. Fiocco, Department of Medicine DIMED, Rheumatology Unit, University of Padova (Italy).

•

Prof. G. Semenzato, Department of Medicine, Hematology and Clinical Immunology Branch, University of Padova (Italy).

•

Prof. A. Arcangeli, Department of Experimental Pathology and Oncology, University of Firenze (Italy).

•

Prof. S. Marmiroli, Department of Anatomy and Histology, University of Modena and Reggio Emilia (Italy).

•

Prof. M. Zollo, Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II (Italy) and Ceinge Biotecnologie Avanzate (Italy).

•

Prof. LA. Liotta and Prof. E. Petricoin III, Center for Applied Proteomics and Molecular Medicine, George Mason University, VA (USA).

•

Prof. K. Sakamoto, Division of Hematology/Oncology, Department of Pediatrics, Stanford University School of Medicine, CA (USA).

•

Prof. P. Van Vlierberghe, Center for Medical Genetics Ghent, Ghent University (Belgium).

79

CV Benedetta Accordi She graduated in 2002 in Biological Sciences at the University of Padova. In 2003 she had the National qualifying examination for Biologist. In 2006 she received her PhD in “Biologia dello Sviluppo e Scienze della Programmazione”.

During her PhD she attended the Laboratory of Pediatric Oncohematology, University of Padova, where she focused her research on molecular biology of pediatric B-ALL. In 2005-2006 she attended the Laboratory headed by Dr. LA Liotta and Dr. E. Petricoin III at the Center for Applied Proteomics and Molecular Medicine, George Mason University (VA, USA), where she was trained on the Reverse Phase rotein rrays tec ni ue. S e or ed on t e p osp oproteomic profilin of pediatric LL supported y a rant from the Istituto Superiore di Sanità.

From October 2006 she is working at the Laboratory of Pediatric Oncohematology, University of Padova, where she set up t e osp oproteomics facility. Her researc topic focuses on p osp oproteomic profilin of pediatric leu emias and other pediatric and adult diseases.

Benedetta Accordi 1 Publications as first/last author 2016 10.43 IF as first/last author 2016 11 H-index (source )

80

RESERCH AREA: Non Hodgkin Lymphoma ≠Molecular Diagnostics

PI:

Lara Mussolin

GROUP MEMBERS

Field of interest Non-Hodgkin lymphoma (NHL) is a heterogeneous group of lymphoid malignancies and it is the fourth most common malignancy across the pediatric age spectrum. Our research area of interest is dedicated mainly to the study and characterization of NHL of childhood. The general approach includes the analysis of molecular mechanisms of tumourigenesis with a translational approach aimed to transfer biological results from the bench to clinical trials. This includes also the study of new tumour specific markers for the diagnosis and the prognosis of various malignancies and the study of minimal disseminated disease.

Summary of recent research activities and of main results achieved Staff Scientists: Federica Lovisa, PhD Elena Pomari, PhD Simona Primerano Technician: Elisa Tosato Student: Elena Cavallari

mic o A and ene e ession si natu e in ediat ic T-cell lymphoblastic lymphoma. The use of intensive treatments has significantly improved the outcome of patients with T-cell lymphoblastic lymphoma (T-LBL), and event-free survival rates of 60% to 70% are now reported. However, the prognosis of relapsed patients still remains poor and the intensive treatment regimens are accompanied by high toxicity with considerable mortality and morbidity. In the last few years a new class of small, regulatory RNAs has been shown to play an important role in the regulation of multiple genes, and since hundreds of miRNA genes are predicted to be present in higher eukaryotes, the potential regulatory circuitry influenced by miRNA is enormous. For this reason, a relevant aim of our research in these last years was to study miRNAs expression in a series of pediatric T-LBL and perform a comparative analysis of the miRNA

81

fig.1

and Gene Expression profiles to define which genes and miRNAs may play a relevant pathogenetic role in tumour initiation and tumour progression, or which genes may be linked to defective tumour response to treatment. Our data was published in Leukemia Journal (Mussolin L et al. 2014).

2. Genome characterization of oncogenic activity of ALK kinase in Anaplastic Large Cell Lymphoma of childhood. The Anaplastic Lymphoma Kinase (ALK) gene codes for a receptor tyrosine kinase (TK) and is fused to nucleophosmin (NPM) as a result of the t(2;5)(p2 ;q 5) chromosomal translocation in the great majority of Anaplastic Large Cell Lymphomas (ALCL) of childhood and adolescence. We performed gene expression analysis of a series of pediatric ALCL tumours and reactive lymph nodes, identifying two novel patient subgroups characterized by differential NPM-AL expression (AL -low and AL -high) and outcome. In particular, genes up-regulated in ALK-high samples included molecules involved in cell cycle progression and division, including Aurora kinases A (AURKA) and B (AURKB). Treatment with AURKA and AURKB inhibitors led to growth inhibition and cell cycle arrest in ALCL cell lines. With our study we identified two novel subgroups of AL ALCL, whose unique biological characteristics may be used to improve tumourclassification, patients’ risk stratification and to exploit new therapeutic strategies. Our data was recently published in Leukemia Journal (Pomari et al. 2016).

3. Minimal Disseminated and Minimal Residual Disease study. Our laboratory has established several new molecular genetic assays for the identification of tumour specific markers to be used in conjunction with histology and immunohistochemistry in the diagnosis of pediatric malignancies. By using newly established molecular markers, such as NPM-ALK transcripts, IgH-MYC rearrangements and T-cell receptor and IgHidiotypic rearrangements we have conducted prospective studies on minimal disseminated and minimal residual disease in Burkitt lymphoma, B-cell acute leukemia and in ALCL, thus demonstrating the clinical relevance of minimal disease in this settings. In addition, we recently established a combination of molecular and immunological assays that can discriminate patients with ALCL at high risk of relapse. Based on this assays, we are planning the next European trial for the diagnosis and treatment of children with ALCL, with the aim to design a tailored therapy which will be most effective but at the same time will spare drug-related toxicity in patients with limited risk of relapse.

Ongoing/Future projects enomics d i en identification o molecula mec anisms and ioma e s in ediat ic ollicula lymphoma.

Follicular lymphoma (FL) is the second most common histological subtype of non-Hodgkin lymphoma (NHL) of adulthood; pediatric FL (pFL) has recently been recognized as a novel variant of FL in the World Health Organization classification of lymphoma, accounting for not more than 2of NHLs in this age group. The molecular mechanisms involved in the pathogenesis of pFL still remain to be elucidated. Starting from this 82

fig.2

scenario and based on the systematic accrual of patients and tumour biopsies in our laboratory for centralized molecular diagnosis of pediatric NHL, the main goals of the present research program will be: a) to analyze the mutational landscape of pFL by exome sequencing of 13 tumour biopsies; b) to investigate the most recurrent mutations in a larger cohort of patients including adult FL and benign hyperplasia; c) to integrate these data with the analysis of gene expression profiles obtained from the same set of samples. enomic and oteomic c a acte i ation o e osomes in Non-Hodgkin Lymphoma of childhood. A field of interest that has grown exponentially in the last few years, impacting various areas of research, is the extracellular vescicles (EVs) study, in particular of small EVs formed inside endosomal compartments (i.e., exosomes). Exosomes are actively released vesicles (carrying RNA, DNA and protein) that can function as inter-cellular messengers. Exosomes and other EVs are particularly interesting as cancer biomarkers since they are stable carriers of genetic material and proteins from their cell of origin. The main goals we aim to achieve are: 1) to isolate EVs, in particular exosomes, from plasma samples in patients with NHL; 2) to characterize the exosomes at a transcriptional level (microRNA, mRNA, lncRNA) comparing them with transcriptional data obtained from primitive tumour cells and 3) to perform an extensive research on the proteolipidic composition of cancer cell-derived exosomes to establish their role in disease progression and/or resistance.

fig.1 A K expression levels identifies two distinct signature in A C . A) Supervised hierarchical clustering performed on 23 paediatric A C tumour biopsies and 4 cell lines gene expression profiles di erentially expressed between A K-high and A K-low cases. B) Boxplot of di erential A K expression in A C tumour biopsies detected by GEP (Welch s t-test, p 0.026). C) NPM-A K expression by RQ-PCR in 42 A C tumour biopsies. Blue and yellow dots represent tumour biopsies evaluated also by GEP analysis. Red dots represent new 1 tumour biopsies (Welch s t-test, p 0.0001). The horizontal lines indicate the median expression level.

fig.2 Validation of isolated plasma-derived exosomes from an A C plasma sample at diagnosis. A) Screenshot from a 60-s video image of a 1:1000 diluted exosome sample ac uired on a NanoSight M10. B) Imaging of exosomes performed by Atomic Force Microscopy (ATM).

3. New prognostic factors in pediatric T-cell lymphoblastic lymphoma. A cooperative international study has been recently initiated by the European Inter-group co-operation on Childhood Non-Hodgkin Lymphoma (EICNHL), to identify biological prognostic factors for a new risk stratification system for T-LBL 83

which serves as the basis for subsequent clinical trials with risk adapted treatment intensity. At the end of this project we will be able to evaluate the potential prognostic role of candidate markers in our national cohort of pediatric T-LBL patients. These candidate markers include not only genetic variants (e.g. PTEN, NOTCH-1, FBXW7, LOH6q and TRG locus rearrangement status), but also regulatory molecules such as microRNAs. Our data will be shared and discussed within the steering committee of the international LBL treatment trial which includes at least one representative of each international participating group which contributes data.

National and international collaborations: • • • • • •

PI: Dr. E. d’Amore PI: Prof. S. Bortoluzzi PI: Prof. Gambacorti-Passerini PI: Dr. V De Re PI: Prof. W. Woessmann PI: Dr. K. Basso/Prof. Dalla Favera

Department of Pathology, Ospedale S. Bortolo, Vicenza, Italy Molecular Biology Department, Padua University, Italy University of Milan-Bicocca, Monza, Italy Bio-ProteomicFacility, CRO Aviano, Italy Justus-Liebig University, Giessen, Germany Columbia University, New York, USA

CV Lara Mussolin, PhD Graduation in Biology with 110/110 at School of Biological Sciences, University of Padua in 1999. Research Doctorate in Oncological Sciences of the Childhood, University of Padua, in 2004 (Doctorate dissertation: “NPM-ALK as molecular marker in ALCL: implications for diagnosis and prognosis”). Specialization in Clinical Pathology with 70/70 with honours at School of Medicine, University of Padua. From 2001 responsible of molecular diagnosis of non-Hodgkin lymphomas (NHL) of childhood in the laboratory, that centralize samples from all national AIEOP (Associazione Italiana di EmatoOncologiaPediatrica) centres. Member of the EICNHL (European InterGroup of Non-Hodgkin Lymphoma) Group for the study of Minimal Residual Disease in non-Hodgkin Lymphomaof childhood. Member of the of non-Hodgkin Lymphoma working Group, member of the Biology working Group and member of the Hodgkin lymphoma working Group of AIEOP Association. Involved in laboratory research aimed to identify new prognostic markers in NHL through modern molecular techniques (i.e. microarray). Invited to numerous national and international conferences as a speaker.Teaching activities in t e ni ersity Master s e ree in ediatric Hematolo y or anized iannual y t e aculty of Medicine of t e ni ersity of the Studies of Rome La Sapienza. Author of over 30 publications on international journals and of over 80 abstracts selected for poster or oral presentations to international and national scientific meetin s. ISI OS H inde 11 I tot 0

Lara Mussolin 2 Publications as first/last author 2016 13.7 IF as first/last author 2016 15 H-index (source )

84

RESERCH AREA: Molecular Genetics

PI:

Martina Pigazzi GROUP MEMBER Post≠Doctoral Fellows: Claudia Tregnago Valeria Bisio Matteo Zampini Elena Manara Technician: Katia Polato

Summary of recent research activities and of main results achieved (2013≠2016) Genetic characterization of acute myeloid leukemia (AML).

Cytogenetic and genetic studies permitted the identification of a great number of molecular markers in the last two decades, that have been used to guide diagnosis and clinical management of pediatric patients. Nowadays, despite a final improvement in the outcome of AML due to the use of intensive multimodality treatment established through prospective clinical trials by cooperative groups of the global pediatric leukemia network, up to 30% of children with AML still experience relapse, and 40 of them die from their disease. The identification of structural alterations, such as inter- and intra-chromosomal rearrangements, remains the most frequent mechanism of mutagenesis occurring in pediatric AML, but for a large proportion of patients (up to 50%) a genetic biomarkers is not present at diagnosis. We characterized new unknown mutations in leukemia at diagnosis, we established their prognostic and biological role for several of them and we created assays for the monitoring of minimal molecular disease, increasing the ability to follow patients during follow up. The new markers have been included in the new AIEOP-AML 2013 trial and will contribute to improve outcome of patients.

Characterization of the cAMP response element binding otein in acute m eloid leu emia A CREB is one of the most important transcription factors that play a crucial role in normal and neoplastic hematopoiesis, governing many critical cellular processes, including proliferation and differentiation of myeloid progenitor cells. Considering that 66 of pediatric AML exhibit high levels of CREB, the dissection of 85

its leukemogenic mechanism has high biological and clinical relevance. We previously demonstrated that enforced expression of CREB in healthy bone marrow cells promoted aberrant growth and survival ability principally through the upregulation of specific downstream target genes. On the contrary, CREB levels downregulation induced in AML cell lines suppressed cell proliferation and lowered clonogenic tumor ability. We generated a zebrafish model overexpressing CREB in the myeloid lineage, which showed an aberrant regulation of primitive hematopoiesis, and in 79% of adult CREB-zebrafish a block of myeloid differentiation, triggering to a monocytic leukemia akin the human counterpart. We provide a new attractive in vivo model for further highthroughput drug screening, which is the main challenge to improve AML therapeutic strategies.

Anti-apoptotic BCL-2 associated AthanoGene 1 (BAG1) protein in pediatric AML. BAG-1 is a pro-survival protein that is frequently used by the tumor cells to sustain their uncontrolled expansion.

In a synergic action with BCL-2, one of the most relevant anti-apoptotic molecules in cancer, BAG-1 impedes the induction of cell death during the use of anti-cancer drugs, decreasing in that way the efficiency of administered therapy. In pediatric AML, we have determined the importance of upregulated BAG-1 protein expression for patients’ overall survival. In addition, using the in vitro approaches, we have established a link between increased BAG-1 level in leukemic cells and their poor response to several cytotoxic drugs such as doxorubicin, VP16 and ABT-737. We demonstrated that after abrogation of BAG1 expression in these cells, the cytotoxicity of anti-cancer drugs was significantly improved. These findings gave further proof that upregulated levels of BAG-1 sustained 86

fig.1 A) Event free survival (EFS) probability of patients with M -rearranged AM . The group of t(11;other) represents t(11;1 )( 23;p13.1), t(11;1 )( 23;p13.3), t(x;11)( 24; 23) and t(11;1 ) ( 23; 25) grouped together, all showing a favorable outcome (EFS 4.1%), whereas the t(6;11)( 2 ; 23) together with t(10;11)(p12; 23) showed a very poor clinical outcome (EFS 23.3%) (P 0.01). Right panel represents the typical AM cells morphology, with an accumulation of clonal blasts at the expense of normal hematopoietic lineages, which appears to be blocked at the same stage of di erentiation.

B) The immuno uorescence shows colocalization of AF6 (red) and RAS (green) in SHI-1 after M AF6 silencing (merged signals, yellow, nuclei blue, 60 zoom). In the negative control (scRNA), the punctuate pattern of AF6 nuclear localization is visible (red AF6, nuclei blue, 60 zoom).

C) CREB-overexpressing zebrafish as a leukemia in vivo model. CREB overexpression in myeloid cells has been obtained by in ecting an EGFPCREB plasmid in zebrafish embryos at 1-cell stage under the control of pu.1 promoter.

EGFP expression in transgenic CREB-zebrafish embryos at 24 hpf is detected in anterior lateral mesoderm (A M) and in intermediate cell mass (ICM), typical sites of primitive hematopoiesis.

Whole mount in situ hybridization reveals an aberrant primitive hematpoiesis in CREBzebrafish embryos, as indicated by increased mpo expression in the ICM at 24hpf, and higher gata1 expression in the A M and posterior blood island (PBI) at 30 hpf. Hematoxilin Eosin histochemical staining of sagittal adult zebrafish sections (scale bar: 500 m), demonstrates an abdominal mass of leukemic cells in AM CREBzebrafish (red box).

D) M -rearranged cell lines treated with increasing concentrations of chemotherapies (Doxo, Ara-C or VP16; 0.01-10 M) showed a similar reduction in proliferation, whereas tipifarnib (0.1–100 M) was specifically reducing M 2 cell proliferation, as indicated by WB analysis showing BC 2-like 11 (apoptosis facilitator) (BIM) and PARP cleavage increased after tipifarnib treatment in M 2.

fig.1

leukemia cells survival and implied that a combined therapeutic strategy with anti-BAG-1 compound and conventional chemotherapics might help in the eradication of tumor cells.

Future/ongoing projects i enetic in A The translocation t(8;21)(q22;q22)RUNX1-RUNX1T1 is a recurrent somatic lesion detected at diagnosis in approximately 12-15% of children with acute myeloid leukemia (AML). Children with this isolated translocation are usually considered at standard risk, but our last multicenter trial revealed a higher than expected cumulative incidence of relapse for these patients. Genetic and epigenetic heterogeneity is emerging as a fundamental property of AML in the dynamic evolution of the clonal architecture. In view of this observation, we hypothesized that within t(8;21) patients there may coexist a complex mosaic of cells containing combinations of the same genetic t(8,21) lesion together with different epigenetic variants, and that epigenetic complexity may play a crucial role in predisposing patients to relapse. We will perform genetic and epigenetic sequencing analysis and then integrated results trying to identify new molecular markers distinctive of t(8,21)-rearranged patients prone to develop relapse in order to improve their cure rate. e t e a eutic o o tunities o ediat ic atients a ected acute m eloid leu emia Over the past decades, significant improvements in survival for children with acute myeloid leukemia (AML) have been reached, with current overall survival of 65-70%. However, despite the use of intensive treatments, a third of children still die from AML, and late effects adversely damage their adult life.

Novel therapies are therefore desperately needed. The goal of this proposal is the development of tridimensional humanized bone marrow niche in vitro model to be implanted in vivo, in NSG mice, to improve patient’s leukemia engraftment and produce Patient derived enografts (Pd ). The Pd s will be used to advance the development of new therapeutic options for kids with AML because they will be used for testing new putative active compounds, pre-selected in vitro. Commercial libraries of FDAapproved molecules will be tested in cell lines or primary AML cultures from patients to define novel potentially active drugs. Their efficacy will be then evaluated in vivo on Pd s. This repurposing approach represents an attractive method for drug discovery, as it can enable rapid clinical translation.

87

National and international collaborations: • • • • • • • • • • •

Prof. Franco Locatelli, Ospedale Bambino Gesù , Roma Dr. Riccardo Masetti, Ospedale Sant’Orsola, Bologna Prof. Andrea Biondi, Università di Milano-Bicocca Prof. Stefano Volinia, Università degli Studi di Ferrara Dr. Stefano Cairo, Lab Head R&D- Xentech Paris – France Dr. Anna Tampieri -Bioceramics and Bio-hybrid Composites CNR - Institute of Science and Technology for Ceramics-Faenza Italy Dr. Dinesh S. Rao, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, LA Dr. Kathleen Sakamoto, Division of Hematology/Oncology Department of Pediatrics, Stanford University School of Medicine, USA Prof. CM Zwaan, Erasmus MC, Rotterdam, The Netherlands Prof. Dirk Reinhardt, Universitätsklinik Essen, Germany Dr. Tanja Gruber, St ude Hospital, Memphis, USA

CV Martina Pigazzi Martina ad finis ed er molecular iolo y de ree in 000 at ni ersity of ado a. S e attended t e Medical Sc ool in Verona where she got specialization in medical genetic in 2004. She obtained the PhD of the Medical School at the ni ersity of ado a in oncolo y ematolo y field in 00 . Currently Martina is rofessor ssistant at t e ni ersity of ado a epartment of oman and c ild Healt OncoHematolo y Clinic and La oratory. S e s in ol ed in asic and translational research concerning pediatric hematological disorders, mainly working on pediatric acute myeloid leukemia (AML). From 2010 she is responsible of the molecular genetics at diagnosis for all de novo AML enrolled in the Associazione Italiana Onco-Ematologia Pediatrica (AIEOP), being our Lab the reference center in Italy. Martina represents the Italian AIEOP-AML group at the AML international group BFM being involved in AML task force group.

