Publications by authors named "Rita De Vito"

73 Publications

GD2 redirected CAR T and activated NK-cell-mediated secretion of IFNγ overcomes MYCN-dependent IDO1 inhibition, contributing to neuroblastoma cell immune escape.

J Immunother Cancer 2021 Mar;9(3)

Department of Hematology/Oncology and Gene and Cell Therapy, IRCCS Bambino Gesù Children's Hospital, Rome, Italy

Immune escape mechanisms employed by neuroblastoma (NB) cells include secretion of immunosuppressive factors disrupting effective antitumor immunity. The use of cellular therapy to treat solid tumors needs to be implemented. Killing activity of anti-GD2 Chimeric Antigen Receptor (CAR) T or natural killer (NK) cells against target NB cells was assessed through coculture experiments and quantified by FACS analysis. ELISA assay was used to quantify interferon-γ (IFNγ) secreted by NK and CAR T cells. Real Time PCR and Western Blot were performed to analyze gene and protein levels modifications. Transcriptional study was performed by chromatin immunoprecipitation and luciferase reporter assays on experiments of mutagenesis on the promoter sequence. NB tissue sample were analyzed by IHC and Real Time PCR to perform correlation study. We demonstrate that Indoleamine-pyrrole 2,3-dioxygenase1 (IDO1), due to its ability to convert tryptophan into kynurenines, is involved in NB resistance to activity of immune cells. In NB, IDO1 is able to inhibit the anti-tumor effect displayed by of both anti-GD2 CAR (GD2.CAR) T-cell and NK cells, mainly by impairing their IFNγ production. Furthermore, inhibition of MYCN expression in NB results into accumulation of IDO1 and consequently of kynurenines, which negatively affect the immune surveillance. Inverse correlation between IDO1 and MYCN expression has been observed in a wide cohort of NB samples. This finding was supported by the identification of a transcriptional repressive role of MYCN on IDO1 promoter. The evidence of IDO1 involvement in NB immune escape and its ability to impair NK and GD2.CAR T-cell activity contribute to clarify one of the possible mechanisms responsible for the limited efficacy of these immunotherapeutic approaches. A combined therapy of NK or GD2.CAR T-cells with IDO1 inhibitors, a class of compounds already in phase I/II clinical studies, could represent a new and still unexplored strategy capable to improve long-term efficacy of these immunotherapeutic approaches.
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http://dx.doi.org/10.1136/jitc-2020-001502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7978286PMC
March 2021

Dysregulated miRNAs in bone cells of patients with Gorham-Stout disease.

FASEB J 2021 Mar;35(3):e21424

Bone Physiopathology Research Unit, Genetics and Rare Diseases Research Division, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Gorham-Stout disease (GSD) is a very rare disease characterized by increased bone erosion with angiomatous proliferation. The mechanisms underlying this disorder have not been deeply investigated. Due to its rarity, no guidelines are currently available for treatment and management of GSD. We recently evaluated the cellular alterations of the bone remodeling in patients showing that osteoclast precursors displayed increased ability to differentiate into osteoclasts and that affected osteoclasts resorb bone more actively than control cells. Moreover, osteoblasts isolated from a patient showed a defective ability to form mineralized nodules. In this paper, we investigated the molecular pathways involved in the cellular defects of GSD bone cells. For this study, we recruited nine patients and performed miRNome analysis of bone cells. Between the 178 miRNAs robustly expressed in GSD osteoclasts, significant modulation of three miRNAs (miR-1246, miR-1-3p, and miR-137-3p) involved in the regulation of osteoclast formation and activity or in the angiomatous proliferation was found in patients' cells. Interestingly, miR-1246 was also up-regulated in serum exosomes from patients. Analysis of miRNAs from patient osteoblasts suggested alteration of miR-204a-5p, miR-615-3p and miR-378a-3p regulating osteoblast function and differentiation. The resulting miRNA pattern may help to understand better the mechanisms involved in GSD and to identify new potential therapeutic targets for this rare disease.
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http://dx.doi.org/10.1096/fj.202001904RRDOI Listing
March 2021

Congenital Craniofacial Plexiform Neurofibroma in Neurofibromatosis Type 1.

