Maura Massimino, Loris De Cecco
Il microbiota in gliomi H3K27M: analisi delle interazioni e implicazioni nella storia di malattia
Report 2021
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Veronica Biassoni, Maura Massimino, Maria Chiara Oprandi, Carlo Alfredo Clerici, Laura Veneroni, Claudia Corti, Elisabetta Schiavello, Filippo Spreafico & Geraldina Poggi
Rehabilitation for children and young people surviving a brain tumor, and their transition to adult services: the main challenges
Expert Review of Quality of Life in Cancer Care, 2017
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Andrea Anichini
Determinazione della presenza, dei livelli, e della loro evoluzione temporale, in relazione alla terapia, di un ampio spettro di molecole solubili (n=26), appartenenti alle famiglie dei fattori di crescita nei DIPG
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Maura Massimino MD, Veronica Biassoni MD, Rosalba Miceli MD, Elisabetta Schiavello MD, Monika Warmuth – Metz MD, Piergiorgio Modena PhD, Michela Casanova MD, Emilia Pecori MD, Felice Ginagaspero MD, Manila Antonelli MD, Francesca Romana Buttarelli PhD, Paolo Potepan MD, Bianca Pollo MD, Raffaele Nunziata MD, Filippo Spreafico MD, Marta Podda MD, Andrea Anichini PhD, Carlo Alfredo Clerici MD, Iacopo Sardi MD, Loris De Cecco PhD, Udo Bode MD, Ferdinand Bach PhD, and Lorenza Gandola MD.
Results of nimotuzumab and vinolrebine, radiation and re-irradiation for diffuse pontine glioma in childhood
Journal of Neuro-oncology 2014, april 7.
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Molecular stratification of high-grade gliomas in children

Molecular stratification of high-grade gliomas in children

The Research

Giangaspero F.

Brain tumours are the main cause of death due to malignant neoplasia in children. Childhood high-grade gliomas identified with the terms anaplastic astrocytoma and glioblastoma, are 15% of all pediatric brain tumours and have high morbidity and survival rate at three years is less than 20%.
Most of our knowledge concerns malignant gliomas in adults, in particular the glioblastoma (GBM) which is the most frequent primitive neoplasia of the central nervous system.
Recent studies on GBM gene expression in adults, helped to identify oncogenetic molecular events and to
stratify patients into prognostic groups. That is to say, the presence or not of some molecular alterations which enable us to predict the more or less aggressive clinical behaviour of these neoplasms. Childhood GBM are histologically similar to those in adults, but have different molecular features.
Our research projects are aimed at characterizing childhood malignant gliomas. Our results show that molecular mechanisms involved in gliomagenesis and progression are different from those in adults. We observed in particular that, differently from what happens in adults, in which mutation of the oncosuppressor gene p53 has no effect on the clinical trend, in childhood neoplasias TP53 gene mutations are associated to reduced survival.
We have also studied protein YKL-40 expression in child tumours that in adults is associated with a worse
response to radiotherapy and shorter overall survival. Our results showed that the expression of such protein in childhood malignant gliomas is very common but has no effect on prognosis.
Likewise, our studies as well as those of other groups show that IDH1 mutations, present in 80% of glial neoplasias in adults, are practically absent in childhood (Antonelli et al. Prognostic significance of histological grading, p53 status, YKL- 40 expression, and IDH1 mutations in pediatric high grade gliomas, Journal of Neuro-Oncology, in press). Another study we carried out aimed at assessing MGMT mutation in pediatric gliomas. When such gene is inactivated, chemotherapic agents act more effectively on tumour cells. Inactivation of this gene in adult gliomas has proven to be a favourable event with a considerable increase in survival. Our research on pediatric gliomas shows that gene MGMT inactivation is less frequent than in the adult and its correlation with response to treatment is not statistically significant (Buttarelli et al. Evaluation status and prognostic significance of MGMT in pediatric high grade gliomas, submitted).
The results of these studies together with those of others in literature strengthen the concept that, despite histological similarities, molecular mechanisms involved in the pathogenesis and neoplastic progression seem to be totally different in the two age groups. While knowledge of these tumours in adults is advanced, in childhood it is still very scarce. It should be pointed out that understanding such mechanisms is the first step towards the definition of new therapies based on specific molecular targets.
Our future research aims to study the expression of molecules involved in the activation of Ras and Akt molecular pathways which are involved in controlling cell growth, differentiation, and survival. Preliminary results highlight a different behaviour between tumors in which these pathways are activated and those in which activation is absent.