Her clinical researc aims at t e identification of ne enetic a errations and mutations it pro nostic alue to e used as iomar er to impro e dia nosis definition, and ris stratification of patients for a tailored adapted t erapy. Her asic translational research commits to understand leukemogenesis, with particular interest at driver oncogenes and activated pat ays identification, for promisin alternati es or complementary t erapeutics to current c emot erapy. S e s aut or of 35 manuscripts published in the more important onco-hematological peer reviewed Journals.

Martina Pigazzi 3 Publications as first/last author 2016 31.29 IF as first/last author 2016 12 H-index (source )

88

RESERCH AREA: Gene Expression (GEP) NGS

PI:

Geertuy Te Kronnie GROUP MEMBER Post≠doctoral Fellows: Gulia Anselmi Silvia Bresolin Matteo Pizzuto PhD Student Annagiulia Bonizzato

Summary of recent research activities and of main results achieved (2012≠2016) Animal model studies In collaboration Dr. Luca Aresu, University of Padova, we studied canine leukemia and lymphoma. Our studies revealed epigenetic silencing of TFPI-2 in canine diffuse large B-cell lymphoma and chromosomal copy number aberrations associated with clinical outcome. In canine AML four recurrent single nucleotide variants were found in DNMT3L, a gene directly implicated in the function of DNMT3A a key-factor in human AML. These study contribute to understand genetic modifications of cDLBCL and cAML, leading up to studies that will elucidate the role of these alterations in the pathogenesis of AML and DLBCL in dogs and human patients.

Little is known about the molecular mechanisms mediating lymphoblast CNS infiltration in acute lymphoblastic leukemia and Lymphoblastic leukemia, and such insights are difficult to obtain from patient samples. We studied the molecular mechanism of CNS infiltration in two zebrafish (D. rerio) T-cell malignancy models. Comprehensive CNS histological analyses revealed that both models develop high rates of CNS disease resembling end stages of the disease in human patients. Further molecular studies showed that cxcr4 positively correlated with the extend of CNS invasion, indicating this pathway may be crucial for T-lymphoblast growth in the CNS. Biomarkers for ALL to improve therapeutic strategies Screening of Philadelphia positive (Ph ) acute lymphoblastic leukemia by next generation amplicon deep sequencing refined the mutational status of IKZF1. In this collaborative study that involved five leukemia research centers in Europe of 98 89

fig.1

patients with no indications of IKZF1 deletions 12 patients were found to carry a deleterious point mutation in IKZF1.

This refinement of I F1 mutational status increased the percentage of Ph patients with aberrant I F1 associated to an inferior clinical response to more than seventy.

Within the EU network ENNCA (European Network for Cancer research in Children and Adolescents) together with leukemia research centers (WP9) we aimed to improve therapeutic strategies using predictive biomarkers in ALL: an activity within the EU network to introduce newly discovered molecular information into clinical practice.

Newly discovered were P2RY8-CRLF2 fusions as marker of poor prognosis in children with intermediate risk B-cell precursor acute lymphoblastic leukemia and IKZF1 status in BCR-ABL1 positive childhood ALL. Italian collaborative projects (Gianni Cazzaniga, Monza, IT) aim to interpret the future challenge in leukemia research, which resides in the possibility to dissect the complexity of events in the tumor pathogenesis and response to treatment for which we use gene expression profiling, Flow Cytometry, Next-Generation Sequencing and MLPA genomic screening.

In collaboration with Cristina Mecucci and Roberta La Starza we used whole transcriptome analyses to find specific signatures of cytogenetic subgroups of T-ALL. We found a specific genetic profile of T-lineage acute lymphoblastic leukemias with MYC translocations in pediatric and adult patients and specific signatures of some MLLT10 rearranged leukemias that place this leukemia in the HOXA subgroup of T-ALL leukemias.

Microvesicles (MVs) as carriers on oncogenic messages As part of a larger EU project (Project reference: 228971funded under: FP7-NMP) aiming to develop and fabricate 90

fig.1 ocation and type of IK F1 mutations; blue s uares define the coding exons of IK F1; purple s uares represent the inc Finger domains. ocalization and mutation types (missense point mutations in red, frameshift mutations in light-blue) are depicted. Each circle represents a detected mutation ( eukemia 2015, ana et al.) fig.2 A. Mutation profile of 0 JMM patients; B. Distribution of missense alterations at the functional domains of JAK3 and SETBP1 proteins; C. Scheme ofthe in vio assay indicating the patient sample xenografted.

fig.2

molecular motor driven nano-devices for molecular diagnosis we studied MVs as prototype disease markers. In the context of leukemia research we analyzed RNA transcript cargo of MVs. We showed that MVs shed from K562 CML cells carry oncogenic BCR-ABL1 pathway transcripts and messengers related to basal cellular functions just like their parental cells. Analysis also showed a remarkable enrichment of transcripts related to membrane activity, cell surface receptors and extracellular communication in MVs compared to parental K562 cells. Co-culture of K562 derived MVs with healthy mesenchymal cells showed transfer of the oncogenic message and confirmed M -dosage dependent increase of target cell proliferation. ene e ession si natu e o ediat ic m elod s lastic s nd omes The project focuses on bone marrow characterization of 2 MDS patients by gene expression profiling.

Applying DC model classifier to MDS specimens, 59. of them received an AML-like call, on the contrary 37.5% a non AML-like call. We observed that patients with an AML-like call had a higher probability to evolve into AML. Moreover in collaboration with the McMaster University (Dott. M. Bhatia) we identified that GS - - and GS - -regulated pathways can be responsible for stepwise transition to MDS and subsequent AML, thereby providing potential therapeutic targets of disease evolution. dentification o ne ioma e s in In collaboration with the EWOG-MDS group, researchers from France and Belgium (University of Gent) we studied the gene expression profiling of 82 MML at diagnosis, we recognized a subgroup of patients with over-expression of LIN28B highly correlated with poor clinical outcome and known prognostic markers in JMML and recognized a novel fetal-like subgroup of this disease. The high expression on LIN28B is regulated by lncRNA H19. Moreover screening of Italian AIEOP JMML we identified 11.4 of patients that harbor secondary mutations in SETBP1 and A occurring in early precursor cells and a associated with a different cell propagating capacity.

Patient-derived induced pluripotent stem cells recapitulate hematopoietic abnormalities of juvenile myelomonocytic leukemia In collaboration with the Hematology Division of Children Hospital of Philadelphia (Prof. M.Weiss, Dr. Deborah French) we generated induced pluripotent stem cells (iPSCs) from malignant cells of two MML patients with PTPN11 mutation. In vitro differentiation of iPSCs produced myeloid cells able to recapitulate JMML features with high proliferative rate. This is demonstrated to be a good in vitro model to explore pathophysiology and treatment option of JMML 91

ansc i tional si natu e o emato oietic di e entiation in The study focuses on a “in silico” sorting approach based on gene expression of surface markers of normal hematopoiesis. We compared the gene expression profiles between cancer samples and the subpopulations at different maturation stages during normal hematopoiesis with the goal to improve the knowledge of the disease and to identify putative enrichment transcriptional module changes underlying the malignant phenotype. We showed that JMML cells mapped closely to different normal hematopoietic hierarchy levels and that this different classification reflected the different survival rate of MML patients. MML patients enriched of stem cells features showed a worse survival compare to JMML patients with activation of modules typical of late maturation stages of normal hematopoiesis. iolo o omato enetic A c itectu e And lonal olution o u enile elomonoc tic Leukemia This project investigates the somatogenetic architecture of MML to understand the grade of tumor heterogeneity in the bone marrow of patients and the sequential acquisition of mutations in the hematopoietic cell hierarchy to reconstruct the clonal phylogeny and its correlation with the distinct groups of JMML patients. The study focuses on a large comprehensive genomic study of JMML to discover molecular mechanisms that contribute to the pathogenesis and the clinical progression of the disease. We will analyze hematopoietic subclonal heterogeneity of the disease and their dynamic modulation during the course of the disease using also a xenotransplant mouse model and iPSCs. The project is part of “My First AIRC Grant”(MFAG 15674, Silvia Bresolin). i cula A ci c A in no mal emato oiesis Recent studies made clear that circular RNAs (circRNAs) are particularly stable transcriptome members with distinctive features. CircRNA biogenesis and functions that we recently reviewed showed circRNAs modulate host gene expression, but also participate in circuits competing for binding of miRNAs, RNA-binding proteins (RBPs) or translation initiation, and are part of key oncogenic axes. RNA-seq identified thousands of circRNAs with developmental stage- and tissuespecific expression and our first results of a collaborative project with Prof. Stefania Bortoluzzi showed that circRNAs are abundantly expressed in the hematopoietic compartment. Analysis of specific circRNA expression in hematopoietic cellular subtypes, validation of RNAseq data and prediction of functional features e.g. interactions with miRNAs, proteins and other regulatory elements are un-going.

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Mutational Screening of high risk resistant B-ALL In the context of B-ALL, although the treatment of pediatric leukemia is considered one of the best achievements in medicine, about 30% of patients present treatment resistance and relapse. In this project we perform whole exome sequencing and gene expression profiling of samples of B-ALL patient with resistance to therapy or that experienced relapse. Identified new genomic and transcriptome aberration will guide leukemia research towards personalized oncohematology.

Inherited ALL Clinicians have known since decades that families with ALL in subsequent generations may point to hereditary components most likely genetic, even if ALL in the vast majority of the cases is considered an acquired disease of somato-genetic origin. Some point mutations in PAX5, ETV6 and IKZF1 were recently reported to be present in families with recurrent ALL and confer an increased risk to ALL development. These studies provide the rational to identify disease related mutations in families with recurrent ALL. Inherited or de novo acquired constitutional mutations in genes are likely to impact the phenotype of the leukemia altering the biology of the aberrant cells, the systemic response to current therapies and the long term disease free survival of patients. Thus therapies may require adjustments and/or provide opportunities to develop specific therapies for these cases. Being inherited ALL a rare variant of a common disorder the only way to meaningfully address these issues is through direct collaborations that were recently launched.

National and international collaborations: • • • • • • • • • • • •

Dr. Luca Aresu (UniPD, Padova-IT) Prof. Silvio Bicciato (UniMO, Modena.IT) Prof. Stefania Bortoluzzi (UniPD, Padova-IT) Dr. Gianni Cazzaniga (Monza, IT) Prof. Sabina Chiaretti (La Sapienza, Rome-IT) Prof. Kimble Frazer (Oklahoma University, Oklahoma-US) Dr. Deborah French (Children Hospital of Phialdelphia-USA) Prof. Fabio Grassi (IRB, Bellinzona-CH) Dr. Stefano Indraccolo (IOV, Padova-IT) Dot.ssa Roberta La Starza (UniPG, Perugia-IT) Prof. Alf Manson (Linnaeus University, Kalmar-SE) Prof. Rolf Marschalek (Goethe University, Frankfurt-G) 93

• • • • • •

Prof. Cristina Mecucci (UniPG, Perugia-IT) Dr. Lueder H. Meyer (UniUlm, Ulm-Germany) Prof. Barbara de Moerlose (University of Gent, Belgium) Prof. Stefano Piccolo (UniPD, Padova-IT) Prof. Pieter Van Vlierberghe (University of Gent, Belgium) Prof. Mitchell Weiss (St ude Children’s Hospital-USA)

EWOG-MDS European Working Group of Myelodysplastic Syndromes (MDS) and JMML in ChildhoodGdL AIEOP MDS-JMML; GdL AIEOP LAL; iBFM BD committee; I-BFM ALL/NHL host genetic variation working group.

CV Geertuy te Kronnie Since 2001 Dr. Geertuy te Kronnie, senior researcher in molecular genetics of Pediatric Onco ematolo y, focused er scientific researc on t e molecular c aracterization of leu emia su roups it omic scale tec nolo ies from enome to transcriptome analysis of acute lymp o lastic and myelo lastic leu emias 0 pu lications in peer re ie ed international ournals from 00 1 cum cit inde . S e esta lis ed colla orations with national and international leukemia researchers also involving studies of several model organisms of leukemias from ze rafis to do s it natural occurrin leu emia in addition to murine eno raft models of uman acute leu emias. In the framework of collaborative research projects Dr. te Kronnie supervised master students and PhD candidates and many of them became independent researchers. Latest discoveries led Dr. te Kronnie to direct part of her research to studies of host genetic variations in childhood acute lymphoblastic leukemia with particular emphasis on hereditary mutations in families with recurrent hematopoietic malignancies.

Geertuy te Kronnie 2 Publications as first/last author 2016 9.09 IF as first/last author 2016 2 H-index (source )

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RESERCH AREA: MLL Leukemia

PI:

Luca Trentin GROUP MEMBER Student: Tamara Verbeek

Summary of recent research activities and of main results achieved Ended Projects aluation o a ne e i enetic t eatment t e a o MLL-AF4+ leukemia.

In this collaborative study with Dr. Michela Bardini (University of Milano-Bicocca) we used an in vivo MLL-AF4 mouse model to investigate the effect of I-BET151, a new epigenetic drug, on the proliferation capacity of MLL-AF4 leukemia cells. We identified the molecular mechanisms and gene expression profiles modifications caused by the drug treatment in leukemia cells. Furthermore, we showed that I-BET151 treatment significantly reduces cells proliferation and induces leukemia cell apoptosis. Given the aggressiveness of the disease and the lack of an ultimate cure, this study poses the rationale for future clinical applications. alutation o t e A mutation landsca e in A patients.

In this study we took advantage of the next generation sequencing technique to investigate the presence of KRAS and NRAS mutations frequencies in MLL-AF4 patients. We showed that 64% of patients at diagnosis present a RAS mutation at subclonal level; furthermore, we identified specific RAS-mutated gene and protein expression signatures. Finally, we showed that the MEK1/2 inhibition could be a promising therapeutic strategy for the treatment of MLL-AF4 RAS mutated patients. This study contributes to the evaluation of the impact of RAS mutations as secondary hits in MLL-AF4 leukemia.

95

fig.1 - A,B

The hystone deacetylase inhibitor Givinostat for the t eatment o ea an ed cell ecu so acute lymphoblastic leukemia.

Alterations of Cytokine Receptor-like Factor 2 (CRLF2) is a new negative prognostic factor in pediatric BCP-ALL. Rearrangements of CRLF2 lead to the deregulation of JAK/STAT and PI3K/mTOR pathways causing hyperactive signaling. In this study in collaboration with Dr. Angela Savino and Dr. Giovanni Cazzaniga (University of Milano-Bicocca), we used the hystone deacetylase inhibitor Givinostat alone or in combination with conventional chemotherapy and we showed in vitro and in vivo that this compound is able to inhibit the proliferation of CRLF2 rearranged cells and to modulate the transcription of genes involved in the JAK/ STAT pathway leading to the inactivation of this signaling network.

Ongoing/future projects n i o unctional identification o c ucial enes o t e A leu emia e ansion We have identified in our previous works new putative genes involved in MLL-AF4-mediated leukemia; using a library of shRNAs we will functionally identify, in collaboration with Dr. Michela Bardini (University of Milano-Bicocca), the genes that are really crucial for leukemia development. This approach will allow us to discover putative targets for the development of new treatment strategies.

HDACi for the treatment of the MLL-AF4 leukemia.

The use of the HDACi has shown promising application as new therapeutic drug in several cancers. In collaboration with Dr. Ronald Stam (Erasmus University, Rotterdam,

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fig.1 A, B. Evaluation of a new epigenetic drug for the treatment of M -AF4+ leukemia patients.

Gene expression profile analysis (A) showing the transcriptomic profile alteration of leukemia patients harboring the M -AF4 translocation upon treatment with the epigenetic drug I-BET151. The heatmap shows the upregulated genes in red and the down-regulated genes in green. Gene ontology (GO) analysis (B) of the di erentially regulated genes upon treatment with I-BET151. Genes belonging to similar molecular and biological processes are grouped together allowing a functional interpretation of the gene expression data; the color scale re ects the statistical significance of the analysis.

fig.2 - C, D

Netherlands) we are investigating the effects of a HDACi therapy in MLL-AF4 leukemia cells in vitro and in vivo.

Furthermore, using gene expression profile analysis we aim to identify the molecular pathways modified upon treatment and resulting in a reduced leukemia propagation. ic o As mi As e ulation o A leukemia cells growth. miRNAs are non-protein-coding small RNAs that regulate the expression of over 60% of all human genes by inhibition of translation or destabilization of target mRNAs. In our analysis, we have identified a specific miRNA-gene network that we are currently investigating to clarify its role in the regulation of MLL-AF4 leukemia cells growth and proliferation.

National and collaborations: • • • •

• • • •

fig.2 C, D. Analysis of the RAS mutational status in M -AF4 leukemia patients. The plot (C) shows the K-RAS and N-RAS variant allele fre uency (VAF) in pediatric M -AF4 patients at diagnosis measured by ultra deepse uencing analysis. Viability analysis (D) of M -AF4+ RAS mutated (FAIS) and RAS wildtype cell lines (RS4;11, SEM) upon treatment with a compound inhibiting the RAS pathway.

The myeloid cell line THP-1 harboring a N-RAS mutation was used as a positive control.

The mutated cell lines showed a reduced proliferation compared to the RAS wild type cell lines after 2h of treatment. z

international

Dr. M. Bardini, University of Milano-Bicocca, Milan, Italy Dr. Angela Savino, University of Milano-Bicocca, Milan, Italy Dr. Gianni Cazzaniga, University of Milano-Bicocca, Milan, Italy Dr. Franco Fais, Department of Experimental Medicine, University of Genoa, Genoa, Italy; IRCCS AOU San Martino - IST Istituto Nazionale per la Ricerca sul Cancro, Genova,Italy.

Dr. L.H. Meyer, University of Ulm, Ulm, Germany Prof. K.M. Debatin, University of Ulm, Ulm, Germany Dr. R.W. Stam, Erasmus University, Rotterdam, Netherlands Dr. Patricia Garrido Castro, Erasmus University, Rotterdam, Netherlands

97

CV Luca Trentin Born in 1978, Dr. Luca Trentin obtained his graduation degree in Biological Science in 2004 at University of Padova.

From 2005 to 2010 he worked in the Laboratory of Oncohematology, Departments of Pediatrics directed by Prof. G. asso at first as a inner of a sc olars ip and t en as a student. urin t e pro ram, is researc pro ect was mainly focused on the study of the MLL rearranged leukemia. He got his PhD degree on 2010 and he carried out his postdoctoral training in Ulm (Germany) from August 2010 to February 2014 working in the Department of Pediatrics and dolescent Medicine directed y rof. .M. e atin. urin is post doc trainin e or ed on t e identification and characterization of the leukemia initiating cells. In 2013, he won a Futuro in Ricerca 2013� (FIRB) fellowship awarded by t e Ministero dell Istruzione, dell ni ersit e della icerca MI and startin from Marc 014 to present e is or in as a researcher in the Laboratory of Oncohematology, Departments of Pediatrics University of Padova directed by Prof.

G. Basso on new therapeutic approaches for the treatment of MLL rearranged leukemia.

Luca Trentin 2 Publications as first/last author 2016 9.39 IF as first/last author 2016 7 H-index (source Sco us)

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Laboratory of Solid Tumor Biology GROUP LEADER:

Giuseppe Basso

REFERENT:

Gianni Bisogno

Field of interest

The laboratory has been established in 1993 by prof. Angelo Rosolen and is part of the Laboratory of Pediatric Hematology Oncology directed by Prof. Giuseppe Basso.