Diagnostics (Basel) 2021 Feb 2;11(2). Epub 2021 Feb 2.

Oncological Neuroradiology Unit, Department of Imaging, Bambino Gesù Children's Hospital, IRCCS, 00165 Rome, Italy.

We present a case demonstrating the performance of different radiographical imaging modalities in the diagnostic work-up of a patient with neurofibromatosis type 1 (NF1) and plexiform neurofibroma (PN). The newborn boy showed an expansive-infiltrative cervical and facial mass presented with macrocrania, craniofacial disfigurement, exophthalmos and glaucoma. A computer tomography (CT) and a magnetic resonance imaging (MRI) were performed. The CT was fundamental to evaluate the bone dysmorphisms and the MRI was crucial to estimate the mass extension. The biopsy of the lesion confirmed the suspicion of PN, thus allowing the diagnosis of NF1. PN is a variant of neurofibromas, a peripheral nerves sheath tumor typically associated with NF1. Even through currently available improved detection techniques, NF1 diagnosis at birth remains a challenge due to a lack of pathognomonic signs; therefore congenital PN are recognized in 20% of cases. This case highlights the importance of using different radiological methods both for the correct diagnosis and the follow-up of the patient with PN. Thanks to MRI evaluation, it was possible to identify earlier the progressive increasing size of the PN and the possible life threatening evolution in order to perform a tracheostomy to avoid airways compression.
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http://dx.doi.org/10.3390/diagnostics11020218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913090PMC
February 2021

Clinicopathologic and Molecular Characterization of Four Cases of Pediatric Salivary Secretory Carcinoma (SSC), One with ETV6-RET Fusion.

Head Neck Pathol 2021 Jan 18. Epub 2021 Jan 18.

Ospedale Pediatrico Bambino Gesu (OPBG), and Istituto Ricovero E Cura a Carattere Scientifico, Rome, Italy.

Salivary gland secretory carcinoma (SSC) is a neoplasm with characteristic histologic features, similar to those of secretory carcinoma of the breast. Only a few pediatric SSC cases have been reported, all with ETV6-NTRK3 fusion. We present four new pediatric SSC examples, one with a novel ETV6-RET fusion. Four cases of SSC were diagnosed between 2010 and 2020: 2 boys, 7 and 9 year-old with parotid tumors (1.5 and 1.3 cm, respectively); and two 14 year-old girls: one with a submandibular tumor (2.1 cm), and one with a parotid lesion (1.2 cm). Histologically, all tumors were similar: well-circumscribed lesions composed by mid-size, monotonous cells with eosinophilic and sometimes vacuolated cytoplasm. The nuclei are oval to round with open chromatin and a single nucleolus. There are duct-like structures and microcysts with colloid-like material. Immunohistochemically, tumor cells are positive for S100, CK7, mammaglobin and GATA3. A classic ETV6-NTRK3 translocation was confirmed in the three parotid tumors; an ETV6-RET fusion was demonstrated in the submandibular lesion. All patients underwent complete surgical resection and are alive without tumor recurrence after a follow-up time ranging from one to 4 years. Pediatric SSC is extremely rare but their characteristic morphology and immunohphenotype facilitate their diagnosis. We describe the first pediatric case with the recently reported ETV6-RET fusion. Similar to adult cases, this tumor is morphologically undistinguishable from those carrying the classic ETV6-NTRK3 translocation. Thus, in pediatric cases with morphology suggestive of SSC and negative ETV6-NTRK3 by RT-PCR, other possible fusions should be investigated.
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http://dx.doi.org/10.1007/s12105-021-01288-7DOI Listing
January 2021

Identification of neuroblastoma cell lines with uncommon TAZ/mesenchymal stromal cell phenotype with strong suppressive activity on natural killer cells.

J Immunother Cancer 2021 Jan;9(1)

Immunology Area, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy

Background: Neuroblastoma (NB) is the most common, extracranial childhood solid tumor arising from neural crest progenitor cells and is a primary cause of death in pediatric patients. In solid tumors, stromal elements recruited or generated by the cancer cells favor the development of an immune-suppressive microenvironment. Herein, we investigated in NB cell lines and in NB biopsies, the presence of cancer cells with mesenchymal phenotype and determined the immune-suppressive properties of these tumor cells on natural killer (NK) cells.