Prof. Felice Giangaspero
Laboratorio Neuropatologia
Policlinico Umberto I, Roma
Università Sapienza Roma
Viale del Policlinico, 155, 00197 Roma
Tel/fax: +390649979175
E mail: felice.giangaspero@uniroma1.it


Buttarelli FR, Massimino M, Antonelli M, Lauriola L, Nozza P, Donofrio V, Arcella A, Oliva MA, Di Rocco C, Giangaspero F.
Evaluation status and prognostic significance of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation in pediatric high grade gliomas.
Childs Nerv Syst. 2010 Aug;26(8):1051-6. Epub 2010 Jun 16.

Antonelli M, Buttarelli FR, Arcella A, Nobusawa S, Donofrio V, Oghaki H, Giangaspero F.
Prognostic significance of histological grading, p53 status, YKL-40 expression, and IDH1 mutations in pediatric high-grade gliomas.
J Neurooncol. 2010 Sep;99(2):209-15. Epub 2010 Feb 21.

Manila Antonelli, Maura Massimino, Isabella Morra, Maria Luisa Garrè, Marina Paola Gardiman, Francesca Romana Buttarelli, Antonietta Arcella, Felice Giangaspero
Epression of pERK and pAKT in pediatric high grade astrocytomas: Correlation with YKL40 and prognostic significance.
Neuropathology 2011 Aug doi:10.1111/j.1440-1789.2011.01252.x.
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Manuela Badiali, Vincent Gleize, Sophie Paris, Loredana Moi, Selma Elhouadani, Antonietta Arcella, Roberta Morace, Manila Antonelli, Francesca Romana Buttarelli, Dominique Figarella- Branger, Young-Ho Kim, Hiroko Ohgaki, Karima Mokhtari, Marc Sanson, Felice Giangaspero
KIAA1549-BRAF Fusions and IDH Mutations Can Coexist in Diffuse Gliomas of Adults.
Brain Pathology (2012) ISSN 1015-6305.
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Manila Antonelli, Manuela Badiali, Francesca Romana Buttarelli, Caterina Baldi, V. Gleize, S. Paris, Loredana Moi, Paolo Nozza, Antonietta Arcella, Maura Massimino, Marc Sanson, Felice Giangaspero.
KIAA1549: BRAF fusion gene in pediatric brain tumors of different histogenesis.
Submitted to Brain Pathology (2013).
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Morphine treatment permits doxorubicin hydrochloride to cross blood brain barrier in rat model

Morphine treatment permits doxorubicin hydrochloride to cross blood brain barrier in rat model

The Research

Iacopo Sardi, *Giancarlo la Marca, *Maria Grazia Giovannini, °Sabrina Malvagia, °Renzo Guerrini, °Lorenzo Genitori, ^Maura Massimino, Maurizio Aricò

Department of Onco-Hematology, Meyer Children’s Hospital, Florence Italy
*Department of Pharmacology, University of Florence Italy
°Department of Neuroscience, Meyer Children’s Hospital, Florence Italy
^Department of Pediatrics, IRCCS Foundation, National Cancer Institute, Milan, Italy

The blood brain barrier discriminates the access of several molecules into the brain. This hampers the use of some drugs, as doxorubicin, potentially active for treatment of brain tumors. We explored the feasibility of active modification of the blood brain barrier protection, by using morphine pretreatment, to allow doxorubicin accumulation into the brain in an animal model. Rats were pretreated with different doses of intraperitoneal morphine before injection of doxorubicin (12 mg/kg). Quantitative analysis of doxorubicin was performed by mass spectrometry. Acute hearth and kidney damage was analyzed by measuring doxorubicin accumulation, LDH activity and malondialdehyde plasma levels. The concentration of doxorubicin was significantly higher in all brain areas of rats pretreated with morphine than in control tissues (P <0.001). This was evident only at therapeutic morphine dose (10 mg/kg, three times over 24 hours), while lower doses (2.5 and 5 mg/kg) were not associated with doxorubicin accumulation. Pretreatment with morphine did not induce an elevation of LDH activity or of lipid peroxidation compared to controls. Our data suggest that morphine pre-treatment is able to allow doxorubicin penetration inside the brain, by modulating the blood-brain barrier. This is not associated with acute cardiac or renal toxicity. These preliminary results will enable us to generate novel therapeutic approaches to refractory or recurrent brain tumors, and might be useful in other human diseases of the central nervous system in which molecules usually stopped by the blood brain barrier may have a therapeutic impact.