The laboratory works closely with the physicians that follows children with different types of solid tumors at the Pediatric Hematology Oncology Division in Padova. Over the years it has developed a particular interest in diagnostics and basic and translational research for children with soft sarcoma and other rare tumors.

The main activities are: • To provide the necessary molecular biology investigations to support the diagnosis of solid tumor. The laboratory is the reference laboratory of the AIEOP soft tissue sarcoma working group and offer these services at a national and international level. • To collect, handling and store biological samples from patients with solid tumors treated in the Pediatric Hematology Oncology Division of Padova. Sample are also collected from all children with soft tissue sarcoma enrolled by Italian Centres in the national and international protocol dedicated to children with soft tissue sarcomas. • To promote research projects dedicated to study the biological characteristics of pediatric soft tissue sarcomas and biomarkers that can have a direct clinical application

Summary of recent research activities and of main results achieved According to its aims to improve the diagnostic capabilities the panel of genetic alteration correlated with different soft tissue sarcoma histotypes have been progressively expanded and

99

the laboratory is now able to search for specific genetic alteration. The laboratory is also searching for new specific alteration able to support the diagnosis and indicate the patient prognosis. This recently lead to the discovery, in collaboration with the Pathology departemte of teh Memorial Sloan Kettering Cancer Center, of a novel and recurrent GLL gene rearrangement specific in infantile cases of rhabdomyosarcoma.

These specific genetic alterations have been used to study the minimal disease in peripheral blood and bone marrow in a European multicentre study coordinated by our laboratory. The analysis of collected data is ongoing.

A main research interest of our group is the identification of pediatric soft tissue sarcoma novel tumor biomarkers that can be used to establish tumor diagnosis and prognosis, and the search for driver genes and proteins that can be considered for targeted therapy.

A series of studies utilizing “liquid biopsies” are underway with the aim to identify biomarkers predictive of tumor resistance and metastatic potential. This is a coohprensevie approach aiming to study in eth same patients circulating tumor cells and tumor derived product (DNA, protein, antigens and so on).

International and national cooperation have been established or strengthened in the last years.

New collaborations with other research Groups working in the IRP have been recently established (D.ssa Pozzobon group to study tumor matrix, dr. Agostini’s Group). On a national level a pediatric “biosarc group” has been established to foster collaboration among Italian laboratories interested in pediatric soft tissue sarcoma and facilitate the elaboration of common studies. Finally the laboratory is part of the Biology Group of the European pediatric soft tissue sarcoma study group.

Research Projects: Closed Funded Projects [2012-2015]: • “Dna Methylation and Epigenetic Therapy Approaches in Human Rhabdomyosarcoma” [AIRC 2012-2015] • “Genome and proteome-wide characterization of oncogenic activity of ALK kinase in anaplastic large cell lymphoma of childhood” • [Fondazione CARIPARO 2012-2015] Ongoing Funded Projects: • “Novel Strategies in Metastasis Research and Biomarker Discovery in Rhabdomyosarcoma” [AIRC 2014-2017] • “Development of Advanced Plasmonic Biosensors for Non-invasive Detection of Circulating MicroRNAs as Biomarkers in Cancer Diagnosis and Prognosis” • Pediatric Rhabdomyosarcoma Biomarkers in Liquid Biopsies” [BIRD 2016] • Use of liquid biopsies in pediatric soft tissue sarcomas for diagnosis and non-invasive disease monitoring [Cariparo 2017-2019]

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National and international collaborations: • • • • • • • • • •

COG – Children Oncology Group EpSSG – European Paediatric Soft Tissue Sarcoma Study Group TCC – Innovative Therapy for Children with Cancer IOV – Istituto Oncologico Veneto INT – Fondazione IRCCS, Istituto Nazionale Tumori - Milano CWS - Cooperative Weichteilsarkom Study Group CINECA – Consorzio Interuniversitario, Bologna SIOPE – International Society of Paediatric Oncology, European branch EXPeRT - European Cooperative Study Group for Pediatrci rare Tumours ExPO-r-NeT – European Expert Paediatric Oncology Reference Network for Diagnostics and Treatment

10 Selected Publications Stachowicz-Stencel T, Orbach D, Brecht I, Schneider D, Bien E, Synakiewicz A, Rod J, Ferrari A, Cecchetto G, Bisogno G

Thymoma and thymic carcinoma in children and adolescents: a report from the European Cooperative Study Group for Pediatric Rare Tumors (EXPeRT).

Eur J Cancer. 2015 Nov;51(16):2444-52.

Bisogno G, Brennan B, Orbach D, Stachowicz-Stencel T, Cecchetto , Indolfi , ien , errari , Dommange-Romero F

Treatment and prognostic factors in pleuropulmonary blastoma: an EXPeRT report

Eur J Cancer. 2014 Jan;50(1):178-84.

Ferrari A, De Salvo GL, Dall'Igna P, Meazza C, De Leonardis F, Manzitti C, De Ioris MA, Casanova M, Carli M, Bisogno G

Salvage rates and prognostic factors after relapse in children and adolescents with initially localised synovial sarcoma

Eur J Cancer. 2012 Dec;48(18):3448-55.

Ferrari S, Ruggieri P, Cefalo G, Tamburini A, Capanna R, Fagioli F, Comandone A, Bertulli R, Bisogno G, almerini , l er ini M, arafioriti A, Linari A, Picci P, Bacci G.

Neoadjuvant chemotherapy with methotrexate, cisplatin, and doxorubicin with or without ifosfamide in nonmetastatic osteosarcoma of the extremity: an Italian sarcoma group trial ISG/OS-1

J Clin Oncol. 2012 Jun 10;30(17):2112-8.

Bisogno G, Soloni P, Conte M, Podda M, Ferrari A, Garaventa A, Luksch R, Cecchetto G.

Esthesioneuroblastoma in pediatric and adolescent age. A report from the TREP project in cooperation with the Italian Neuroblastoma and Soft Tissue Sarcoma Committees.

BMC Cancer. 2012 Mar 25;12:117.

Bisogno G, Compostella A, Ferrari A, Pastore G, Cecchetto G, Garaventa , Indolfi , e Sio L, Carli M.

Rhabdomyosarcoma in adolescents: a report from the AIEOP Soft Tissue Sarcoma Committee.

Cancer. 2012 Feb 1;118(3):821-7

Bisogno G, Ferrari A, Prete A, Messina C, Basso E, Cecchetto G, Indolfi , Scarzello , n elo , e Sio L, Di Cataldo A, Carli M

Sequential high-dose chemotherapy for children with metastatic rhabdomyosarcoma

Eur J Cancer. 2009 Nov;45(17):3035-41.

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Oberlin O, Rey A, Lyden E, Bisogno G, Stevens MC, Meyer WH, Carli M, Anderson JR.

Prognostic factors in metastatic rhabdomyosarcomas: results of a pooled analysis from United States and European cooperative groups

J Clin Oncol. 2008 May 10;26(14):2384-9.

Bisogno G, Riccardi R, Ruggiero A, Arcamone G, Prete A, Surico G, Provenzi M, Bertolini P, Paolucci P, Carli M

Phase II study of a protracted irinotecan schedule in children with refractory or recurrent soft tissue sarcoma.

Cancer 2006 Feb 1;106(3):703-7

Bisogno G, Ferrari A, Bergeron C, Scagnellato A, Prete A, Alaggio R, Casanova M, D'Angelo P, Di Cataldo A, Carli M

The IVADo regimen--a pilot study with ifosfamide, vincristine, actinomycin D, and doxorubicin in children with metastatic soft tissue sarcoma: a pilot study of behalf of the European pediatric Soft tissue sarcoma Study Group

Cancer. 2005 Apr 15;103(8):1719-24.

CV Gianni Bisogno Gianni Bisogno is Associate Professor in Pediatrics, University of Padova, and Consultant Pediatric Oncologist and responsible for the Solid Tumor and New Drugs Program at the Division of Hematology-Oncology, Dpt of Mother’s and Child’s Health, University Hospital of Padova.

His clinical and research activity is mainly dedicated to childhood solid tumors. In particular he has been the Coordinator of the Italian Soft Tissue Sarcoma Committee (STSC) from 2007 to 2015 and still maintains a leading position in this Group.

In these years the STSC has progressively increased its activity with the launch of several clinical trials and the creation of a multidisciplinary team that supports the activity of Pediatric Oncology Centres providing central review of diagnosis, molecular iolo y c aracterization, real time ad ice on difficult cases and pro idin treatments not a aila le in ot er Italian Centres. He is also coordinating the laboratory dedicated to the study of pediatric STS molecular biology.

As a result of these activities nearly 100% of the Italian children with rhabdomyosarcoma (RMS) are registered in STSC protocols and 5-years survival has increased from 62% to 81%.

In 2004 GB has been among the founders of the European paediatric Soft tissue sarcoma Study Group (EpSSG), and is the PI of the trial for localized RMS that registers patients from approximately 150 European Centres. He has been elected pSS C air in 01 and reconfirmed in 01 .

GB is one of the coordinators of the TREP (Tumori rari in età pediatrica) project, a national network established in 2000 to promote research in favour of children with very rare tumors (nasopharingeal carcinoma, pediatric melanoma, pleuropulmonary blastoma, pancreatic cancer and many more).

Similar initiatives have been subsequently adopted by other European countries and this led to the foundation of EXPeRT (European Cooperative Study for Pediatric Rare Tumors) in Padova, June 2008.

In 2013 he has been elected by the chairs of the European Trials Groups as their representative in the SIOP Europe Board.

GB has been leader or partner in several projects including European funded projects such as EPOC, ENCCA and EXPORNET and 18 projects dedicated to new drugs sponsored by Pharmaceutical Companies or Research Institutes.

Publication activity includes 174 papers published on international Journals and indexed on PubMed, 16 paper published on Italian Journal, more than 230 abstracts presented at international and national Congresses, and 9 book chapters Total Impact factor: : 690,915 (last update: 19.04.17) H-index (last update: 19.04.17) : 28 (ISI web of science); 34 (ResearchGate)

Gianni Bisogno 10 Publications 2016 35,38 IF 2016 34 H-index (source RG)

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STUDY AREA: Basic Signal Trasduction

PI:

Field of interest Novel Biomarkers in Pediatric Solid Tumors.

A main research interest of our group is the identification of novel driver genes and proteins in childhood cancer that can

Paolo Bonvini GROUP MEMBER Post≠doctoral Fellows: Elena Pomari

be considered for targeted therapy. Project’s goal is to use novel tumor markers to better stratify children with cancer and individualize their treatment.

Preclinical Pharmacology in Childhood Cancer.

The laboratory of Solid Tumor Biology is interested in the

Elena Poli

evaluation of novel cancer therapeutics as natural upshot of

Research Assistant:

the biomarker discovery program. During the past years we

Marica Peron

have paid particular attention to tyrosine kinase inhibitors,

Students:

based on the concept that tumors carrying deregulated

Marco Frigo

tyrosine kinases are particularly susceptible to their inhibition.

Technician

We have tested several kinase-specific and group-selective

Elisa Tosato

agents over the years, using heterogeneous and oncogeneaddicted pediatric malignancies as in vitro models.

Cellular Stress Response in Cancer Therapy.

Tyrosine kinase inhibitors (TKI) are among the most promising therapeutic agents to treat high-risk cancers. Recent studies, however, suggest that T I efficacy may be hampered by several resistance mechanisms, including point mutations within the protein kinase domain. To bypass such a hindrance, inhibition of Hsp90 molecular chaperone may be an alternative approach to cure cancer, since many oncogenic protein kinases strictly depends on Hsp90 for their folding

103

fig.1

and activity. Accordingly, we have shown that oncogenic NPM-ALK kinase relies on Hsp90 for proper folding and activity in T-cell lymphoma ALCL cells but the principles of such interaction, the specific NPM-AL domains or motifs recognized by Hsp90 remain to be established.

Mutational Analysis of ALK Oncogene in Pediatric Solid Tumors.

Tyrosine kinase inhibitors have become the gold standard therapy of tumor types expressing oncogenic forms of protein tyrosine kinases. However, clinical studies indicate that a significant portion of patients treated with this class of molecules develop resistance due to the selection of cancer cells carrying point mutations within critical domains or motifs of kinases. Early detection of resistance mutations is important to predict response to treatment, but also for the impact their identity, frequency and evolution have on clinical course. In addition, their identification and characterization may be helpful to identify the most appropriate therapy for each patient, preventing either over-treatment or relapse.

Immuno Response in Childhood Cancer.

Many disease states, including autoimmune diseases and cancer, are characterized by the presence of antibodies directed against self-antigens. Circulating antibodies and antigens serve as potential biomarkers for diagnostic and prognostic purposes, since there is a strong evidence that tumors undergoing immune surveillance are characterized

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fig.1 Dose-response curves of M0- 1 (ETV6NTRK3-positive) leukemic cells after 48h treatment with entrectinib ( ) or crizotinib( ) at low nanomolar concentrations (0.1-3 nM).

(B) M0- 1 cell cycle analysis at the indicated doses of crizotinib and entrectinib for 24 hours. (C) E ect of 25 nM entrectinib on ETV6-NTRK3 phosphorylation (pEN). Altered cell homeostasis and apoptosis induction are indicated by the increase in green uorescent intensity of JC-1 mitochondrial membrane potential indicator.

(F) Representative images of Wright-Giemsa-stained cytospin preparations of M0- 1 cells treated with increasing concentrations of entrectinib, as indicated in figure (x63). (E) Representative brightfield (upper) and uorescent (lower) images of embryos transplanted with CMDil-labeled M0- 1 cell line at 24 hpt with 1.0 uM of entrectinib or vehicle (left panel). White arrows indicate distribution of CM-Dil stained M0- 1 cells in representative 2 hpf embryos after 24h of treatment. Schematic of in vivo xenotransplantation assay (middle panel). In vivo proliferation of CM-Dil M0- 1 cells after pooling of at least 20 transplanted zebrafish and enumerated by ow cytometry analysis for the expression of both human CD33 antigen and CM-Dil (right panel). Mean SD of three independent experiments. hpf, hours post fertilization; hpt, hours post treatment.

by a continuous monitoring by the immune system that recognizes changes in protein sequence, expression and folding. We aim at identifying novel protein-based circulating biomarkers in blood samples of children with soft tissue sarcoma and lymphoma, by exploring their autoantibody profiles.

Autoantibodies may help to define the presence of specific tumor antigens at early stages of disease, as well as to monitor disease progression, recurrence or metastasis.

Summary of recent research activities and of main results achieved Novel Biomarkers in Pediatric Solid Tumors.

To this regard, our group has focused on the expression and activity of ALK tyrosine kinase in RMS and ALCL tumors, to provide a rationale for the use of ALK inhibitors in these two malignancies.

We have provided evidences that in RMS patients ALK is expressed as full-length receptor at the plasma membrane, but lacks of constitutive tyrosine kinase activity, whereas ALCL patients express a constitutive active truncated form of ALK, namely NPM-ALK, which is responsible for cell transformation and tumor development. Nonetheless, we have shown that under some circumstances ALK has a negative prognostic impact in RMS as well, in particular when AL gene is amplified or overexpressed.

Based on these findings, we have suggested that cancer patients genotyped in early-phase clinical trials for AL

gene amplification, overexpression or assessed for constitutive protein kinase activity

are at high likelihood for response to ALK inhibitors, whereas patients where ALK is expressed at physiological levels would benefit most likely of combinatorial treatments or multitargeted therapeutic agents.

Preclinical Pharmacology in Childhood Cancer.

Recently, we have investigated preclinical activity of entrectinib tyrosine kinase inhibitor in pediatric tumors harboring ALK and TRK gene rearrangements and shown that tyrosine kinase inhibitors are moderately active in childhood cancers with no continuous signaling of the primary intended target (i.e. full-length ALK receptor in RMS), whereas in tumors bearing gene fusions, such as NPM-ALK in anaplastic large cell lymphoma (ALCL), or ETV6-NTRK3 in acute myeloid leukaemia (AML) and 105

congenital infantile fibrosarcoma (CFS), this class of molecules exerts superior inhibitory potency (figure 1). Main goal of this program is to demonstrate that protein kinases represent ideal therapeutic targets in childhood cancer, and future drug development programs need to take into account that tumor markers occurring frequently in rare cancers or at a low incidence in more common tumors deserve the same attention and consideration.

Cellular Stress Response in Cancer Therapy.

Goal of this research program is to define the exact manner by which NPM-AL

is recognized by

Hsp90, to therapeutically hamper this event. With respect to this, computational efforts coupled to in vitro functional analyses have demonstrated that: 1) AL

kinase domain is necessary and sufficient

to bind Hsp90; 2) Hsp90 binds to NPM-ALK regardless of its kinase activity; 3) inactive NPM-ALK kinase is trapped more avidly to Hsp90 and is more susceptible to Hsp90 inhibition. Based on these findings we have proposed a model in which NPM-AL

propensity to unfold affects Hsp90 binding

and the allosteric properties of NPM-ALK n-lobe and c-lobe interface regulate such an interaction. On the basis of this model, targeting the NPM-ALK/Hsp90 interaction is now under investigation.

Mutational Analysis of ALK Oncogene in Pediatric Solid Tumors.

In this context, we have employed a high sensitive sequencing approach to detect minor mutated cell populations in children with cancer, for whom biopsy sampling remains a critical challenge. We have assessed NPM-ALK pre-therapeutic mutations in children with ALCL and found that these mutations occur at very low frequency at diagnosis, most likely within the ALK tyrosine kinase domain. All the identified mutations affected NPM-AL

activity and included both point mutations and insertion/

deletions (INDELs) rearrangements. Functional validation of selected NPM-ALK mutants coupled to structure-based computational analysis demonstrated that NPM-ALK point mutations impair ATP binding and protein kinase activity, whereas INDELS impact mostly on NPM-ALK signaling output through the formation of non-functional protein heterocomplexes. On the basis of these premises, we have suggested that NPM-ALK mutations are rare events in children with ALCL at diagnosis.

However, if occurring in the absence of drug selection, they lead to altered oncogene signaling and transforming capacity.

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Immuno Response in Childhood Cancer.

With respect to this topic, we have recently started a research program that uses minimal amount of plasma from children with cancer as source of autoantibodies and tumor-associated antigens, to improve disease staging and patients’ risk stratification. By using proteome-scale technologies we have demonstrated that cancer-related antigens with a statistically different immune reactivity between patients and healthy subjects do exist, and most likely represent functional proteins involved in tumor growth and differentiation. We have provided evidence that antibodies against tumor antigens evoke an immune response and can be used to distinguish pediatric patients from healthy subjects, as well as patients belonging to different risk groups. Since early disease detection is a critical factor for successful treatment of patients, our autoantibody screening and autoantigen discovery approach may have value in the diagnosis of cancer but may also be worthwhile for the management of children with this type of malignancies.

National and international collaborations: •

Prof. Gianni Bisogno Solid Tumor Biology Group Coordinator, Department of Women’s and Children’s Health, Hematology Oncology Division, Hospital-University of

Padova, Italy

•

Dott.ssa Rita Zamarchi, IOV- Istituto Oncologico Veneto, Padova, Italy.