Methods: We assessed the mesenchymal stromal cell (MSC)-like phenotype and function of five human NB cell lines and the presence of this particular subset of neuroblasts in NB biopsies using flow-cytometry, immunohistochemistry, RT-qPCR, cytotoxicity assays, western blot and silencing strategy. We corroborated our data consulting a public gene-expression dataset.

Results: Two NB cell lines, SK-N-AS and SK-N-BE(2)C, exhibited an unprecedented MSC phenotype (CD105/CD90/CD73/CD29/CD146/GD2/TAZ). In these NB-MSCs, the ectoenzyme CD73 and the oncogenic/immune-regulatory transcriptional coactivator TAZ were peculiar markers. Their MSC-like nature was confirmed by their adipogenic and osteogenic differentiation potential. Immunohistochemical analysis confirmed the presence of neuroblasts with MSC phenotype (CD105/CD73/TAZ). Moreover, a public gene-expression dataset revealed that, in stage IV NB, a higher expression of TAZ and CD105 strongly correlated with a poorer outcome.Among the NB-cell lines analyzed, only NB-MSCs exhibited multifactorial resistance to NK-mediated lysis, inhibition of activating NK receptors, signal adaptors and of NK-cell cytotoxicity through cell-cell contact mediated mechanisms. The latter property was controlled partially by TAZ, since its silencing in NB cells efficiently rescued NK-cell cytotoxic activity, while its overexpression induced opposite effects in non-NB-MSC cells.

Conclusions: We identified a novel NB immunoregulatory subset that: (i) displayed phenotypic and functional properties of MSC, (ii) mediated multifactorial resistance to NK-cell-induced killing and (iii) efficiently inhibited, in coculture, the cytotoxic activity of NK cells against target cells through a TAZ-dependent mechanism. These findings indicate that targeting novel cellular and molecular components may disrupt the immunomodulatory milieu of the NB microenvironment ameliorating the response to conventional treatments as well as to advanced immunotherapeutic approaches, including adoptive transfer of NK cells and chimeric antigen receptor T or NK cells.
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http://dx.doi.org/10.1136/jitc-2020-001313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7813384PMC
January 2021

Interaction between SNAI2 and MYOD enhances oncogenesis and suppresses differentiation in Fusion Negative Rhabdomyosarcoma.

Nat Commun 2021 01 8;12(1):192. Epub 2021 Jan 8.

Greehey Children's Cancer Research Institute, Department of Molecular Medicine, University of Texas Health Sciences Center, San Antonio, Texas, USA.

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis.
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http://dx.doi.org/10.1038/s41467-020-20386-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794422PMC
January 2021

Cellular and gene signatures of tumor-infiltrating dendritic cells and natural-killer cells predict prognosis of neuroblastoma.

Nat Commun 2020 11 25;11(1):5992. Epub 2020 Nov 25.

Department of Paediatric Haematology/Oncology and of Cell and Gene Therapy, Ospedale Pediatrico Bambino Gesù, IRCCS, 00146, Rome, Italy.

Tumor-infiltrating lymphocytes play an essential role in improving clinical outcome of neuroblastoma (NB) patients, but their relationship with other tumor-infiltrating immune cells in the T cell-inflamed tumors remains poorly investigated. Here we show that dendritic cells (DCs) and natural killer (NK) cells are positively correlated with T-cell infiltration in human NB, both at transcriptional and protein levels, and associate with a favorable prognosis. Multiplex imaging displays DC/NK/T cell conjugates in the tumor microenvironment of low-risk NB. Remarkably, this connection is further strengthened by the identification of gene signatures related to DCs and NK cells able to predict survival of NB patients and strongly correlate with the expression of PD-1 and PD-L1. In summary, our findings unveil a key prognostic role of DCs and NK cells and indicate their related gene signatures as promising tools for the identification of clinical biomarkers to better define risk stratification and survival of NB patients.
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http://dx.doi.org/10.1038/s41467-020-19781-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689423PMC
November 2020

Neuroblastoma-secreted exosomes carrying miR-375 promote osteogenic differentiation of bone-marrow mesenchymal stromal cells.