Research project on pediatric brain tumors:
“Alteration of the blood-brain barrier (BBB) using drugs that interact with the P-glycoprotein.
New perspectives for the treatment of “poor-responder” brain tumors of the pediatric age”.
Progress Report June 2011.
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Assessment of the cytotoxic effects of the chemotherapeutic drugs used in the treatment of high-risk brain tumors of the pediatric age.
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L’Ondasetron facilita il passaggio della doxorubicina attraverso la barriera ematoencefalica in modello murino.
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Alterazioni della barriera Emato-Encefalica mediante farmaci che interagiscono con la MDR. Nuove prospettive per la cura dei tumori intrinseci del tronco encefalico.
Stato di avanzamento Novembre 2013.
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Dendritic cell immunotherapy of glioblastoma multiforme

Dendritic cell immunotherapy of glioblastoma multiforme

The Research

Finocchiaro G.

Current standard treatment of glioblastoma multiforme (GBM) is based on the surgical resection of the tumour mass followed by radio- and chemotherapy (Stupp et al., 2005). In spite of novel therapeutic approaches, the prognosis remains dismal and relapse is almost inevitable. We thus clearly need to produce long term, well tolerated and tumourspecific treatments able to eliminate the (remaining) tumour cells infiltrated in the nearby brain areas.
The immune system provides important tools in this direction. Preclinical trials for the development of vaccines against high-grade gliomas, based on the use of potent immune cells i.e. dendritic cells, exposed to a mix of tumour antigens deriving from the glioma (Pellegatta et al., 2005 and 2006), have been carried out in both rat models (Liau et al., 1999; Siesjo et al., 1996; Witham et al., 2002) and mouse models (Akasaki et al., 2001; Heimberger et al., 2000; Insug et al., 2002; Kikuchi et al., 2002; Ni et al., 2001; Okada et al., 1998), showing their capacity to produce a tumour-specific immune response.
Mature DC with autologous tumour lysate have been used to treat patients with relapsed malignant tumours (De Vleeschouwer et al., 2004; Rutkowski et al., 2004; Wheeler et al., 2004; Yamanaka et al., 2003; Yu et al., 2004); no significant adverse effects have been reported. In Italy we have been able to repeat this experience with the treatment of six patients affected by relapsing GBM, using dendritic cells prepared at the Istituto Neurologico Besta. Median survival of these patients from time of relapse is currently of 11,5 months (Finocchiaro et al, manuscript in preparation).
The results of the use of immunotherapy on GBM patients are promising, but further investigation is necessary to identify a safe and more effective combination of immunotherapy with radio- and chemotherapy after tumour resection.

Dott. Gaetano Finocchiaro, M.D.

Director, Unit Neurology 8- Molecular Neuro-Oncology
Director, Department of Neuro-Oncology
Fondazione IRCCS Istituto Neurologico Besta
via Celoria 11, 20133 Milano
Phone +39 02 2394 2769
fax +39 02 2394 2453

Group-leader IFOM-IEO Campus
via Adamello 16, 20139 Milano
Phone +39 02 9437 5113