•

Dott.ssa Elisabetta Rossi (Novel Biomarkers in Pediatric Solid Tumors)

•

Dr. Leonard M. Neckers, Urologic Oncology Branch NCI/CCR, NIH, Bethesda, MD (USA) (Cellular Stress Response in Cancer Therapy)

•

Dr. Luke Whitesell, Whitehead Institute for Biomedical Research, Cambridge, MA,

•

(USA) (Cellular Stress Response in Cancer Therapy)

•

Prof. Stefano Moro, Department of Pharmaceutical and Pharmacological Scienc

•

Dr. Giorgio Cozza, University of Padua, Italy

•

(Mutational Analysis of ALK Oncogene in Pediatric Solid Tumors)

•

Prof. Silvio Tosatto, Biocomputing Laboratory, Department of Biomedical Sciences,

•

Dr. Giovanni Minervini, University of Padua, Italy (Mutational Analysis of ALK Oncogene in Pediatric Solid Tumors) 107

•

Dr. Michela Pozzobon, Laboratory of Stem Cells and Regenerative Medicine, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padova, Italy

•

(Novel Biomarkers in Pediatric Solid Tumors)

•

Dr. Manuela Cattellan, Department of Statistical Sciences, University of Padova, Italy (Immuno Response in Childhood Cancer)

•

Dr. Gary Li, IGNYTA, Inc.11111 Flintkote Ave San Diego, CA, USA

•

Dr. Kristen M. Smith, (Preclinical Pharmacology in Childhood Cancer)

CV Paolo Bonvini Degree in Biology, University of Padova (1993) PhD in Developmental Medicine, University of Padova (2002) Specialization in Medical Genetics, University of Padova (2008) Dr. Bonvini is a research member of the Hematology and Oncology Clinic of Pediatrics since 1993, where, under the supervision of Prof. A. Rosolen, started focusing on pathophysiology, signal transduction and drug response of paediatric solid tumours. Moreover, Dr. Bonvini has spent 3 years at NIH (National Institutes of Health, Bethesda, MD, USA) as research scientist, where he studied novel pathways and regulators of malignant transformation in adult cancers. Dr. Bonvini has more than 20 years’ experience in many molecular and cellular biology techniques applied to both cancer genomic and proteomics and his main research interests include mechanisms of oncogene expression and mutation, oncogene addiction and iomar er disco ery. In particular, r. on ini is interested in t e identification of no el predicti e and pro nostic biomarkers in childhood cancer and the preclinical evaluation of novel targeted agents, such as protein kinase inhibitors, cell cycle and checkpoint inhibitors, proteasome inhibitors, apoptosis inducers and heat shock protein inhibitors. Since 2009, Dr. Bonvini is senior researcher at the Istituto di Ricerca Pediatrica (IRP) Città della Speranza and primarily involved in tar eted t erapy and cancer immunoresponse. r. on ini is aut or of many ori inal researc papers and e s scientific responsible of several material transfer agreements (MTA) with both research institutions and pharmaceutical companies.

He is a member of the American Association for Cancer Research (AACR), the Italian Society of Pediatric Hematology and Oncology (AIEOP) and the Italian Society of Biology (ONB). More recently, Dr. Bonvini joined the European Soft Tissue Sarcoma Group (EpSSG) as member of the biology committee.

Paolo Bonvini 2 Publications 2016 16.79 IF 2016 15 H-index (source RG)

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STUDY AREA: Solid Tumor Genomics

PI:

Lucia Tombolan GROUP MEMBERS Post≠ Doctoral Fellow: Elena Poli

Field of interest Her field of interest is the molecular characterization of childhood soft tissue sarcoma by genome-wide approaches such as microarray and next-generation sequencing.

Advances in genome- wide technologies have enabled the rapid identification of somatic genomic alterations in individual tumors, and nowadays they offer important information both for patients’ risk stratification and treatment assignment.

Our studies have contributed to demonstrate that different subgroups of rhabdomyosarcoma (RMS) show a peculiar gene expression profiling as a different miRNAs signatures, highlighting the deregulation of novel markers in rhabdomyosarcoma. Finally, in the last year her research topic has focused on discovery novel biomarkers in pediatric soft tissue sarcoma applying genomic techniques to “liquid biopsies”. The identification of biomarkers in “liquid biopsy” (i.e. plasma, bone marrow, urine) predictive of tumor resistance and metastatic potential represents a non-invasive tool that can improve patients’ management, with important implications for prognosis and treatment assignment.

Summary of recent research activities and of main results achieved Rhabdomyosarcoma (RMS) is the most frequent sarcoma in children. Alveolar RMS (ARMS) has a dismal prognosis, significantly worse than embryonal RMS (ERMS), and is characterized in 75% of the cases by the PAX3/FOXO1 fusion gene. Previous studies have reported the differential

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fig.1

gene expression profiles that distinguish ERMS, PA / FOXO1 positive and PAX3/FOXO1 negative ARMS. By microarray analysis and other techniques, we identified several biological differences among distinct subgroups of RMS (De Pittà et al, 2006; Tombolan et al,2015) Despite several recent reports highlighting numerous differences among apparently histologically and clinically identical RMS, there is an increasing need to refine the definitions of ARMS and ERMS and possibly of additional newly identified biologically-driven entities.

DNA methylation in pediatric rhabdomyosarcoma With the goal to distinguish RMS patients with different prognosis, we have studied the methylation status of different subgroups of RMS using a genome-wide approach such as microarray and next-generation sequencing.

Study results demonstrated that DNA methylation patterns distinguish not only alveolar and embryonal RMS but also between metastatic and non-metastatic RMS and suggest that epigenetic regulation of specific genes could represent a novel therapeutic target that could enhance the efficiency of RMS treatments (Tombolan L et al 2016).

Identification of circulating tumor DNA (ctDNA) and miRNAs in plasma of children with rhabdomyosarcoma Despite prognosis of RMS patients has improved considerably in the last decades, children with metastasis at diagnosis or disease relapse after therapy have a particularly dismal prognosis. Current therapeutic strategies include surgical resection, radiotherapy and chemotherapy tailored on the patients’ risk profiles determined on the basis of clinical prognostic factors (i.e. tumor site and size, nodal involvement, metastasis at diagnosis). There is an urgent need to improve our ability to predict each patient’s 110

fig.1 NE 1 expression correlates with rhabdomyosarcoma (RMS) clinical prognostic factors. NE 1 mRNA levels assessed by RTPCR showed a significant correlation with fusion status (A), histology (C) and advanced stage of disease (stage IV) (D), while no correlation was observed with tumor size and gender (B, E). In a multivariate analysis (Cox regression model) NE 1 expression show a trend of correlation with known RMS risk factors (F). (G) Receiver operating characteristic curve (ROC) analysis showed the sensitivity and specificity of NE 1 mRNA concentration as a parameter to classify RMS patients (n 5) on the basis of the fusion gene status (RMS-fusion positive vs RMS-fusion negative; Cut-o RQ 14.24). Kaplan– Meier and log-rank analysis for progression-free (PFS) (H) and overall survival (OS) (I) of RMS patients (n 5) based on specific uantitative NE 1 mRNA cuto value (RQ 14.24) identify two subgroups of patients with significantly di erent outcomes (PFS, P 0.011; OS, P 0.050). All statistical analyses were performed with R software and Prism6. P 0.05; P 0.01; P 0.001.

Abbreviations: RQ, relative uantification; AUC area under the curve.

outcome in order to better tailor treatments and increase his chance of cure. Aim of this project is to implement strategies able to identify biomarkers associated with the risk of treatment failure in patients with RMS. To this purpose we plan to use a next-generation sequencing approach to perform a DNA whole exome sequencing of selected RMS tumor specimens collected at diagnosis and at relapse and a parallel sequencing of circulating tumor DNA (ctDNA) obtained from plasma of matched RMS samples. The mutational profiling of ctDNA and its correlation with mutational pattern of primary tumors could be a useful approach to the comprehension of genetic abnormalities acquired or accumulated during tumor evolution.

Micro-RNAs (miRNAs) have emerged as tiny but potent molecules that regulate cell differentiation and proliferation. Deregulated miRNAs have been reported in several types of cancer, and both tumor suppressor and oncogenic functions have been attributed to miRNA. Recently, the discovery that microRNAs (miRNAs) are stable in body fluids such as plasma, presents the opportunity to use circulating miRNA as biomarkers of disease and predictors of outcome.

Taken together the evaluation of such circulating poor prognosis-associated biomarkers is expected to provide important information on the molecular characteristics of resistant RMS, which might be used to improve diagnosis, treatment and prognosis of the affected children.

National and international collaborations: • •

• •

• • • •

Prof. Gianni Bisogno, Department of Women’s and Children’s Health, Hematology Oncology Division.

He is responsible of the national center for diagnostic review and protocol coordination for different pediatric oncologic malignancies. He is the coordinator of the European Pediatric Soft Tissue Sarcoma Study Group (EpSSG) and PI of AIRC Grant entitled “NOVEL STRATEGIES IN METASTASIS RESEARCH AND BIOMARKER DISCOVERY IN RHABDOMYOSARCOMA” Dott.ssa Rita Zamarchi and Dott.ssa Elisabetta Rossi IOV- Istituto Oncologico Veneto The IOV-IRCCS CTC-Lab is the reference center of North-East Italy for the circulating tumor cells (CTCs) investigation for clinical purposes and since 2006 it has developed integrated CTC assays now included in multicenter clinical trials.

The collaboration has the aim to identify circulating tumor cells (CTCs) in rhabomyosarcoma blood samples.

Prof. Chiara Romualdi, Department of Biology, University of Padova.

Collaboration field: statistical analysis of NGS data Dott. Cristiano De Pittà Department of Biology, University of Padova.

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• • • • •

Collaboration field: miRNAs in solid tumors Dott.ssa Tiziana Cesca Department of Physics and Astronomy (DFA), University of Padova Collaboration field: Detection of circulating micro-RNAs as biomarkers in cancer diagnosis and prognosis Dott.ssa Michela Pozzobon. Laboratory of Stem Cells and Regenerative Medicine, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padova, Italy Collaboration field: Identification and analysis of CAFs in rhabdomyosarcoma

CV Lucia Tombolan Lucia Tombolan graduated in Biological Sciences at University of Padova in 2003. In the same year she had the national qualifying examination for Biologist. In 2007 she received the specialization in Medical Genetics at the University of Verona.

Lucia Tombolan 1 Publications as first/last author 2016 3.23 IF as first/last author 2016 5 H-index (source RG)

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STUDY AREA: Soft Tissue Sarcomas Molecular Diagnosis ≠Biobank

PI:

Angelica Zin

Field of interest She is responsible of molecular diagnosis and Minimal Residual Disease of STS in childhood and Biobanking of Soft Tissue Sarcoma and other Solid Tumors. During these years she has been involved in research on the identification of new diagnostic and prognostic biomarkers for different solid tumors of childhood. In many malignant tumors the molecular analysis has showed the presence of the specific genetic anomalies especially for some istotypes such as mutations, deletions, amplification and chromosomal translocations.

In particular, in soft tissue sarcomas the most important genetic abnormality is translocation chromosome and these genetic anomalies represent tumor-associated markers and may be used to confirm the histological diagnosis, for the evaluation of biological characteristics and their prognostic significance, and to detect the minimal dissemination of disease and response to therapy. From 1993 the Laboratory of Biology of Solid Tumors is the national reference laboratory for the research of specific molecular markers and other studies on Soft Tissue Sarcomas Soft tissue for AIEOP group. Each year are collected biological samples (tissue biopsies, bone marrow, peripheral blood, pleural and peritoneal fluid, serum, etc.) for about 150 new patients and performed more than 1000 molecular analysis. Recently, the Biobank accepts biological samples for other pediatric solid tumor such as neuroblastoma and brain tumors. She is involved in research project for identification of CTC and circulating biomarkers from a simple blood test and without invasive procedures might permit a rapid identification of the tumour biology and guide treatment and give a better upshots to molecular targeted therapies.

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fig.1

Summary of recent research activities and of main results achieved A Molecular Study of Pediatric Spindle and Sclerosing Rhabdomyosarcoma: Identification of Novel and Recurrent VGLL2≠related Fusions in Infantile Cases. Sclerosing rhabdomyosarcoma (ScRMS) and spindle cell rhabdomyosarcoma (SRMS) have been recently reclassified as a stand-alone pathologic entity, separate from embryonal RMS. Genetically, a subset of the congenital cases display different gene rearrangements to compare tumors occurring in older children or adults. Despite these recent advances, a significant number of tumors lack known genetic alterations. In this study we sought to investigate a large group of pediatric SRMS/ScRMS, spanning a diverse clinical and pathologic spectrum, by using a combined fluorescence in situ hybridization, targeted DNA, and whole-transcriptome sequencing methodology for a more definitive molecular classification. Ten congenital/infantile SRMS showed recurrent fusion genes: with novel VGLL2 rearrangements seen in 7, including VGLL2-CITED2 fusion in 4 and VGLL2-NCOA2 in 2 cases. Three cases harbored the previously described NCOA2 gene fusions, including TEAD1-NCOA2 in 2 and SRF-NCOA2 in 1. All fusion-positive congenital/infantile SRMS patients with available long-term follow-up were alive and well, none developing distant metastases. Among the remaining 15 SRMS patients older than 1 year, 10 showed MYOD1 L122R mutations, most of them following a fatal outcome despite an aggressive multimodality treatment. In conclusion, despite a relatively similar histomorphology,

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fig.1 Novel VG -CITED2 fusin gene discovery in congenital infantile SRMS by RNA se uencing and TopHat-se data analysis. A, Diagrammatic representation of the 6 22- 23 region breaks at the VG 2 and CITED2 loci, followed by an inversion and fusion of the 3 CITED2 to the 5 VG 2 portion. B, Fusion candidates were validated by RT-PCR showing fusion of the 1020 bp of VG 2 intron 3 to CITED2 exon 2 (SRMS6). C, FISH validation of VG 2-CITED2 fusion, first by the VG 2 breakapart assay (left panel) (arrows show constant split signal, in keeping with an intrachromosomal inversion break; red, centromeric; green, telomeric) and followed by the VG -CITED2 3 color fusion assay (right panel) (arrows show red-yellow fused signals; red, centromeric of CITED2; yellow, VG 2). D, Western blotting showing lack of the 28 kDa wild-type VG band in the 2 VG -CITED2 fusion-positive cases (SRMS and SRMS) compared with the control skeletal muscle. Instead, they show a lower band, possibly truncated protein at 22 kDa molecular weight.

fig.2

pediatric SRMS is a heterogenous disease genetically as well as clinically.

Recurrent BCOR Internal Tandem Duplication and YWHAE≠NUTM2B Fusions in Soft Tissue Undifferentiated Round Cell Sarcoma of Infancy: Overlapping Genetic Features With Clear Cell Sarcoma of Kidney.

Soft tissue undifferentiated round cell sarcoma (URCS) occurring in infants is a heterogenous group of tumors, often lacking known genetic abnormalities. We have investigated the possibility of shared genetic abnormalities in CCSK and soft tissue URCS. Most CCSKs are characterized

by BCOR exon 16 internal tandem duplications (ITDs), whereas a smaller subset shows YWHAE-NUTM2B/E fusions. Because of overlapping clinicopathologic features, we have also investigated these genetic alterations in the so-called primitive myxoid mesenchymal tumor of infancy (PMMTI). Among the 22 infantile URCSs and 7 PMMTIs selected, RNA sequencing was performed in 5 and 2 cases, with frozen tissue, respectively. The remaining cases with archival material were tested for YWHAENUTM2B/E by fluorescence in situ hybridization (FISH) or reverse transcription-polymerase chain reaction (RTPCR), and BCOR ITD by PCR. Histologically, URCS with both genotypes and PMMTI shared significant histologic overlap, with uniform small blue round cells with fine chromatin and indistinct nucleoli. A prominent capillary network similar to CCSK, rosette structures, and varying degree of myxoid change were occasionally seen. BCOR ITD-positive tumors occurred preferentially in the somatic soft tissue of the trunk, abdomen, and head and neck, sparing the extremities. RNAseq showed high BCOR mRNA levels in BCOR ITD-positive cases, compared with

fig.2 The index URCS case with YWHAENUTM2B FUSION. Di use sheets of monotonous round cells with fine chromatin, indistinct nucleoli, fre uent apoptosis, and occasional mitoses (A, hematoxylin and eosin). Representative karyotype showing a 3-way translocation t(10;1 ;14) ( 22.1;p13.3; 24) indicated by the arrows (B). FISH showing YWHAE rearrangement, with break-apart green (telomeric) and red (centromeric) signals (C, white arrows). Sanger se uencing of RTPCR product demonstrating YWHAE exon 5 fused to NUTM2B EXON 2 (D).

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fig.3

other URCSs. In summary, we report recurrent BCOR exon 16 ITD and YWHAE-NUTM2B fusions in half of infantile soft tissue URCS and most PMMTI cases, but not in other pediatric sarcomas. These findings suggest a significant overlap between infantile URCS and CCS , such as age at presentation, histologic features, and genetic signature, thus raising the possibility of a soft tissue counterpart to CCSK. Our data also suggest that the detection of BCOR-ITD by PCR or YWHAE gene rearrangement by FISH can be used in clinical practice for confirming this diagnosis in challenging cases of soft tissue tumors in infants.

Role of CIC-DUX4 and BCOR-CCNB3 fusions in edefinin ediat ic undi e entiated sa coma (UND) and translocation negative Ewing sarcomas (ES). Ewing sarcomas are primitive round cell tumours of bone and soft tissue of uncertain histogenesis. They are characterized by recurrent balanced translocations involving EWSR1 gene and members of the ETS gene family. Recently, a sub-group of UND with Ewing-like features and specific transcripts have been identified. CICDUX4 and BCOR-CCNB3 fusions have been detected in 68% and 4% of UND respectively. Whether they represent variants of ES, or distinct entities is so far unknown. In this study we investigated the presence of these transcripts in a series of paediatric sarcomas with histologic characteristics of ES or UND. About 300 paediatric cases (age range 0.5-18 yrs) with histological diagnosis of ES or UND were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) assays to specifically detect EWS/FLI1, EWS-ERG and EWS/ETV4 transcripts in freshfrozen and formalin-fixed, paraffin-embedded tissues. In 20 translocation negative cases one (0.4%) ES harboured 116

fig.3 Classical ES histology with uniform round cells and round to ovoid nucleus (A). CIC-DUX4 sarcoma elongated cells and clear cytoplasm (Case 1; B, C). Case 2 (BCOR-CCNB3) with elongated cells suggestive of a malignant peipheral nerve sheath tumor (F). RT-PCR analysis of CIC-DUX4 and BCORCCNB3 fusion transcript in a series of tumor biopsies.

a CIC-DUX4 fusion, and 8 (0.3%) UND the BCOR-CCNB3 transcript. These tumours showed a heterogeneous morphology, with elongated cells suggestive of a malignant peripheral nerve sheath tumour in one and a frankly round cell morphology with chondroid areas in the other, reminiscent of mesenchymal chondrosarcoma. CIC-DUX4 and BCOR-CCNB3 sarcomas should be considered in the spectrum of differential diagnoses of paediatric UND. These molecular investigations should be included in the diagnostic work-up of these tumours. Moreover, the morphologic heterogeneity of BCOR sarcomas may represent a diagnostic pitfall, simulating MPNST and mesenchymal chondrosarcoma, like in our cases. to enetics molecula and isto at olo ical c a acte i ation o tumo io sies a o in a a e usion t ansc i t Rhabdomyosarcoma account for 4-5% of all childhood malignancies, with two main histotypes: embryonal (ERMS) with intermediate prognosis and alveolar (ARMS) more aggressive. ERMS are genetically heterogeneous with LOH at 11p15.5 and various gene mutations, ARMS harboring in 75-80% of cases a gene fusion between PAX3 or PAX7 and FOXO1. Recent studies demonstrate the presence of rare fusion transcripts, PAX3/NCOA1, PAX3/NCOA2 and SRF4/NCOA2, in 1 or 2 cases of the patients cohort studied. Five PAX3-NCOA1 ARMS were identified in the Italian cohort from 2002. The aim of this study is to define the molecular features of these tumors in order to identify specific morphology, biology and clinical course for this subgroup of ARMS.