J Extracell Vesicles 2020 Jun 3;9(1):1774144. Epub 2020 Jun 3.

Department of Pediatric Onco-Hematology and Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Bone marrow (BM) is the major target organ for neuroblastoma (NB) metastasis and its involvement is associated with poor outcome. Yet, the mechanism by which NB cells invade BM is largely unknown. Tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (MSCs) have been recognized as a fundamental part of the associated tumour stroma. Here, we show that BM-MSCs isolated from NB patients with BM involvement exhibit a greater osteogenic potential than MSCs from non-infiltrated BM. We show that BM metastasis-derived NB-cell lines secrete higher levels of exosomal miR-375, which promotes osteogenic differentiation in MSCs. Of note, clinical data demonstrate that high level of miR-375 correlates with BM metastasis in NB patients. Our findings suggest, indeed, a potential role for exosomal miR-375 in determining a favourable microenvironment in BM to promote metastatic progression. MiR-375 may, thus, represent a novel biomarker and a potential target for NB patients with BM involvement.
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http://dx.doi.org/10.1080/20013078.2020.1774144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448845PMC
June 2020

Proteomic Profiling of Retinoblastoma-Derived Exosomes Reveals Potential Biomarkers of Vitreous Seeding.

Cancers (Basel) 2020 06 12;12(6). Epub 2020 Jun 12.

Department of Pediatric Hematology/Oncology and Cell and Gene Therapy, IRCCS, Ospedale Pediatrico Bambino Gesù, Piazza Sant'Onofrio 4, 00165 Rome, Italy.

Retinoblastoma (RB) is the most common tumor of the eye in early childhood. Although recent advances in conservative treatment have greatly improved the visual outcome, local tumor control remains difficult in the presence of massive vitreous seeding. Traditional biopsy has long been considered unsafe in RB, due to the risk of extraocular spread. Thus, the identification of new biomarkers is crucial to design safer diagnostic and more effective therapeutic approaches. Exosomes, membrane-derived nanovesicles that are secreted abundantly by aggressive tumor cells and that can be isolated from several biological fluids, represent an interesting alternative for the detection of tumor-associated biomarkers. In this study, we defined the protein signature of exosomes released by RB tumors (RBT) and vitreous seeding (RBVS) primary cell lines by high resolution mass spectrometry. A total of 5666 proteins were identified. Among these, 5223 and 3637 were expressed in exosomes RBT and one RBVS group, respectively. Gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified in RBVS exosomes upregulated proteins specifically related to invasion and metastasis, such as proteins involved in extracellular matrix (ECM) remodeling and interaction, resistance to anoikis and the metabolism/catabolism of glucose and amino acids.
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http://dx.doi.org/10.3390/cancers12061555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352325PMC
June 2020

Ectopic ACTH Secretion in a Child With Metastatic Ewing's Sarcoma: A Case Report.

Front Oncol 2020 28;10:574. Epub 2020 Apr 28.

Department of Paediatric Haematology/Oncology, IRCCS Bambino Gesù Children's Hospital, Rome, Italy.

Ectopic ACTH syndrome is rare in pediatric age. Sarcomas that cause Ectopic ACTH Syndrome (EAS) are even more uncommon. Currently, only three cases of EAS caused by Ewing' sarcoma have been reported. We detail a 10-year-old boy with Cushing's syndrome symptoms caused by ectopic ACTH production by a metastatic Ewing's sarcoma of the right ischio-pubic and ileo-pubic branches. The rapid appearance of cushingoid symptoms, with significant weight gain, acne, hirsutism, and hypercortisolism were implications of ectopic ACTH production as paraneoplastic Cushing's Syndrome. The very high levels of ACTH and non-suppression at the high dose dexamethasone test confirmed the clinical suspicion. We underline the possibility EAS was caused by an ACTH-secreting tumor, including soft tissue sarcomas.
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http://dx.doi.org/10.3389/fonc.2020.00574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198717PMC
April 2020

CD28.OX40 co-stimulatory combination is associated with long in vivo persistence and high activity of CAR.CD30 T-cells.