E mail: finocchiaro@istituto-besta.it


Serena Pellegatta, Barbara Savoldo, Natalia Di Ianni, Cristina Corbetta, Yuhui Chen, Monica Patané, Chuang Sun, Bianca Pollo, Soldano Ferrone, Francesco DiMeco, Gaetano Finocchiaro, Gianpietro Dotti
Constitutive and TNF?-inducible expression of chondroitin sulfate proteoglycan 4 in glioblastoma and neurospheres: Implications for CAR-T cell therapy.
Science Translational Medicine, 10, eaao2731 (2018) 28 February 2018.
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Serena Pellegatta, Marica Eoli, Valeria Cuccarini, Elena Anghileri, Bianca Pollo, Sara Pessina, Simona Frigerio, Maura Servida, Lucia Cuppini, Carlo Antozzi, Stefania Cuzzubbo, Cristina Corbetta, Rosina Paterra, Francesco Acerbi, Paolo Ferroli, Francesco DiMeco, Laura Fariselli, Eugenio A. Parati, Maria Grazia Bruzzone & Gaetano Finocchiaro
Survival gain in glioblastoma patients treated with dendritic cell immunotherapy is associated with increased NK but not CD8+ T cell activation in the presence of adjuvant temozolomide
OncoImmunology, Volume 7, 2018 – Issue 4
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Serena Pellegatta, Pietro Luigi Poliani, Elena Stucchi, Daniela Corno, Chiara Agnese Colombo, Francesca Orzan, Maria Ravanini, and Gaetano Finocchiaro.
Intra-tumoral dendritic cells increase efficacy of peripheral vaccination by modulation of glioma microenvironment.
Neuro-Oncology 12(4) -377-388, 2010.
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Gabriele Cantini, Federica Pisati, Sara Pessina, Gaetano Finocchiaro and Serena Pellegatta.
Immunotherapy against the radial glia marker GLAST effectively triggers specific antitumor effectors without autoimmunity.
Oncoimmunology 1:6, 884-893; September 2012.
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Serena Pellegatta, Marica Eoli, Simona Frigerio, Carlo Antozzi, Maria Grazia Bruzzone, Gabriele Cantini, sara Nava, Elena Anghileri, Lucia Cuppini, Valeria Cuccarini, Emilio Ciusani, Marta Dossena, Bianca Pollo, Renato Mantegazza, Eugenio A. Parati and Gaetano Finocchiaro.
The natural killer cell response and tumor debulking are associated with prolonged serviva in recurrent glioblastoma patients receiving dendritic cells loaded with autologous tumor lysates.
Oncoimmunology 2:3, e23401; March 2013.
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Pellegatta S, Poliani PL, Corno D, Menghi F, Ghielmetti F, Suarez-Merino B, Caldera V, Nava S, Ravanini M, Facchetti F, Bruzzone MG, Finocchiaro G.
Neurospheres Enriched in Cancer Stem-Like Cells Are Highly Effective in Eliciting a Dendritic Cell-Mediated Immune Response against Malignant Gliomas.
Cancer Res. 2006 Nov 1;66(21):10247-52.
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Gabriele Cantini, Federica Pisati, Alfonso Mastropietro, Véronique Frattini, Yoichiro Iwakura, Gaetano Finocchiaro, Serena Pellegatta
A critical role for regulatory T cells in driving cytokines profiles of Th17 cells and their modulation of glioma microenvironment.
Cancer Immunol. Immunother. 2011 Jul DOI 10.1007/s00262-011-1069-4.
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Gabriele Cantini, Federica Pisati, Sara Pessina, Gaetano Finocchiaro and Serena Pellegatta
Immunotherapy against the radial glia marker GLAST effectively triggers specific antitumor effectors without autoimmunity
OncoImmunology 1:8, 1-10; November 2012; © 2012 Landes Bioscience.
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Véronique Frattini, Federica Pisati, Maria Carmela Speranza, Pietro Luigi Poliani, Gianmaria Frigé, Gabriele Cantini, Dimos Kapetis, Manuela Cominelli, Alessandra Rossi, Gaetano Finocchiaro and Serena Pellegatta
FOXP3, a novel glioblastoma oncosuppressor, affects proliferation and migration
Oncotarget, Advance Publications, September 2012.
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Antonella De Rosa, Serena Pellegatta, Marco Rossi, Patrizia Tunici, Letizia Magnoni, Maria Carmela Speranza, Federico Malusa, Vincenzo Miragliotta, Elisa Mori, Gaetano Finocchiaro, Annette Bakker
A Radial Glia Gene Marker, Fatty Acid Binding Protein 7 (FABP7), Is Involved in Proliferation and Invasion of Glioblastoma Cells
PLOS ONE, December 2012, Volume 7, Issue 12, e52113.
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Sara Nava, Marta Dossena, Simona Pogliani, Serena Pellegatta, Carlo Antozzi, Fulvio Baggi, Cinzia Gellera, Bianca Pollo, Eugenio A. Parati, Gaetano Finocchiaro, Simona Frigerio
An Optimized Method for Manufacturing a Clinical Scale Dendritic Cell-Based Vaccine for the Treatment of Glioblastoma
PLOS ONE, December 2012, Volume 7, Issue 12, e52301.
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Generation of immortalized cell lines of human medulloblastoma through the expression of a dominant negative form of TP53 gene

Generation of immortalized cell lines of human medulloblastoma through the expression of a dominant negative form of TP53 gene

The Research

Salsano E.