National and international collaborations: • • • • • • •

Gianni Bisogno, MD Pediatric oncologist, Division of Hematology-Oncology, Dpt of Mother and Child Health, University Hospital of Padova Prof. Rita Alaggio, Department of Pathology, University of Padova, Padova, Italy Dr. Cristina Antonescu, Department of Pathology, Memorial Sloan Kettering Cancer Center, New York Dr. Marina Gardiman, Department of Pathology, University of Padova, Padova, Italy Dr. Michela Pozzobon, Laboratory of Stem Cells and Regenerative Medicine, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padova, Italy Dr. Rita Zamarchi / Dr. Elisabetta Rossi / Dr. Mariangela Manicone, IOV- Istituto Oncologico Veneto, Padova, Italy Dr. Roberta La Starza, Laboratory of molecular medicin, University of Perugia, Italy

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CV Angelica Zin In October 1998 she started to work as technician in the Laboratory of Hematology-Oncology, Department of Woman’s and Child’s health, University of Padua, and in 2007 she obtained the degree in Health Biology at the University of Padova. From 2006, she is responsible of molecular diagnosis and Minimal Residual Disease of soft-tissue sarcomas (STS) in childhood in the laboratory of Solid Tumor Biology, that centralize samples from all national AIEOP centres (Associazione Italiana di Emato-Oncologia Pediatrica). She is molecular biology consultant for the Italian Soft Tissue Sarcoma AIEOP group and, from 2008, member of the Biology panel for the European Pediatric Soft Tissue Sarcoma Group (EpSSG). She attended the PhD school in Developmental Medicine and Planning Sciences, Adress Rare diseases, at the University of Padova where in the 2012 she was proclaimed PhD.

Angelica Zin 3 Publications 2016 13.52 IF as first/last author 2016 6 H-index (source RG)

118

RESEARCH AREA: Flow cytometry/Sorting

PI:

Chiara Frasson

Field of interest Flow cytometry is an extremely powerful technology that allows the individual measurement of physical and chemical characteristics of particles as they pass one by one through a light source.

Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry characterized by the same technology of the flow cytometer, but further it allows the physical separation of a cell or particle of interest (based upon the specific light scattering and fluorescent characteristics of each cell) from a heterogeneous population. If a population can be identified in an flow cytometer, it may be separated using a flow sorter.

In many situations in which a complex population is under investigation, there is frequently a need to isolate unique populations for further studies. Respect to other cellular separation instruments, the sorter is useful when multiple criteria of separation are requested.

There are two instruments in the Laboratory: the MoFlo (Beckman Coulter) and the FACS Aria III (Becton Dickinson). Both of these instruments are characterized by a high functionality and speed both in the analysis and in the sorting step. They are equipped by 3 lasers: a 488nm laser, a 375nm near UV laser and a 633nm red laser, with a configuration that allows to analyze potentially 12 parameters at the same time. They both are high speed instruments, they guarantee the maintenance of the high vitality of the sample during all the working steps, and they assure an elevated cellular recovery. The high definition and the versatility of the two sorters always guarantee optimal performances.

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Summary of recent research activities and of main results achieved

Sorter is an instrument available for both the internal and the external groups of the Laboratory. For this reason the types of experiments are several and various, according to the requirements of each different group. The request to sort cells is dependent on the need to deepen and to better characterize two or more different cell populations.

National and international collaborations: • • • • • • • •

Prof.ssa Marmiroli Sandra: Cellular Signaling Unit, Department of Surgery, Medicine, Dentistry and Morphology, University of Modena and Reggio Emilia, Modena, Italy.

Prof.ssa Parolin Cristina: Dipartimento di Medicina Molecolare, Università di Padova Dr. Indraccolo Stefano: Immunology and Molecular Oncology Unit; Istituto Oncologico Veneto-IRCCS; Padova, Italy Dr.ssa De Martin Sara: Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy.

Prof. Piccolo Stefano: Department of Molecular Medicine, University of Padua, Padua, Italy Dr.ssa Basso Daniela/Dr.ssa Fogar Paola: Department of Laboratory Medicine; University-Hospital of Padova; Padova, Italy.

Dr.Francesco Paolo Russo: Dipartimento di Scienze Chirurgiche, Oncologiche e Gastroenterologiche -DiSCOG, Università di Padova Dr.ssa Trevisan Marta: Dipartimento di Medicina Molecolare, Università di Padova

CV Chiara Frasson Biology degree, Research line: Physiopathology, University of Padua Clinical Pathology postgraduate school graduation, University of Padua PhD in the Doctoral School in Developmental Medicine and Planning Sciences - Research line: Haemato-Oncology and Immunology, University of Padua Personal skills and competence. She graduated in Biology at the University of Padova. Title of the thesis: “Characterization of cellular models for the study of the damages by oxidative stress in mitochondrial pathologies “ S e mo ed at t e aediatric epartment and e an er trainin in t e Onco ematolo y fields ein in ol ed in t e projects: “Immunotherapy strategies and immunologic characteristics in paediatric acute myeloid leukemia (AML)”.

From 2001 she attended the Medical school at the University of Padova to be specialized in Clinical Pathology in the 2005.

During this period she attended the Laboratory of Immunology and Transplant I.R.C.C.S. Policlinico S. Matteo, Pavia, Title of t e pro ect: Lymp o lastoid cell line eneration and specific C L lines eneration a ainst pstein arr irus .

Then, she attended the PhD school in Developmental Medicine and Planning Sciences - Research line: Haemato-Oncology and Immunology, University of Padua, where in the 2010 she was proclaimed PhD, Title of the thesis “ Tumoral stem cells: the role of Side Population in solid tumours”. Since 2008 she is the group leader for the sorting facility in the laboratory.

Chiara Frasson 5 Publications 2016 | 45.54 IF 2016 | 16 H-index (source RG) 120

RESEARCH AREA: Zebrafish and Leukemia

PI:

My research interests focus on understanding the genetic and molecular mechanisms of pediatric leukemias by integrating several complementary approaches including zebrafish genetic models, in vitro work, patient-derived primary cells and high-resolution genomic studies.

Giuseppe Germano

Summary of recent research activities and of main results achieved Projects ongoing 1) Functional analysis of genes deregulated in MLLrearranged AML MLL translocations lead to development of a very aggressive subtype of pediatric acute myeloid leukemia (AML), in which the conventional chemotherapies result often ineffective, indicating that more effective molecular mechanism-based therapies are needed. Gene expression profiling has demonstrated that leukemias bearing MLL translocations comprise a distinct disease characterized by a unique coexpression of early lymphoid progenitor-associated genes as well as myeloid-specific genes (Armstrong et al., 2002), suggesting immortalization of an early multipotent progenitor that remain blocked an undifferentiated stage of blood development.

The goal here is to characterize deregulated genes involved in stem cell maintenance and repression of differentiation-associated genes that may be potentially linked to MLL-induced leukemogenesis, and thereby gain insight into the mechanisms cause this subtype of AML in children and in developing new treatment approaches.

We recently identified a strongly upregulation of the transcription factor ZNF521 in MLL-rearranged AML, and demonstrated that it contributes to the differentiation arrest of leukemic cells.

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fig.1

fig.1 2) High risk T-cell acute lymphoblastic leukemia in Scheme of combinatorial analyses to study e afis model leukemia devolepment by zebrafish model T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subset of ALL that affects children and adults, and often is associated with primary non-responsive and relapse. Patients that relapse are largely unresponsive to additional therapy, for which there is a need to identify new therapeutic strategies.

Recently, our laboratory focused in dissecting the genetic bases of pediatric acute leukemia that currently do not respond to therapy revealing a large database of information, including deregulated transcriptional and proteomic profiles. We identified a strictly correlation between very high-risk T-ALL patients to relapse and the upregulation of the homebox gene HHE , which is involved in hematopoietic differentiation. In this project, I am developing robust transgenic zebrafish models overexpressing HHE in thymocytes to generate T-cell leukemia to screen small molecule libraries to identify candidates for more effective targeted therapy of T-ALL. ndi idual es onse to t e a in e afis enot ans lantation model Determine the best treatment that will be effective for the primary tumor and avoid drug resistance is challenging. This ongoing research includes the zebrafish embryos xenotransplantation with the long-term goal to develop a zebrafish xenograft assay for rapidly evaluating drug responses of leukemia cells from individual patients in an in vivo setting. Human fluorescently labeled cancer cells can be transplanted into zebrafish embryos and directly observed in the transparent zebrafish larva and rapidly evaluated after few days for the response to the treatment.

National and international collaborations:

Carolyn Felix, Division of Oncology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. Stefano Indraccolo, Immunology and Molecular Oncology Unit, Istituto Oncologico Veneto-IRCCS, Padova, Italy.

CV Giuseppe Germano Dr Giuseppe Germano has obtained his graduate degree in biological science (BS) at the University of Padua in 1996, followed by specialization in Clinical Pathology in 2004. He in 1998-2000 attended at the Foundation M. Tettamanti of University of Milan, where he achieved the expertise in monitoring of the minimal residual disease by quantitative PCR analysis of Ig and TCR gene receptor rearrangements in children with acute lymphoblastic leukemia. He received his PhD from the University of Padua in 2007, where he had the opportunity to attend for 2 years at the Children’s Hospital of Philadelphia during his doctorate, in which t e pro ram included studies on patient deri ed materials and rele ant animal models mouse and ze rafis in area of leu emia pathobiology driven by MLL fusion oncogenes.

He currently works as a postdoctoral fellow in the hematology and oncology research program at the Istituto di Ricerca Pediatrica ondazione I Citt della Speranza. He is t e mana er of t e Ze rafis acility at I , in c ar e of all aspects of maintain t e ze rafis colony in ood ealt . He is currently responsi le of t e elfare and care of t e animals in t e esta lis ment. en e is not in the lab, he enjoys running, playing tennis, reading, and most of all spending time with his wife and son.

Giuseppe Germano 2 Publications 2016 | 12.82 IF 2016 | 6 H-index (source RG) 122

RESEARCH AREA: Gene Sequencing

PI:

Maddalena Paganin GROUP MEMBER PhD Student: Francesca Grillo Student: Maria Saitta

Summary of recent research activities and of main results achieved The T immunophenotype is considered to be an unfavorable prognostic feature in childhood ALL and T-ALL patients commonly are treated with high-risk protocols. Even if the contemporary clinical trials have improved the survival of childhood acute lymphoblastic leukemia, further advances in survival and quality of life oblige a better understanding of acute lymphoblastic leukemia pathology.

•

•

•

Acute lymphoblastic and myeloid leukemia are neoplasms characterized by abnormal proliferation and development of hematopoietic cells. We studied the genetic profile of a T-ALL to AML lineage switched leukemia case. The genetic study revealed that these leukemias shared somatic gene mutations/deletions and TCR rearrangements The genetic abnormalities recurrently present in the T-cell leukemia cells could have prognostic or therapeutic relevance but still there is no consensus on the specific genotypes for eventually a treatment stratification in T-ALL. Specifically we analyzed variants in NOTCH1, FBXW7, IL7R and PTEN in a cohort of 200 T-ALL patients treated with AIEOP protocols.

Our genetic study aimed to clarify the consequent outcome with the presence of genetic mutations.

Each human cell contain in the genes the instruction that utilize to growth, divide and develop in a normal way. When a cell acquire genetic mutations the tumor develop as the consequence of a functionality chaos of the cell. Our group focused on the identification of new somatically mutated genes in T-ALL patients, non ETP and not responding

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completely to the initial treatment protocol. The prospective is to use the detection of the somatic mutations for therapy decision.

Future Projects The cure rate of childhood acute lymphoblastic leukemia is near to 90% merit of improved combined therapy, more precise risk stratification, and personalized treatment based on the characteristics of leukemic cells and hosts. Minimal residual disease (MRD) is the level of tumor residual cells not identifiable with the light microscopy. The prognostic significance of MRD during therapy has been already clearly demonstrated in paediatric ALL. Actually the MRD is quantifiable by sensitive and specific technique such as real-time quantitative PCR of immunoglobulin/T cell receptor or detection of aberrant immunophenotypes by flowcytometry. We are going to study if the somatic mutations can be used as genetic marker for each specific leukemia clone in T-ALL patients and if the high-throughput ultra deep next generation-sequencing allow the precise e sensitive detection of the single cancer cell in a bulk of normal cells and the analysis of the MRD during therapy.

CV Maddalena Paganin Degree in Biological Science, University of Padua; PhD in Medicine of Development and Science of Programming, University of Padua.

She graduated in Biological Science at University of Padua in 2004, studying and working one year abroad at the Imperial College of London. Since 2005 she has been working at the laboratory of Oncology and Haematology in Hospital of Padua, Pediatric Department, University of Padua. Since 2005 to 2014 she was a member of the International BFM Study Group MRD Task Force. From 2006 to 2009 she did her Phd in Medicina dello Sviluppo e Scienze della Programmazione in University of Padua and she was responsible of the Antigen Receptor Group. From to 2009 to 2010 she was a post-doc in the laboratory of Prof. A.A. Ferrando in the Institute for Cancer Genetics, Columbia University, Irving Cancer Centre, e or , or in on t e molecular c aracterization of cell lymp o lastic leu emia. Specifically s e performed a i density mutation screening with next-generation sequencing technique. Sine 2011 she has worked in the laboratory of the Clinic of Paediatrics Oncoematology in the University of Padua directed by Prof. Giuseppe Basso to head the Antigen eceptor roup and to study molecular enetics of LL or in in t e t ree researc pro ects: 1 enomic profilin of acute leukaemia T-cell lineage switched in acute myeloid leukaemia, 2) Improving cure rates for children with cancer-focus on patients that currently do not respond to therapy, 3) Validation of impact of genetic aberrations and host genetic variation in childhood acute lymphoblastic leukaemia for integration into clinical practice.

In the 2015 she won the Grant Young Researcher from Ministero della Salute in Italy, to develop the research project “A new strategy for the detection of residual leukemia cells� combining her knowledge on minimal residual disease in acute lymphoblastic leukemia and the detection of genomic mutation with next-generation sequencing techniques.

Maddalena Paganin 1 Publications as first/last author 2016 124

2.39 IF as first/last author 2016

9 H-index (source RG)

STUDY AREA: Brain Tumors

PI:

Luca Persano GROUP MEMBER Post≠doctoral Fellows: Chiara Frasson Elena Rampazzo Elena Porc˘ Francesca Maule Daniele Boso

Summary of recent research activities and of main results achieved Deciphering the role of developmental signalling pathways in modulating Glioblastoma aggressiveness and phenotype.

Glioblastoma multiforme (GBM) is the highest grade glioma, characterized by a rapid growth rate and an extensive infiltration into the surrounding brain tissue. In this context, the Brain Tumors group focused in the last years on unveiling the mechanisms by which GBM tumor microenvironment (i.e. hypoxia) influences the activation of many developmental pathways and how their modulation impacts GBM biology.

In particular, they unveiled the interplay between the hypoxic GBM microenvironment and the -catenin/TCFs complex as key determinants of GBM cell aggressiveness and phenotype.

Moreover, they evaluated the role of Annexin A2 in GBM proliferation and dissemination, exploiting the possibility to target Annexin A2 intracellular signalling pathway to counteract GBM infiltrative nature. Historically, the research group focused its efforts on the pro-differentiating strategy on cancer stem cells as potential therapeutic approach for GBM management. In this regard, they are testing the use of Bone Morphogenetic Proteins, as both recombinant proteins or synthetic small peptides BMP-mimetic, for their ability to induce GBM cell differentiation and sensitization to chemotherapy.

Characterization of Medulloblastoma Cancer Stem Cells and their impact on therapy response.

Medulloblastoma (MDB) is the most common malignant

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brain tumor of childhood. Although survival has slowly increased in the past years, the prognosis of these patients remains unfavourable. In this context, their research on paediatric Medulloblastoma has been focused in the study of intracellular signaling pathways, commonly activated during embryonic cerebellar development, that are often deregulated in MDB. In particular, they exploited the fundamental role of microenvironmental hypoxia in maintaining MDB stem-like cells, considered to be responsible for treatment resistance and recurrence. Moreover, the group recently characterized the Notch pathway and the PI3K/AKT/mTOR intracellular axis as leading intracellular signalling implicated in MDB cell proliferation, survival, growth rate, and protein synthesis. As a result, they demonstrated that a specific targeting of the stem cell compartment in MDB tumours can boost standard chemotherapy efficacy.

Future Perspectives

GBM tumours are notoriously resistant to current therapies with a median survival of patients of only 17 months after diagnosis. For this reason, the identification and setup of innovative strategies to counteract GBM resistance are urgently needed. In this context the Brain Tumors Team is developing two projects: 1.In the first project they aim to identify the molecular mechanisms involved in EGFRvIII mutation driven gliomagenesis. Indeed, EGFR mutations are a common feature of GBM tumors and are thought to play a fundamental role in supporting the aggressive phenotype of these tumors, by sustaining cell proliferation and resistance to treatments. For these reasons, they will generate an EGFRvIII-driven GBM ebrafish ( F) model and, after its characterization, they will exploit a series of F strains reporter for the major developmental signalling pathways in order to identify the leading molecular signalling activated during GBM onset. The identified pathways will be then considered as potential targets for GBM therapy and used to screen an FDA-approved drug library in order to test already known drugs for their potential repositioning. This project has been funded by the University of Padova with a Senior investigator Grant - University of Padova - Bando Giovani Studiosi to Elena Rampazzo in 2016.

2.In the same attempt to move a step forward towards personalized therapy, they recently submitted to the Ministry of Health a project aimed to validate a drug repurposing workflow in an “ORGANized model� of GBM tumors by classifying patients into four distinct molecular subtypes according to their transcriptional profile. Moreover, specific compounds for each GBM subtype will be evaluated for their specificity and effectiveness in novel tridimensional ORGANoid cultures, already described as a new reliable tool to model the complexity and heterogeneity of the brain microenvironment.

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National and international collaborations: • • • • • • • •

Dr. Alessandro Della Puppa, Neurosurgery Unit, University-Hospital of Padova, Padova, Italy.

Dr. Carlo Zanon, Neuroblastoma Laboratory, Istituto di Ricerca Pediatrica Città della Speranza, Padova, Italy.

Dr. Francesca Pistollato, European Commission, Joint Research Centre, Ispra, VA, Italy.

Dr. Stefano Indraccolo, Istituto Oncologico Veneto IOV-IRCCS, Padova, Italy.

Prof. Antonio Rosato, Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy.

Prof. Monica Dettin, Department of Industrial Engineering, University of Padova, Padova, Italy.

Prof. Paolo Bonaldo, Department of Molecular Medicine, University of Padova, Padova, Italy.

Dr. Flora Cimmino, Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II”, CEINGE Advanced Biotechnology, Naples, Italy.

CV Luca Persano Since his PhD studentship in Oncology and Surgical Oncology at University of Padova, Dr. Persano’s studies have been focused on dissecting the molecular pathways underlying cancer progression and resistance to therapy, with particular focus on the role played by tumour microenvironment. Indeed, in the Laboratory of Molecular Immunology and Gene Therapy directed by Dr. Indraccolo his project was committed to study the process of tumour angiogenesis and exploit its potential in i ition as a t erapeutic strate y in different tumors includin prostate, o arian, esop a eal and colon cancers.

In addition, since 2009 Dr. Persano’s studies have been focused on brain tumour biology with particular emphasis in unveiling t e mec anisms y ic rain tumor microen ironment i.e. ypo ia in uences t e acti ation of many de elopmental pat ays includin one Morp o enetic roteins, nt and otc si nalin . In t is conte t, in t e recent years t e rain Tumors Team developed a multilayer model of Glioblastoma in which they characterized the activation of the hypoxic signalling and its crosstalk with Glioblastoma cancer stem cells. Dr. Persano’s studies on Glioblastoma have been granted by the University of Padova (Young Investigator Grant 2010) for the development of a two-year project as independent PI it full scientific and financial responsi ilities. Moreo er, from 010 e is in c ar e for t e coordination of t e acti ities of the Brain Tumors Unit in the Laboratory of Pediatric Oncohematology, University of Padova, being the tutor for four Master Degree and four PhD students whose projects were all addressed to the study of brain tumor biology.

Luca Persano 1Publications as first/last author 2016 6.36 IF as first/last author 2016 17 H-index (source RG)

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RESERCH AREA: Experimental Pharmacology

Summary of recent research activities and of main results achieved

PI:

Past projects Giampietro Viola GROUP MEMBERS Post≠ Doctoral Fellows: Roberta Bortolozzi Elena Porc˘ Elena Mariotto Elena Mattiuzzo PhD Student: Annagiulia Bonizzato

Design and development of new potential antitumor drugs The microtubule system of eukaryotic cells is a critical element in a variety of fundamental cellular processes such as cell proliferation, mitotic spindle formation, maintenance of cell shape, regulation of motility, cell signaling, secretion, and intracellular transport.

Its important role in mammalian cell have made it an attractive target for the development of anticancer drugs. Because the central role of microtubules dynamic in mitosis progression and thus in cell proliferation, drugs that affect microtubule assembly are important component in combination chemotherapy for the treatment of pediatric and adult cancer. In this context we have evaluated both in vitro and in vivo different new compounds for their antimitotic activity.

Development of new vascular disrupting agents The tumour vasculature is an excellent therapeutic target as it is easily accessible to blood-borne drugs and tumor cells generally die unless they are continually supplied with oxygen and nutrients from the blood. The two approaches to inhibiting vascular function are to inhibit angiogenesis — the formation of new blood vessels — using anti-angiogenic agents and to destroy the integrity of existing tumour vasculature using vascular-disrupting agents. Formation of new blood vessels involves proliferation and migration of endothelial cells and both of these processes seem to be extraordinarily sensitive to microtubule-targeted drugs., With the purpose to hit both the tumor cells and the vasculature, we have studied the efficacy of new antimitotic molecules on angiogenic models.

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fig.1

Role of FOXM1 in B-ALL progression Forkhead transcription factors are a superfamily of evolutionary conserved proteins that function in several physiologic processes including cellular differentiation, tumor suppression, metabolism, cell cycle arrest, resistance, and apoptosis. In particular we have identify FOXM1 as a target for leukaemia therapy. Our findings showed that FO M1 expression is higher in B-ALL patients and in B-cell lines compared CD19+ cells from healthy donors. Furthermore, blocking FOXM1 activity in B-ALL cell lines by either knockdown or treatment with the FO M1 inhibitor thiostrepton, causes significant decreases in the cell viability in both cell lines. Moreover, thiostrepton synergises with chemotherapeutic agents commonly used in B-ALL therapy increasing their efficiency and overriding drug resistance, suggesting that targeting FOXM1 could be an attractive strategy for leukaemia therapy and for overcoming drug resistance.

fig.1 The aim of our research is to study the function of antiproliferative compounds at the molecular level, and to develop novel active compounds in the field of cancer pharmacology. Moreover, we are also studying the function of selected compounds and targets ex vivo and in vivo using animal models and genetically modified mice. Most of our pro ects are interdisciplinary, using computer modeling and medicinal chemistry in con unction with molecular pharmacology.

e a eutic otential o a is in ediat ic acute lymphoblastic leukemias Another transcription factor potentially involved in leukemogenesis is FOXO3a. We investigated whether therapy resistance in childhood T-ALL cells correlates with inactivation of FOXO3. We have found that cells from prednisone-resistant T-acute lymphoblastic leukemia (T-ALL) patients showed cytoplasmic localization of FOXO3 in comparison to prednisonesensitive patients suggesting its inactivation. We uncovered, that FOXO3 activates apoptosis by induction of TRAIL and Noxa and found that the expression of the frequently mutated tumor suppressor p16INK4A in T-ALL represses endogenous FOXO3, suggesting that these two tumor suppressor proteins cooperate to prevent childhood leukemia. The involvement of FOXO3a in glucocorticoid resistance was also studied in B-acute lymphoblastic leukemia (B-ALL). We have demonstrated that in response to dexamethasone, FOXO3a translocates into the nucleus and elicits the expression of downstream targets

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important for proliferative arrest and cell death. FOXO3a activation by dexamethasone is mediated through its Ser-7 phosphorylation and Lys-242/5 acetylation. These findings expose FO O a and its post-translational modifications as essential for dexamethasone response, which can be exploited for B-ALL diagnosis and treatment.

Ongoing/Future Projects Potential application of new choline kinase inhibitors.

Choline kinase (Cho ) is one of the most important enzymes for the generation of two major membrane phospholipids, phosphatidylcholine (PC) and sphingomyelin (SM), and subsequently for the cell division.

Choline kinase is a homodimeric enzyme that catalyses the Mg.ATP-dependent phosphorylation of choline to generate phosphocholine (PCho) as the first step in the pathway. In mammalian cells, only the Cho isoform (synthesized by Chk gene) has a central role in sustaining PCho biosynthesis, indeed Cho alone cannot compensate this activity. Furthermore, a large number of studies in cancer cells suggests that Cho expression and activity are directly associated not only with increased cancer cell proliferation but also with malignancy, making it a potential prognostic marker for some cancers, such as non-small-cell lung cancer and breast cancer as well as a potential novel target for image-guided cancer therapy. The cholinic phenotype, characterized by increased Cho activity and expression, renders this enzyme an attractive new therapeutic target. The feasibility of Cho inhibition as antitumoral therapy is being pursued in our laboratory through the development of chemical inhibitors of its enzymatic activity. In this context we are evaluating the antitumor activity of new choline kinase inhibitor in breast cancer and in T-acute lymphoblastic leukemia (T-ALL). inome ofile o A T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic disorder resulting from the malignant transformation of T-cell progenitors that represent about 15 of pediatric ALL. Significant improvements in chemotherapeutic protocols for childhood acute lymphoblastic leukemia, achieve 5-year survival rates of about 80%, however relapse is frequently observed in T-ALL and prognosis after relapse remains poor with survival rate from 10 to 40 . This project will develop an innovative approach for molecular target and drug discovery, based on the identification of kinases aberrantly switched on in malignant cells. Moreover, focusing our study on resistant T-ALL patients, we will provide important resource for T-ALL molecular characterization and for the development of molecular targeted therapies. This approach represent a new promising strategy to give an effort in the fighting of drug resistance in T-cell acute lymphoblastic leukemia.

The knowledge already at diagnosis that one patient could benefit from the use of a specific phosphoprotein inhibitor more than from the use of a conventional drug, or stratify patients to the most correct treatment protocol avoiding dangerous overtreatment or uneffective therapies, will allow a patient-adapted treatment more efficient and less toxic, thus improving therapy response and patients quality of life.

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National and international collaborations: • • • • • • • • • • • • • •

Prof. Romeo Romagnoli, Department of Pharmaceutical Science, University of Ferrara.

Synthesis of new molecule with antiproliferative activity.

Dr. Maria Grazia Ferlin/Prof. Stefano Moro Department of Pharmaceutical and Pharmacological Sciences, University of Padova.

Synthesis of new molecule with antiproliferative activity.

Prof. Marco Presta, Department of Biological Science and Biotechnology, University of Brescia.

In vivo study of angiogenesis.

Prof. Michael J. Ausserlechner, Tyrolean Cancer Research Institute, Department of Pediatrics II, Medical University, Innsbruck, Austria.

Study of FOXO3a transcription factor.

Prof. Eric W.F. Lam Imperial College London Study of FOXO3 and FOXM1 in pediatric ALL Prof. Heiko Ihmels, Organic Chemistry II, University of Siegen, Adolf-Reichwein-Str. 2, D-57068 Siegen, Germany.

Synthesis of new molecule with antitelomerase activity.

Prof. Luisa Carlota Lopez Cara University of Granada (Spain) Synthesis of new Choline kinase inhibitors.

CV Giampietro Viola Giampietro Viola was graduated in Pharmacy and in Chemistry and Pharmaceutical technology at the University of Padova.

At the same university he also obtained a PhD in Pharmaceutical Sciences. He started his career as researcher at FIDIA Research Laboratories and then joined the University of Padova as research assistant. Actually he is Principal Investigator at t e epartment of oman s and C ild s Healt . He spent a lar e num er of period it ualified researc structures abroad. His research interests concern the study of new molecules endowed with antiproliferative activity both in vitro and in i o and in particular of t e mec anisms t at lead to cell deat . In t e last years is researc efforts a e een de oted to t e identification of ne tar ets in c ild ood acute lymp o lastic leu emia and cancer it particular attention on dru resistance. Moreover he is also involved in the screening and biological evaluation of newly synthesized compounds. He is aut or of more t an 1 scientific paper in ualified peer re ie ournals. His pu lications a e recei ed until no more t an 4000 citations and H 0 is is Hirsc inde . He currently ser e as re ie er for many ournal in t e field of medicinal chemistry and pharmacology and he is Associate Editor of Biochemical Pharmacology. He is also holder of Italian and European patents concerning the synthesis and biomedical application of new potential antitumoral molecules.

Giampietro Viola 2 Publications as first/last author 2016 .0 IF as first/last author 2016 30 H-index (source RG) 131

Laboratory of Neuroblastoma GROUP LEADER AND REFERENT:

Gian Paolo Tonini

GROUP MEMBERS

Post Doctoral Fellows: Sanja Aveic Diana Corallo Maria Rosaria Esposito Pina Fusco Medical Science: Carlo Zanon Technician: Marcella Pantile

Field of interest The main research programs are focused to neuroblastoma and the main projects of the Laboratory are: 1.

Discovery of mutations associated with aggressiveness of High Risk Neuroblastoma (HR-NB) patients using Whole Exome Sequencing (MRE; MP). The analysis of several neuroblastoma cases in all clinical stages by Whole genome (WGS) or exome sequencing (WES) revealed structural alterations affecting the PTPRD, OD , TRIO, DLC1, CSMD1, ATR , ARID1A and ARID1A genes and a few high-frequency recurrent somatic variants in the AL , CHD9, PT 2, NA , NA 1, F D1, ARID1A, ARID1B, TIAM1, SOS1, ARHGAP10, PTPN11, OR5T1, PDE6G, M CN and NRAS genes. There is no evidence of genes associated with rapid disease progression and we believe that NB aggressiveness is associated with a long “tail” of many genes mutated that deregulate specific biological pathways. This project is aimed to identify gene mutations and gene pathways abnormalities in HR-NB. 2. Pharmacological studies: Seeking for novel therapeutic combinations for HR-NB (AS; DC; MP). Currently adopted strategies for neuroblastoma treatment vary greatly depending on patients risk stratification. Therefore, for the patients with low risk and localized tumors either tumors regress spontaneously or the surgery is an approach sufficient for tumor mass elimination. Instead, the intermediate risk requires a combination of surgery and chemotherapy. On the other side, the worst-case scenario is written for HR-NB patients for whom treatment includes multimodal therapy, which has an important “weight” on the child’s life and their general wellness.

In spite of these treatments the overall survival for the children with HR-NB is still excessively low below 40 . For this reason, exploration of novel drugs that are more efficient and therapies that could increase a possibility for the cure is highly required. In

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fig.1

our laboratory we aim to perform a pre-clinical phase of drug testing in order to select new potentially applicable drugs against important molecular targets in neuroblastoma (Figure 1). Among these targets are: AL (inhibitors Entrectinib and Crizotinib); AURORA A (inhibitor Alisertib), and receptor tyrosin kinase inhibitors (with inhibitors: Afatinib, Sorafenib and TP-090 ). Single treatments or combined therapy are applicable in order to select the best possible therapeutic strategy.

3. Study of transcriptome instability (TIN) (CZ; GPT). Genome instability is a hallmark of cancer, but its transcriptional counterpart is rarely taken into consideration as a relevant factor in cell destabilization. To investigate the transcriptome instability (TIN) in neuroblastoma we devised an index to quantify TIN (TIN-index). The TIN-index has been studied in a large cohort of cases to evaluate its prognostic value.

4. Role of Lin28 in neuroblastoma development (DC; AS; MP). Lin28 gene is participating to the aggressiveness of neuroblastoma. Lin28 is also express during development of neural crest cells (NCC), the cells from which neuroblastoma is originating. In order to establish at which moment Lin28 might sustain tumorigenesis we are using an in vivo experimental approach. This project is focused to clarify the role of Lin28 in neuroblastoma tumorigenesis.

5. Organoid model of neuroblastoma (MRE; PF).

Recently, it has been described the three-dimensional culture (the so called: organoid) method that can recapitulate features of in vivo tumor cell growth, allowing self-renewal and self-organization. These organoids mimic the tissue of origin in both their composition and architecture, and exhibiting similar organ functionality as the primary tissue.

Therefore, organoid system offer unique possibilities for disease modelling in vitro. In order to assess the role of AP/TA activation, already described in others human cancer, in growth, self-renewal and progression of NB tumor 134

fig.1 E ects of ATRA on cell morphology. Retinoic acid (ATRA) induces di erentiation of neuroblastoma cells. Control (DMSO treated) cells form aggregates over time whereas ATRA treated cells form intense neurite outgrowth 2h post-treatment. Magnification, 20.

fig.2

we established organoid culture using patient-derived neuroblastoma cells of primary tissue. Organoid culture system is a state-of-art tool to study cellular signalling pathways as they structurally and functionally resemble the tissue of origin, while remaining phenotypically and genetically stable. Additionally, organoids can easily genetically modified with different strategies such as the CRISPR-Cas9 system and lentiviral transduction. We will perform lentiviral transduction of AP and TA , either overexpression or knockdown in the organoid system to investigate the role of AP/TA in neuroblastoma aggressiveness.

6. Importance of Autophagy in drug resistance and metastatic process (AS; DC; CZ; MP). Autophagy has been connected with two important strategies (resistance and metastasis) both of which are frequently used by tumor cells to escape cytotoxic effects of administered drugs. In order to explore in which extent this biologic process is involved in the impediment of drug efficiency in neuroblastoma we are currently testing autophagy inhibitors together with antitumor drugs plausibly applicable in neuroblastoma therapy.

In order to distinguish in which extent autophagy can regulate resistance and invasive feature of neuroblastoma cells we apply in vitro, in vivo and the ex-vivo neuroblastoma primary cells model systems.

fig.2 Bioinformatic pipeline and study design used for somatic variants identification. The owchart summarizes the work ow followed for the identification of group-exclusive somatic variants and mutated pathways.

Summary of recent research activities and of main results achieved

1. Discovery of mutations associated with aggressiveness of High Risk Neuroblastoma (HR-NB) patients using Whole Exome Sequencing. To identify biological pathways associated with poor disease outcome we realized WES on 2 subgroups of HR-NB patients (Figure 2): long survivor (LS) with slower disease course and an OS over 60 months; short survivor (SS) irresponsive to therapy 135

fig.3

and OS within 60 months from diagnosis. In the latter we found significantly affected G-protein-coupled-receptors (GPCR) and GPCR ligand binding signaling. GPCR pathway regulates AP/TA activity through the upstream Rho/Ras cascade and actin cytoskeleton. According to previous NB reports describing an up-regulation of Hippo- AP in tumor at relapse, we believe that the activation of AP/TA , already described in others human cancer, plays a critical role in growth, self-renewal and HR-NB progression.

2. Pharmacological studies: Seeking for novel therapeutic combinations for HR-NB. Pre-clinical examination of the efficiency of novel AL inhibitor Entrectinib gave very promising results. This drug, proposed by Ignyta, demonstrated the effectiveness in blocking neuroblastoma cells’ proliferation (Figure 3). Beside cytostatic effects, Entrectinib showed also the toxic potential when applied at higher concentrations (5 M). Of big importance was our discovery that the activation of autophagy occurred due to the use of AL inhibitor Entrectinib. This event was then correlated with decreased efficiency of Entrectinib in impeding the growth of neuroblastoma cells with confirmed F1174L mutation. In accordance, combined use of Entrectinib and chloroquine, an inhibitor of autophagy, improved significantly the cytotoxic effects of drug itself giving a new possible therapeutic option for neuroblastoma patients. The obtained pre-clinical results remain to be tested in clinical practice.

3. Study of transcriptome instability (TIN). We evaluated the TIN-index in 500 neuroblastoma samples and its correlation with a number of clinical parameters. We found that metastatic tumors from HR patients are characterized by high TIN-indexes opposed to localized tumors which show low TIN-index values and we also identified a TIN-related gene signature (TIN-signature) capable of predicting patient’s outcome with high confidence. Furthermore, we showed that this global transcriptome alteration interferes with the integrity of chromatin domains involving super-enhancers 136

fig.4

fig.3 Colony formation. Semi-solid medium assay shows significant reduction of a number of the colonies for Entrectinib (5 M) treated cells with respect to DMSO control. Magnification, 10. fig.4 Genome-wide (left panel) and chromosome 1 (right panel) gene expression correlation heatmaps. Genes are ordered from left to right by chromosome (left panel) and position.

Correlations are calculated for IR (top panel) and HR samples (middle panel), respectively.

High correlation values (dark-red) characterize blocks of neighboring genes emerging as small triangular-shaped domains at the bottom of the heatmaps. Higher order triangular-shaped patterns are also visible, identifying larger fields within which the average correlations are higher compared to longer-range interactions.

Blue values represent low correlations, characterizing insulation regions separating high-correlation blocks. The bottom heatmap represents the arithmetic di erence between the HR and the IR heatmaps (HR minus IR); positive values (orange) identifies blocks in which the expression correlation is higher in HR samples compared to IR samples.

Negative values (cyan) show regions of higher interaction in IR compared to HR ones.

fig.5

active in neuroblastoma cell lines. This novel approach to gene expression analysis broadens the perspectives of genomic instability investigations towards their structural and functional aspects (Figure 4).

4. Role of Lin28 in neuroblastoma development. In zebrafish and enopus embryos, we are characterizing the expression of Lin28 paralogs during embryonic development.

Moreover, we are developing a suitable system that allows the recapitulation of neuroblastoma development starting from the embryonic origin of the disease, through Lin28b overexpression specifically in the peripheral sympathetic nervous system. With this aim, we are studying the precise mechanism by which Lin28b participates in the modulation of neural crest cells migration and differentiation toward the sympathoadrenal cell lineage (Figure 5). Moreover, we are performing a parallel analysis with the transgenic Tg(M CN:EGFP) zebrafish line which is a well-known in vivo model of neuroblastoma.

5. Organoid model of neuroblastoma. We are performing ex vivo organoid model of neuroblastoma. We were able to derive single cells from NB patient tumor tissues to realize organoid model (Figure 6) and we are planning to characterize the model. An organoid is defined as an ex vivo D cellular cluster derived exclusively from primary tissue, ESCs or iPSCs, capable of self-renewal and self-organization, and exhibiting similar organ functionality as the tissue of origin.

We attempt to obtain a novel model to recapitulate tissue tumor architecture, microenvironment and the complex nature of biological processes associated to neuroblastoma.

6. Importance of Autophagy in drug resistance and metastatic process. We are evaluating whether autophagy inhibition might improve effects of several RT inhibitors that have been proposed in pre-clinical studies for neuroblastoma treatment (e.g. sorafenib, afatinib, TP-090 , Entrectinib) (Figure 7). At second line, we are evaluating in vitro and in vivo anti-A L compound TP090 (able to reverse

fig.5 Analysis of neural crest cells (NCCs) migration path in in28-b overexpressing embryos.

In control embryos, many NCCs (red) have already migrated to a position near the dorsal aorta (green), where sympathetic ganglia normally arise. This region appeared depleted of NCCs cells in in28b transgenic embryos (white asterisks).

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fig.6

EMT process) for its possibility to inhibit neuroblastoma cells growth. Particular focus will be given in studding how these compounds might influence neuroblastoma tumor initiating cells (TICs) to form tumors.

All projects were supported by Fondazione Italiana per la Lotta al Neuroblastoma

National and international collaborations: •

• •

• • • •

PI: Dr. Mario Capasso - Università degli Studi di Napoli Federico II, Dipartimento di Medicina Molecolare e Biotecnologie Mediche, CEINGE Biotecnolgie Avanzate, Naples, Italy PI: Prof. Stefania Bortoluzzi - Department of Molecular Medicine University of Padua, Italy PI. Dr. Luca Longo - U.O. Biotherapy, IRCCS AOU San Martino-IST, National Cancer Research Institute, Genoa, Italy, Pathology Unit “Giannina Gaslini Institute/Italian Neuroblastoma Foundation”, Genoa, Italy Prof. Michela Ori Unit of Cellular and Developmental Biology, Department of Biology, University of Pisa.