Medulloblastoma (MB) is the most frequent brain tumour in children. Despite the advances in surgery, radiotherapy and chemoterapy, mortality rate at 5 years is still 50-70% of cases and many children who survive, show damages affecting the central nervous system and other organs due to the adverse effects of the therapies.
The key aim of our project is to generate human medulloblastoma cell lines which can be as faithful as possible to the medulloblastomas they derive from, enabling in this way the creation of a useful in vitro model to study the pathogenesis of the tumour and to find more efficient and targeted therapeutic approaches. To date, obtaining medulloblastoma line cells from the correspondent primary tumour is not easy and the few medulloblastoma cell lines available seem not to be very representative of the tumours they derive from. For example, the DAOY, a cell line commonly used to investigate in vitro the biology of medulloblastomas and to prove the effectiveness of antimedulloblastoma chemiotherapic agents, shows a deletion of the oncosuppressor gene CDNK2A, a non-documented biological feature in human medulloblastomas.
To reach the aim of the project, we try to express a dominant-negative form of the oncosuppressor gene TP53 in human medulloblastoma cells obtained from surgical samples. In fact TP53 was mutated in the most malignant and rare form of medulloblastoma, the so called anaplastic medulloblastoma, and this mutation could be necessary to generate immortalized medulloblastoma cells in vitro, as we observed in our laboratory.
The project should permit to assess: 1) whether and how it is possible to introduce and express genetic material in primary human medulloblastoma cells in vitro; 2) whether TP53 mutations are able to immortalize these tumor cells (normally unable to survive in vitro for more than a few passages).
We suppose that this approach might be a novel strategy to routinely establish human medulloblastoma cell lines, useful for both in vitro and in vivo investigations (the latter after being implanted into laboratory animals).

Dr. Ettore Salsano
Unità Operativa Neurologia 8-Neuro-Oncologia molecolare
Fondazione IRCCS, Istituto Neurologico Carlo Besta
Via Celoria 11, 20133 Milano, Italia
T. +39.02.2394.2285; F. +39.02.2394.2453
E mail: ettore.salsano@istituto-besta.it

Results (in Italian)

Data la difficoltà tecnica di trasfettare cellule di medulloblastoma umano con una forma dominante negativa del gene TP53 in modo da garantirne un’espressione quantitativamente adeguata e stabile come proposto inizialmente, abbiamo deciso di verificare se le linee cellulari di medulloblastoma umano derivate da soggetti operati presso l’Istituto Neurologico C. Besta di Milano nel corso di due anni avessero un’alterazione molecolare a carico del gene TP53 o di un gene correlato che ne potesse compromettere il funzionamento, al pari di quanto accade in presenza di forme dominanti negative del suddetto gene. È stato possibile ottenere l’immortalizzazione di solo due linee cellulari su venti. Le due linee cellulari immortalizzate sono state chiamate MB4 ed MB16, sono state prodotte rispettivamente da un medulloblastoma classico e da un medulloblastoma anaplastico pediatrici e hanno dimostrato di continuare a replicarsi dopo 20 passaggi in condizioni serum-free (DMEM/F12 + B27 w/o vitamina A) con l’aggiunta del solo FGF2 (20ng/mL) (la presenza dell’EGF, infatti, si è dimostrata superflua). In accordo con la nostra ipotesi di lavoro, nella linea MB16 abbiamo trovato una mutazione loss-of-function in eterozigosi nel gene TP53 (IVS9+1G>T) associata ad una delezione del braccio corto del cromosoma 17 (17p) dove è ubicata l’altra copia del gene TP53; nella linea MB4, invece, è stata identificata un’amplificazione del gene MDM2, il cui prodotto è capace di inibire TP53. Da notare che la mutazione in eterozigosi del gene TP53 non è stata documentata nel DNA estratto dal frammento del corrispondente tumore di origine, suggerendo che le condizioni di coltura possano avere favorito la selezione in vitro delle sole cellule che ne erano provviste. La linea MB16 si è inoltre dimostrata tumorigenica in vivo, data la formazione di una neoplasia similmedulloblastoma dopo trapianto in topi nudi di 3 ? 105 cellule. Ancorché molto preliminari, questi dati sono a supporto dell’importanza della disregolazione del TP53 quale meccanismo chiave per le generazione di linee cellulari immortalizzate di MB umano in condizioni standard di coltura serum-free e suggeriscono che tale disregolazione può avvenire o in modo diretto come nel caso della linea MB16 o in modo indiretto come nel caso della linea MB4.


Ettore Salsano, Rosina Paterra, Miriam Figus, Francesca Menghi, Emanuela Maderna, Bianca Pollo, Carlo Lazzaro Solero, Luca Massimi, Gaetano Finocchiaro
Expression Profile of frizzled receptors in human medulloblastomas
J Neurooncol 2011 Aug DOI 10.1007/s11060-011-0682-6.
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