Prof. Simona Candiani, Laboratory of Developmental Neurobiology. DISTA , Università di Genova CNR Rome Dr. Francesca Degrassi, Institute of Molecular Biology and Pathology, IBPM National Research Council, CNR c/o Department of Biology and Biotechnology Sapienza University, Rome

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fig.6 ight microscopy of organoids derived from TICs. In the figure showed organoid growth derived from primary cultures of NB. Ac uisitions made at three time points: time 0, after and 14 days after culturing with Nikon Eclipse TS100 microscope (Nikon Eclipse TS 100, Southern Micro Instruments, Marietta, GA) with a camera Nikon Coolpix. fig.

Autophagy activation. Entrectinib induces autophagy in neuroblastoma cells. The presence of the autophagosomes isconfirmed as GFP-puncta formation only in treated cells (right image) and not in DMSO controls (left image). Magnification, 60.

fig.

5-10 Selected Publications Neuroblastoma (Peripheral neuroblastic tumours)

Luksch R, Castellani MR, Collini P, De Bernardi B, Conte M, Gambini C, Gandola L, Garaventa A, Biasoni D, Podda M, Sementa AR, Gatta G, Tonini GP

Crit Rev Oncol Hematol. 2016 Nov; 107:163-181.

Combating autophagy is a strategy to increase cytotoxic effects of no el L in i itor entrectinib in neuroblastoma cells

Aveic S, Pantile M, Seydel A, Esposito MR, Zanon C, Li G, Tonini GP

Oncotarget. 2016 Feb 2;7(5):5646-63

e ze rafis as a model for studying neuroblastoma

Corallo D, Candiani S, Ori M, Aveic S, Tonini GP

Cancer Cell Int. 2016 Nov 3; 16:82

Genome instability model of metastatic neuro lastoma tumorigenesis by a dictionary learning algorithm

Masecchia S, Coco S, Barla A, Verri A, Tonini GP

BMC Med Genomics.

2015 Sep 10;8:57

o ards a turnin point of neuroblastoma therapy

Tonini GP, Nakagawara A, Berthold F

Cancer Lett. 2012 Dec 30;326(2):128-34

Dissecting the genomic complexity underlying medulloblastoma

ones , er , ool M, Zic ner , Hutter , Sultan M, Nature. 2012 Aug Cho YJ, Pugh TJ, Hovestadt V, StĂźtz AM, Rausch T, Warnatz 2;488(7409):100-5.

H , yz o a M, ender S, Sturm , leier S, Cin H, faff , Sieber L, Wittmann A, Remke M, Witt H, Hutter S, Tzaridis T, eisc enfeldt , aeder , ci M, mstisla s iy , Zapat a M, Weber UD, Wang Q, Lasitschka B, Bartholomae CC, Schmidt M, on alle C, st , La erenz C, ils , a e , enes , an Sluis , oster , olc mann , S i , etts M , ussell , Coco S, onini , Sc ller , Hans , raf , im , Monoranu C, o endorf , nter er , Herold Mende C, Milde , ulozi , on eimlin , itt O, Maass , ssler J, Ebinger M, Schuhmann MU, FrĂźhwald MC, Hasselblatt M, a ado , ut o s i S, on ueren O, illiamson , Clifford SC, McCa e M , Collins , olf S, iemann S, Le rac H, rors , Sc eurlen , els er , eifen er er , ort cott , aylor M , Meyerson M, omeroy SL, aspo ML, or el O, ors uno , ils , fister SM, Lic ter .

Age-dependent accumulation of enomic a errations and dere ulation of cell cycle and telomerasen genes in metastatic neuroblastoma

Coco S, eissen , Scaruffi , Sti liani S, Moretti S, Oberthuer A, Valdora F, Fischer M, Gallo F, Hero B, Bonassi S, Berthold F, Tonini GP

Int J Cancer. 2012 Oct 1;131(7):1591-600

Transcribed-Ultra Conserved Region expression is associated with outcome in high-risk neuroblastoma

Scaruffi , Sti liani S, Moretti S, Coco S, e ecc i C, Valdora F, Garaventa A, Bonassi S, Tonini GP

BMC Cancer. 2009 Dec 15; 9:441

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Identification of lo intratumoral gene expression heterogeneity in neuroblastic tumors by genomewide expression analysis and game theory

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Cancer. 2008 Sep 15;113(6):1412-22

Identification of L as a ma or familial neuro lastoma predisposition gene

Moss , Laudensla er M, Lon o L, Cole , ood A, Attiyeh EF, Laquaglia MJ, Sennett R, Lynch JE, Perri , Laureys , Speleman , im C, Hou C, Ha onarson H, Torkamani A, Schork NJ, Brodeur GM, Tonini GP, Rappaport E, Devoto M, Maris JM

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CV Gian Paolo Tonini ian aolo onini ot t e de ree in iolo y 1 at t e ni ersity of eno a, Italy t e Specialty in iolo y 1 , at t e ni ersity of Milano, Italy and in 1 t e Specialty in enetics, at t e ni ersity of a ia, Italy. rom 1 to 1 e as in t e staff of ransplantation Ser ice, S. Martino Hospital, eno a, Italy. rom 1 to 1 e as in t e staff t e La oratory of Hematolo y Oncolo y, . aslini C ildren s Hospital, enoa, Italy and from 000 to 01 e as enrolled in t e staff of ational Cancer esearc institute of enoa and as in c ar e as ead of t e ranslational ediatric Oncolo y nit. Since 01 e is ead of t e Italian euro lastoma oundation La oratory at t e ediatric esearc Institute, Citt della Speranza, adua. Since 1 4 e as een in ol ed in t e study of enetics and molecular aspects of neuro lastoma. He as een in c ar e as coordinator of Italian euro lastoma roup of I O ssociazione Italiana di mato Oncolo ia ediatrica and e represented t e Italian iolo y roup at t e SIO International Society of aediatric Oncolo y uropean euro lastoma . He as een director of International Sc ool of Medical Sciences, ttore Ma orana Centre for Scientific Culture, rice, rapani, Italy. ditor of scientific oo s and e is in c ar e as re ie er of i impact ational and International ournals. He spent se eral years as isitin fello in t e follo in Institutes: ational Cancer Institute, rederic , S C ildren s Hospital of iladelp ia, iladelp ia, S erman Cancer Center, Heidel er , ermany Ci a Cancer Center, apan. He or ed on: a ad anced dia nostic of neuro lastoma, disco ery of ne molecular pro nostic mar er for i ris neuro lastoma patients, c set up of t e first ational euro lastoma issue an , d disco ery of L ene mutations: t e first neuro lastoma predisposition ene. He is aut or co aut or of o er 1 0 pu lications on peer re ie ed ournals it a total H inde of .

Gian Paolo Tonini 5 Publications as first/last author 2016 16. 2 IF as first/last author 2016 39 H-index (source )

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Bio Nano Characterization

GROUP LEADERS

Field of interest Impact of Nanotechnologies on cancer research.

Nanotechnology — the science and engineering of controlling matter, at the molecular scale, to create devices with novel

Filippo Romanato

chemical, physical and/or biological properties — has the potential to radically change how we diagnose and treat cancer. Although scientists and engineers have only recently

GROUP MEMBERS

developed the ability to industrialize technologies at this scale, there has been good progress in translating nano-based cancer therapies and diagnostics into the clinic and many more are in development. Nanoscale objects—typically, although not exclusively, with dimensions smaller than 100 nanometers— can be useful by themselves or as part of larger devices containing multiple nanoscale objects. Nanotechnology is being applied to almost every field imaginable including biosciences, electronics, magnetics, optics, information

Post≠ doctoral Fellows: Giulia Borile Michele Massari Gianluca Ruffato

technology, and materials development, all of which have an impact on biomedicine.

If you are a cancer biologist looking for new solutions to your research questions or a scientist working with nanomaterials whose applications to cancer research are yet unknown, you

Erfan Mafakheri

soon became aware of the opportunities that nanotechnology

Enrico Gazzola

relies on the intersection of expertise from many science and

Pietro Capaldo

engineering disciplines. Nanotechnology research in cancer

PhD Students: Andrea Filippi Michele Gintoli Roberto Rossi

spans the range from exploratory research to technology innovation and involves a broad spectrum of disciplines such as photonics, nanofabrication, micro and nano engineering, mathematics, computer science, and the physics and materials sciences. The BioNanoCharacterization Group in IRP is structured synergistically to foster integration among the

141

fig.1

physical, biological, and clinical sciences.

Nanotechnology can provide rapid and sensitive detection of cancer-related targets, enabling scientists to detect molecular changes even when they occur only in a small percentage of cells. Nanotechnology also has the potential to generate unique and highly effective therapeutic agents. As an example, liquid biopsy that analyses liquid biological samples from patients is becoming a powerful tool in cancer diagnosis and stratification. In addition to circulating proteins and RNAs, also exosomes have been shown to act as messengers in intercellular communication, with a key role in cancer metastasis. Exosome can be isolated from all liquid biopsies, including serum, plasma, saliva, urine and cerebrospinal fluid and exhibit peculiar proteic signature, related to the producing cells and to the signaling cascade activated in the target cell, such as cell replication, immune response or angiogenesis. Exosomes nanometric size has so far limited their study by traditional optical methods, fundamentally limited by diffraction. Also, their detection and sorting by protein signature is still not available in a single integrated system. Our imaging and lab-on-chip systems intend to overcome these limitations following innovative technological solutions that conjugate physics with biology and medicine.

Imaging Current imaging methods can only readily detect cancers once they have made a visible change to a tissue, by which time thousands of cells will have proliferated and perhaps metastasized.

And even when visible, the nature of the tumor—malignant or benign—and the characteristics that might make it responsive to a particular treatment must be assessed through biopsies.

Imagine instead if cancerous or even pre-cancerous cells could somehow be tagged for detection by conventional scanning devices. Two things would be necessary

something that specifically

identifies a cancerous cell and something that enables it to be seen

and both can be achieved

through nanotechnology. For example, antibodies that identify specific receptors found to be overexpressed in cancerous cells that can be detected by our optical microscope techniques.

Once inside the body, the antibodies on these nanoparticles will bind selectively to cancerous cells, effectively lighting them up for the scanner. Nanotechnology will enable the visualization of molecular markers that identify specific stages and types of cancers, allowing doctors to see cells and molecules undetectable through conventional imaging. Nanoparticles such as quantum dots, which emit light of different colors depending on their size, could enable the simultaneous detection of multiple markers. The photoluminescence signals from antibody-coated quantum dots could be 142

fig.2

used to screen for certain types of cancer. Different colored quantum dots would be attached to antibodies for cancer biomarkers to allow oncologists to discriminate cancerous and healthy cells by the spectrum of light they see.

Screening In the fight against cancer, half of the battle is won based on its early detection. Nanotechnology provides new molecular contrast agents and materials to enable earlier and more accurate initial diagnosis as well as in continual monitoring of cancer patient treatment.

Screening for biomarkers in tissues and fluids for diagnosis will also be enhanced and potentially revolutionized by nanotechnology. Individual cancers differ from each other and from normal cells by changes in the expression and distribution of tens to hundreds of molecules. As therapeutics

fig.1 a) Schematic overview of the acoustoplasmonic device on the LN substrate , characterized by three main parts: an IDT for Rayleigh SAW generation (A), 4 nanostructured SPR sensing areas (B) and a PDMS microchannel with two microchambers (C). b) Final Chip with micro uidic chamber. c) Phase interrogation SPR.

fig.2 Two-Photon Excitation, Second and Third Harmonic Generation microscopy. A) TPE and SHG observation of murine gastrocnemius muscle cryosection highlights cell membranes and collagen deposition, respectively. B) SHG and THG on murine heart cryosections highlights collagen deposition, actomyosin structures inside cardiomyocytes and elastin of a coronary vessel. C) Ex-vivo TPE and SHG of murine gastrocnemius muscle shows infiltrate nuclei and sarcomeric structures, respectively. D) Ex-vivo TPE and SHG of murine lung highlights alveoli and collagen fibers, respectively.

advance, it may require the simultaneous detection of several biomarkers to identify a cancer for treatment selection.

Label free detection with extremely high sensitive detection techniques as home developed lab-on-chip system allow to perform on line wide range screening for specific cancer markers detection. A full screen of markers can be performed without long and expensive laboratory pre-treatments.

Our activities cover the design, the fabrication and characterization of nanostructures and nano-objects. Current research interests of the BioNanoCharacterization Group concern the following applications: 1) The developed bio sensor platforms for a variety of biomedical samples (DNA, RNA, proteins, etc.) based on the Surface Plasmon Resonance (SPR). The sensing platforms is combined with microfluidic circuitry to obtain a lab-on-chip device for label free detection of large variety of analytes.

143

fig.3

2) The group has expertise on the development and optimization of microscopy techniques for a wide range of applications. The main interests of the group are the characterization of biological samples and the development of super-resolution systems.

Summary of recent research activities and of main results achieved Biosensing and Opto-Microfluidics Since their first demonstration in the early 1980s, surface plasmon resonance (SPR) sensors have been widely recognized as useful tools for detecting chemical and biological species, and attracted a rapidly growing interest in the past two decades owing to their high sensitivity, label-free operation and possibility of real-time detection. Recent works have suggested that a turning point in SPR sensor research would be the combination of SPR strategies with other technologies in order to improve integration and plasmonic sensitivity. Although these techniques show many advantages and have led to the commercialization of portable and efficient SPR instruments, SPR systems often suffer from low throughput and bulky detection equipment’s. By properly designing microfluidic biochips it is possible to miniaturize the analyte-sensitive areas with an overall reduction of the chip dimension, downscale the liquid reagents and sample volume, improve automation, and increase the number of experiments in a single biochip by multiplexing approaches. However, as the fluidic channel dimensions

144

fig.3 Super-resolution obtained in HeLa cells by visualizing TOMM20 mitochondrial protein stained with Alexa 64 . From left to right:Image of a whole He a cell in 2PE and STED configuration and two details of the same region in 2PE and STED configuration.

fig.4 Exosomes from C2C12 cell line analyzed

by Transmission Electron Microscopy (in collaboration with Rosella Tomanin).

fig.4

approach the micron scale, mixing times are usually determined by diffusion alone, which can be prohibitively long and leads to long-lasting biochemistry experiments. An elegant method that has been introduced to overcome these issues is to actively perturb the liquid laminar flux by exploiting surface acoustic waves (SAWs). SAWs are a powerful technology that in the past two decades was introduced either for microfluidics and sensing purposes. By exploiting their ability to transfer a large amount of momentum into fluids one can perform several tasks, such as moving droplets, heating fluids, pumping liquids in micro-channels, and, importantly, generating efficient mixing. All of these tasks can be performed in low power consumption, portable, battery-operated systems.

We demonstrated a new approach for SPR biosensing based on the combination of microfluidics, SAW mixing and the innovative real-time phase-interrogation grating-coupling SPR technology.

On a single lithium niobate (LN) substrate the nanostructured SPR sensing areas, the interdigital transducer (IDT) for SAW generation and the polydimethylsiloxane (PDMS) microfluidic chambers were fabricated using standard, reproducible and high throughput micro- and nanofabrication techniques. The SPR detection is based on surface plasmon polariton (SPP) excitation via gold metallic grating upon azimuthal orientation and phase interrogation here applied to real-time SPR sensing for the very first time in combination with SAW microfluidics. In addition, a portable bench detection setup was realized and a dedicated software was developed for data acquisition and data processing.

We also design and realize platforms for a variety of biomedical samples (DNA, RNA, proteins, etc.) that can be combined with microfluidic circuitry to obtain a lab-on-chip devices.

Non-Linear Microscopy Two-Photon Microscopy (2PM) has proven an excellent technique for in vivo fluorescence imaging of deep structures and tissues due to low absorption and scattering of infrared light. The combination of two laser beams introduces the possibility of further imaging capabilities, such as simultaneous excitation of three fluorophores at same time (Multiphoton-Multicolor) or sub-diffraction resolution of cellular structures (Stimulated Emission Depletion, STED).

Two-Photon Excitation (2PE) 2PE of a fluorescent molecule is a phenomenon based on the simultaneous absorption of two identical photons with double the wavelength required for the equivalent Single-Photon process (1PE) 1 . isible dyes can then be imaged using near-infrared light, which undergoes significantly 145

less absorption and scattering from the biological tissue, and proves to be ideal for the study of thick samples ( 200 m) and live organisms, without significant optical aberrations or tissue damage.

Insertion of a second (synchronized) pulsed laser source in the optical path permits additional functionalities, like Multicolor-Multiphoton excitation and STED nanoscopy.

We built a cost-effective solution that combines both techniques by use of a homemade optical bench equipped with a pulsed laser split into two beams by an Optical Parametric Oscillator (OPO).

Preliminary experiments were successfully performed with the multiphoton-multicolor technique, setting proper conditions to perform experiments for the study of neutrophil mobilization in skull bone marrow of diabetic mice.

The penetration power of 2PE light in thick tissues can be exploited also by second and third harmonic generation (SHG and THG), optical techniques well suited for label-free imaging of collagen, myelin, cellulose or starches fibers.

Second and Third Harmonic Generation Second and Third Harmonic Generation (SHG and THG) are nonlinear processes in which two (or three) photons interacting with a material combine to generate a “new� photon with twice (or three times) energy, thus half (or one third) wavelength [2].

SH signal generally originates from non-centrosymmetric structures. Among these, collagen emits strong SH signal as well as the actomyosin lattice of muscle cells. SHG microscopy benefits from intrinsic optical sectioning, deep penetration into three-dimensional samples, and the presence of endogenous sources in live, untreated specimens. SHG can be imaged simultaneously with distinct two-photon-excited fluorescence (2PE) signals from one or more endogenous or exogenous labels.

TH signal is generated by refraction index mismatches, highlighting structures that also would come up in phase contrast or interference contrast microscopy. THG, however, is induced only at the focal point of the excitation laser, enabling D-imaging of tissues. THG signals is sufficiently strong for informative imaging of blood vessel walls and blood cells, muscle fiber sarcomeres, nerve fibers and nuclei of some cell types. Moreover in our microscope configuration SHG and THG can be acquired simultaneously.

Spatial co-registration of 2PE and SHG was achieved, setting proper conditions to perform various experiments in ex vivo cryosections, thick fresh preparations and in vivo.

146

fig.5

STED Nanoscopy Confocal optical microscopy resolution is limited by diffraction at, approximately, half the wavelength of the incident light. Various attempts have been made to overcome this limit. A successful approach developed

by Stephan Hell (Chemistry Nobel Prize in 2014) is the Stimulated Emission Depletion (STED), which has been implemented in commercial laser-scanning systems in the last decade, improving the resolution from 200-300 nm to

fig.5 COMSO Multiphysics simulations of (a) the magnetic field s norm and (b) the y-component (perpendicular) of the electric field for a plasmonic grating device with an optimized period (P), metal slit height (H) and duty cycle (DC), illuminated along the y-axis by a planewave ( 1000nm, P 820nm, H 120nm, DC 35%). (c) Scheme of the plasmonic-enhanced device. The metal slits (dark gold) are placed on an oxide nitride spacer layer in order to optimize the distance between the slits and the detector s active layer (dark grey).

30-70 nm [4]. After photon absorption by the excitation beam, fluorophores return spontaneously to the ground state in a typical time of a few nanoseconds, emitting light with wavelength EM. If the light of a second laser source (

EM), is focused during the excited phase

of the fluorophore, this can force the molecules back to the ground state by means of stimulated emission of a photon at

EM.

The process competes with spontaneous emission, thus resulting in a net reduction of the fluorescence signal (depletion). If the depletion beam is focused in a “doughnut� shaped fashion, a ring of molecules will be depleted, leaving excited only a nanometric region of molecules within the spot, thus enhancing the resolution of the system beyond the diffraction limit.

Depletion efficiency of the depletion (STED) beam was tested using droplets of Alexa 647, obtaining a 80

reduction of the fluorescence signal. After spatial

modulation of the beam, the super-resolution capabilities of the system were tested on HeLa cell mitochondria immunostained with Alexa 647. This preliminary tests showed a visible increase of the resolution, in respect to simple 2PE, of about 0 .

147

fig.6

1 W. Denk et al., Science 248, 7 -76 (1990).

2 L. Hyungsik et al., PNAS 111, 18025-180 0 (2014).

P. Mahou et al., Nat. Meth. 9, 815-818 (2012).

4 S.W. Hell et al., Opt. Lett. 19, 780-782 (1994).

Transmission Electron Microscopy and Atomic Force Microscopy In 19 1,

noll (inventor of SEM, 19 5) and Ruska co-

invented electron microscope and demonstrated electron images. They developed the idea of electron lenses into a practical reality and demonstrated electron. This was a most crucial step, for which Ruska received the Nobel Prize. Historically TEMs were developed because of the limited image resolution in light microscopes, which is imposed by the wavelength of visible light. Electrons show both particle and wave characteristics, illustrating one of the great puzzles of quantum physics that we all seem to accept without too much trouble. There are several methods of sample preparations. Depending on what kind of samples are analyzed they can be fixed on a special grating for the electron microscope or be thinned enough (<200 nm) by the pre-curing methods to ensure the electron beam transmission. The accelerated electrons (here 20-120 kV) are passed through a system of lenses and apertures which finally hits the region of interest in the sample.

EM analysis was performed on pellets of purified exosomes loaded on formwar/carbon-coated grids. 2 Ammonium-Molybdate was used as standard negative stain in biological electron microscopy before mounting in the sample position of the microscope, as reported in [1].

Exosomes were appropriately diluted to form a thin layer 148

fig.6 (a) SEM image of a Spiral Phase Plate fabricated by Electron Beam itography. (b) Detail of the center of the SPP. Example of Spiral Phase Contrast Microscopy: (c) hematoxylin eosin staining of a coronary vessel in heart myocardium and (d) same region ac uired by SPCM. In red the features ac uired with 1 SPP and in green the features ac uired with 2.

on the EM grid, in order to afford the transmission of the electron beam. A Tecnai G2 Spirit TEM was used to image exosome samples with the diameters between 0 to 100 nm and a magnification up to 300kX. The biological specimens are composed of light elements and do not show sufficient contrast in the TEM. The contrast can be enhanced using the lower accelerating voltages (80 to 100 k ), applying an aperture (objective aperture), and defocusing the image. Itâ&#x20AC;&#x2122;s worth mentioning that the more coherent electron sources also will make a better contrast. Depending on the samples type, the operator can choose the best conditions to tune the microscope for an appropriate imaging.

Atomic Force Microscopy (AFM) consists of a nanometer-sized tip that scans a sample on a surface at atomic distance. The characteristics of the interaction are converted into information on the sample D morphology, which can then be reconstructed by o ine image processing. For AFM imaging, exosomes are adsorbed to freshly cleaved mica sheets, rinsed with de-ionized water, dried under a gentle stream of Nitrogen and processed with a Veeco CP-II AFM (Department of Physics and Astronomy, University of Padova) [1].

1 Th ry C,et al., Curr Prot Cell Biol .22.1-29(2006).

Improving Silicon Single-Photon Photodetectors via Plasmonic Nanostructures (in collaboration with Fondazione Bruno essler) Silicon photomultipliers (SiPM) has obtained a growing attention as an alternative to the traditional photomultiplier tube in the detection of low photon fluxes thanks to a number of advantages typical of solid state detectors, such as compactness, ruggedness, ease of use, low operational voltage, and insensitivity to magnetic fields 1 .

Fondazione Bruno

essler (FB , Trento) has a well-established history of SiPM development

and characterization, in particular, for the detection of light from scintillators used in time-of-flight positron emission tomography (TOF-PET), which typically ranges between the visible blue and the near-ultraviolet (NU ) region 2 . Recently, they are trying to expand the field of application of their photodetectors, developing models with an operative range shifted to higher wavelengths, toward the visible green or, even, the near-infrared (NIR) region. These new devices could bring all the benefits of the SiPM to the many bio-related microscopy techniques usually working with low photon signals and small S/N ratios, like Second and Third Harmonic Generation Microscopy (SHG, 149

THG) and Stimulated Emission Depletion Nanoscopy (STED). However, while a green-optimized SiPM has already been successfully micro-fabricated, shifting the operative range into the NIR proved to be more challenging, due to longer absorption depths and lower absorption coefficients.

In order to overcome this problem, the first major focus of the project will be the development and integration on a silicon photomultiplier, by means of plasmonic simulations and a combination of micro- and nano-fabrication techniques (specifically, EBL and FIB), of a plasmonic grating working around 1000 nm, capable of enhancing the absorption efficiency of NIR photons by several times its original value [3]. Furthermore, the green-optimized SiPM will be tested on a microscope with HG capabilities, in order to verify the improvement over a traditional photomultiplier.

A very important feature of the SiPM developed by FB

is its photon-counting ability, which is

sufficiently refined to enable the detection of a single incident photon. Working with such kind of sensibility is very appealing, or even necessary, for a plethora of different applications. However, extremely low photon fluxes require very low dark count rate (DCR) and correlated noise (CN), high photodetection efficiency (PE) and very small active area in order to produce a significant S/N ratio.

Since the DCR, CN and PE were already optimized by FB , the second major focus of the project will be the enhancement of the S/N ratio by drastically reducing the active area using a nanofabricated plasmonic Bull’s Eye structure [4] and exploiting the Extraordinary Optical Transmission phenomenon 5 .

1 D. Renker, Nucl. Intrum. Methods A, vol. 567, pp. 48-56 (2006).

[2] C. Piemonte et al., IEEE Trans. Nucl. Sci., vol. 63, pp. 1111-1116 (2016). . u et al., Appl. Phys. Lett. 89, 1511116 (2006).

4 R. Bhat et al., Opt. Express 16, pp. 4588-4596 (2008).

5 S. A. Maier, “Plasmonic: Fundamentals and Applications”, Chap. 8, pp. 144-150 (Springer, 2007).

Spiral-phase plates for structured-light phase-contrast microscopy Since the seminal paper of Allen and coworkers in 1992 1 , the discovery that light beams with a helical phase front carry orbital angular momentum (OAM) has seen applications in many fields ranging from optical manipulation and microscopy to quantum information processing and, recently, free-space information transfer and communications in the near-infrared and micro-wave regimes.

As far as microscopy is concerned, the exploitation of structured light has provided remarkable 150

improvements in resolution and complementary optical techniques for sample inspection and analysis. Among the several methods to generate OAM beams, spiral phase plates (SPP) provide an efficient and robust solution for the transfer of orbital angular momentum to common planar waves.

A spiral phase plate is a helicoidal transmission element imposing an azimuthally-dependent phase delay on an incident optical wavefront, while preserving the direction of the optical axis. It is a transparent plate, looking like a spiral staircase, whose height increases around the central axis for a total thickness given by:

being nSPP the refractive index of the SPP material and

the impinging wavelength, l the topological

charge, denoting the amount of OAM carried by photons. SPPs are built for a specific wavelength at which the maximum fainting inside the dark central singularity is obtained. Therefore the height of the spiral needs to be precisely engineered, depending on the desired topological charge and incident wavelength.

A nanofabrication process to produce these kind of structures must be focused on the precise control of the geometry of the spiral plate in order to accurately fulfil the optical design. In particular, the optical quality of the fabricated SPP mask depends on a good shape definition of the spiral, on the smoothness of the mask and on the resolution of the central singularity and of the step.

Electron beam lithography (EBL) represents a powerful tool to generate this kind of structures, due to the possibility to fabricate continuous surface profiles, high flexibility in the design and good optical quality of the fabricated reliefs. Spiral masks can be realized by shaping a disk of transparent material, with refraction index n, imposing a direct proportionality between the thickness of the material and the azimuthal position. D profiles can be generated modulating the local dose distribution, inducing different dissolution rates in the polymer exposed, giving rise to different resist thicknesses at the end of the development process.

D-spiral phase mask profiles have

been written on a polymethylmethacrylate (PMMA) resist layer over a glass substrate, using the JEOL JBX-6300FS EBL machine at LaNN laboratory, in high-resolution mode: 100 keV of energy, 100 pA of current, 5 nm resolution 2 . Spiral-phase contrast microscopy exploits a spiral phase plate fort Fourier filtering of the object image, providing an efficient and high-resolution method 151

for edge contrast enhancement in light microscopy. The phase plate imprints a helical phase term on the transmitted light field in the Fourier plane. In the image plane, this results in a strong and isotropic edge contrast enhancement for both amplitude and phase samples. Image filtering with this technique results in an intensity distribution which is proportional to the intensity gradient of the original image, conserving the total image intensity, thus providing a higher efficiency with respect to other phase-contrast methods. This promises extremely sensitive detection of phase jumps or edges, which are orders of magnitude smaller than those detectable with the common phasecontrast method [3].

1 L. Allen et al., Phys. Rev. A 45 (11), 8185-8189 (1992).

2 M. Massari et al., App. Opt. 54, 4077-408 (2015).

S. Bernet et al., Opt. Express 14, 792- 805 (2006).

152

10 Selected Publications Sonato, ostini, M uffato, G; Gazzola,E; Liuni, D; Greco, G; Travagliati, M ; Cecchini, M; Romanato F.

A surface acoustic wave (SAW)-enhanced grating-coupling phase-interrogation surface plasmon resonance (SPR) micro uidic iosensor

LAB ON A CHIP, 2016, Volume: 16, Issue: 7, Pages: 1224-1233

uffato, ., Massari, M., Romanato, F.

iffracti e optics for com ined spatial and mode di ision demultiple in of optical ortices: esi n, fa rication and optical characterization

Scientific eports , 24760

Della Giustina, G., Sonato, A., Gazzola, E., (...), Brusa, S., Romanato, F.

S n anced molecular imprinted sol el film: promising tool for gas-phase TNT detection

2016 Materials Letters

Oldoni, M., Spinello, F., Mari, E., (...), Coassini, P., ThidĂŠ, B.

Space di ision demultiple in in or ital an ular momentum ased MIMO radio systems

2015 IEEE Transactions on Antennas and Propagation 63 (10), 07160713, pp. 45824587

Massari, M., uffato, ., intoli, M., Ricci, F., Romanato, F.

a rication and c aracterization of i plates for optical applications

2015 Applied Optics 54 (13), pp. 4077-4083

uality spiral p ase

aroli, ., uffato, ., Zilio, ., ... , Nanoporous gold leaves: Preparation, optical Romanato, F., Cattarin, S. c aracterization and plasmonic e a ior in t e isi le and mid-infrared spectral regions

2015 Optical Materials Express 5 (10), pp.

2246-2256

Meneghello, A., Antognoli, A., Sonato, A., (...), Cretaio, E., Romanato, F

La el free efficient and accurate detection of cystic fi rosis causin mutations usin an azimut ally rotated C S platform

014 nalytical C emistry

Sammito, D., De Salvador, D., Zilio, ., ... , aio, M., omanato, F.

Integrated architecture for the electrical detection of plasmonic resonances ased on i electron mo ility photo-transistors

2014 Nanoscale 6 (3), pp. 1390-1397

Parisi, G., Mari, E., Spinello, F., omanato, ., am urini, .

Manipulatin intensity and p ase distri ution of composite La uerre aussian eams

2014 Optics Express 22 (14), pp. 1713517146

ior is, ., Zilio, ., uffato, ., ... , Zacco, ., omanato, .

Resonance properties of thick plasmonic split ring resonators for sensing applications

2014 Optics Express 22 (22), pp. 2647626486

153

CV Filippo Romanato ilippo omanato is ssociate rofessor of ysics at adua ni ersity Material Science and ysics ere e is t e coordinator of the Nanodevices group.

Since une 1 e is operati e responsi le of ray lit o rap y and and co founder of t e micro and nano fa rication roup of IOM ational La oratory of C at lettra Syncrotron in rieste Italy ere no adays t e lit o rap y facility is constituted of 4 clean room and of a complete set of nanofa rication tools.

He is appointed as director of L ororatory for anofa rication and anode ices L 01 t at founded and de eloped 00 01 at present mana ed y ecamrecert roup. t present e is also CSO C ief Scientific officers of SM Optics and director of t e ano e ices roup of Institute of Pediatric Research of CittĂ della Speranza â&#x20AC;&#x201C; IRP. d in p ysics at adua ni ., e as isitor scientist at MI 1 , senior scientists at S sync rotron 1 , and ssociate rofessor at Sc ool of Material n ineerin of anyan ec nolo ical ni ersity of Sin apore 00 ere e founded a roup for t e de elopment of plasmonic io sensors.

Coordinator and rincipal In esti ator of se eral national and international pro ects. In particular e is unit responsi le in a pro ect, and national pro ects. He is co founder of four spin off related to nanofa rication and telecom.He is responsi le for se eral pro ect de oted to t e de elopment of de ice for nanolit o rap y and nanoptics and la on c ip.

He is currently co aut or of 10 issued patents, 00 scientific re ie ed papers accordin e of Science , inde source RG.

Filippo Romanato 2 Publications as first/last author 2016 .6 IF as first/last author 2016 26 H-index (source esearch ate)

154

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Peptide Patterns as Discriminating Biomarkers in Plasma of Patients With Familial Adenomatous Polyposis.

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A Molecular Study of Pediatric Spindle and Sclerosing Rhabdomyosarcoma: Identification of Novel and Recurrent VGLL2-related Fusions in Infantile Cases

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Evaluation of the Global Lung Initiative 2012 Reference Values for Spirometry in African Children.

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Arigliani M., V. Dolcemascolo, A. Nocerino, E. Pasqual, C. Avellini, and ;A05%<%

A Rare Cause of Acute Abdomen: Omental Infarction

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Untargeted Metabolomic Analysis of Amniotic Fluid in the Prediction of Preterm Delivery and Bronchopulmonary Dysplasia

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Extensive cardiac infiltration in acute T-cell lymphoblastic leukemia: occult extra-medullary relapse and remission after salvage chemotherapy.

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Lessons from genetics: is it time to revise the therapeutic approach to children with steroid-resistant nephrotic syndrome?

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9+:)-05, Enzo MV, Del Bianco P, Pucciarelli S, Nitti D, Agostini M

Diagnostic and prognostic role of cell-free DNA testing for colorectal cancer patients

International Journal of Cancer

Bellagamba M. P., E. Carmenati, R. D’Ascenzo, M. Malatesta, C. Spagnoli, C. Biagetti, I. Burattini, and @A0;A05.&-)+**)

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First steps to define murine amniotic fluid stem cell microenvironment.

Sci Rep. 2016 Nov 15;6:37080.

Bertin Enrics, Martina Piccoli, Chiara Franzin+!<$.-(%!

Nagy, Maria Mileikovsky, ;.%*%03+0 Coppi, Michela Pozzobon

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

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Double blind exploratory study on de novo lipogenesis in preterm infants on parenteral nutrition with a lipid emulsion containing 10% fish oil

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NUP98-fusion transcripts characterize different biological entities within acute myeloid leukemia: a report from the <PMZVL<O)!C-#B;

Leukemia. 2016 Dec 13

9&+'%*)-0D, De Filippi P, Vendemini F, D’Alia M, Zecca M, Meyer LH, Danesino C, Locatelli F, Masetti R20 9.''%0=20(+0E&%--)+0=A

Mutations of SETBP1 and JAK3 in juvenile myelomonocytic leukemia: a report from the italian AIEOP study group

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DNA methylation and targeted sequencing of methyltransferases family genes in canine acute myeloid leukaemia, modelling human myeloid leukaemia

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Amino Acid Intake in Preterm Infants.

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Marangon E, Giodini L, Nitti D, Toffoli G, Traldi P, Agostini M.

Cross-validation of a mass spectrometric-based method for the therapeutic drug monitoring of irinotecan: implementation of matrix-assisted laser desorption/ ionization mass spectrometry in pharmacokinetic 3&(%B-&3&$7%

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Testing the domain-general nature of monitoring in spatial and verbal cognitive domains.

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Neonatal Respiratory Diseases in the Newborn Infant: Novel Insights from Stable Isotope Tracer Studies

Neonatology, vol. 109, no. 4, pp. 325–333, 2016.

Carrera P, Calzavara S, Magistroni R, den Dunnen JT, Rigo F, Stenirri S, Testa F, Messa P, Cerutti R, Scolari F, Izzi C, Edefonti A, F+<&)'%*%0D20 Benetti E+!<965-($.6!O[+!O($B$7(!V+!

Boletta A, Ferrari M.

Deciphering Variability of PKD1 and PKD2 in an Italian Cohort of 643 Patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD)

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Palmgren I, Timdahl K, Hernell O.

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Cassina M, Ruol M, Pertile, Midrio P, Piffer S, Vicenzi V, Saugo O+!Q7#''#!0"+!Y(35(!V+ Clementi M

Prevalence, characteristics, and survival of children with esophageal atresia: A 32-year population-based study including 1,417,724 consecutive newborns

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Cystic Fibrosis carrier screening effects on birth prevalence and newborn screening

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Functional connectivity correlates of response inhibition impairment in anorexia nervosa

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Early T-cell precursor acute lymphoblastic leukaemia in children treated in AIEOP centres with AIEOP-BFM protocols: a retrospective analysis

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EMILIN3, an extracellular matrix molecule with restricted distribution in skin

Exp Dermatol. 2016 Nov 28

5%&.**%03, Candiani S, Ori M, Aveic Q+!(%-)-)0=;

The zebrafish as a model for studying neuroblastoma

Cancer Cell Int. 2016 Nov RD@AJE>

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Giordano A, Nitti D, Agostini!O=

Extracellular Matrix and Colorectal Cancer: How Surrounding Microenvironment Affects Cancer Cell Behavior?

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Crotti S, Agnoletto E, Cancemi G, Di O(-'#!*+0(&.*:)0;, Pucciarelli S, Nitti D, Agostini M

Altered plasma levels of decanoic acid in colorectal cancer as a new diagnostic biomarker

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An NBD derivative of the selective 1 rat toxicant 2 norbormide as a new probe for living cell imaging

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Digito, Massimo Rugge, Donato Nitti, Marco Agostini

Serum miR-125b is a non-invasive predictive biomarker of the pre-operative chemoradiotherapy responsiveness in patients with rectal adenocarcinoma

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Field-assisted paper spray mass spectrometry for the quantitative evaluation of imatinib levels in plasma.

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MRI in the assessment of ischaemic heart disease.

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Hypothermia and Meconium Aspiration Syndrome: International Multicenter Retrospective Cohort Study.

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SPR Enhanced molecular imprinted sol-gel film: A promising tool for gas-phase TNT detection

Materials Letters

Della Puppa Alessandro, · Giuseppe Lombardi, · Marta Rossetto, · Oriela Rustemi1,·Franco Berti,· Diego Cecchin4,· Marina Paola Gardiman, · Giuseppe Rolma, ·Luca Persano, · Vittorina Zagonel, · Renato Scienza

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5-aminolevulinic acid guided resection followed by BCNU wafers implantation: a 3-year follow-up

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The COQ2 genotype predicts the severity of Coenzyme Q10 deficiency

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Doimo M, Lopreiato R, Basso V, Bortolotto R, Tessa A, Santorelli FM, Trevisson E, Salviati L.

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Pharmacogenetics Biomarkers and Their Specific Role in Neoadjuvant Chemoradiotherapy Treatments: An Exploratory Study on Rectal Cancer Patients

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Haupt R, Erminio G, Defferrari R, Mazzocco K, Kohler J, (%-)-)0=;+! )(.&$%7&6$!/+!Q'8-&6&-!Y

Piloting the European Unified Patient Identity Management (EUPID) Concept to Facilitate Secondary Use of Neuroblastoma Data from Clinical Trials and Biobanking

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Emma F, Montini G, Parikh SM, Salviati L.

Mitochondrial dysfunction in inherited renal disease and acute kidney injury

Nat Rev Nephrol. 2016 O(2D@>HFIJ>AGLE?=!

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