Haematologica 2021 Apr 1;106(4):987-999. Epub 2021 Apr 1.

Dept Onco-Haematology, Cell and Gene Therapy, Bambino Gesù Children Hospital, Rome, Italy.

The prognosis of many patients with chemotherapy-refractory or multiply relapsed CD30+ non-Hodgkin Lymphoma (NHL) or Hodgkin lymphoma (HL) still remains poor, and novel therapeutic approaches are warranted to address this unmet clinical need. In light of this consideration, we designed and pre-clinically validated a Chimeric Antigen Receptor (CAR) construct characterized by a novel anti-CD30 single-chain variable-fragment cassette, linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. We found that CAR.CD30 T-cells exhibit remarkable cytolytic activity in vitro against HL and NHL cell lines, with sustained proliferation and pro-inflammatory cytokine production, even after multiple and sequential lymphoma cell challenges. CAR.CD30 T-cells also demonstrated anti-lymphoma activity in two in vivo xenograft immune-deficient mouse models of metastatic HL and NHL. We observed that administration of CAR.CD30 T-cells, incorporating the CD28.OX40 costimulatory domains and manufactured in the presence of IL7 and IL15, were associated with the best overall survival in the treated mice, along with the establishment of a long-term immunological memory, able to protect mice from further tumor re-challenge. Our data indicate that, in the context of in vivo systemic metastatic xenograft mouse models, the costimulatory machinery of CD28.OX40 is crucial for improving persistence, in vivo expansion and proliferation of CAR.CD30 T-cells upon tumor encounter. CD28.OX40 costimulatory combination is ultimately responsible for the antitumor efficacy of the approach, paving the way to translate this therapeutic strategy in patients with CD30+ HL and NHL.
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http://dx.doi.org/10.3324/haematol.2019.231183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018158PMC
April 2021

DNA Methylation Profiling for Diagnosing Undifferentiated Sarcoma with Capicua Transcriptional Receptor () Alterations.

Int J Mol Sci 2020 Mar 6;21(5). Epub 2020 Mar 6.

Department of Pediatric Onco-Hematology and Cell and Gene Therapy, IRCCS Bambino Gesù Children's Hospital, 00165 Rome, Italy.

Undifferentiated soft tissue sarcomas are a group of diagnostically challenging tumors in the pediatric population. Molecular techniques are instrumental for the categorization and differential diagnosis of these tumors. A subgroup of recently identified soft tissue sarcomas with undifferentiated round cell morphology was characterized by Capicua transcriptional receptor () rearrangements. Recently, an array-based DNA methylation analysis of undifferentiated tumors with small blue round cell histology was shown to provide a highly robust and reproducible approach for precisely classifying this diagnostically challenging group of tumors. We describe the case of an undifferentiated sarcoma of the abdominal wall in a 12-year-old girl. The patient presented with a voluminous mass of the abdominal wall, and multiple micro-nodules in the right lung. The tumor was unclassifiable with current immunohistochemical and molecular approaches. However, DNA methylation profiling allowed us to classify this neoplasia as small blue round cell tumor with alterations. The patient was treated with neoadjuvant chemotherapy followed by complete surgical resection and adjuvant chemotherapy. After 22 months, the patient is disease-free and in good clinical condition. To put our experience in context, we conducted a literature review, analyzing current knowledge and state-of-the-art diagnosis, prognosis, and clinical management of rearranged sarcomas. Our findings further support the use of DNA methylation profiling as an important tool to improve diagnosis of non-Ewing small round cell tumors.
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http://dx.doi.org/10.3390/ijms21051818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084764PMC
March 2020

Pediatric gastrointestinal stromal tumor: Report of two novel patients harboring germline variants in SDHB and SDHC genes.

Cancer Genet 2020 02 16;241:61-65. Epub 2019 Dec 16.

Laboratory of Medical Genetics, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract and rarely occur in pediatric patients. 85% of pediatric GISTs and 15% of adult GISTs lack of KIT or PDGFRA mutations. 40% of these "wild-type" GISTs present loss of function mutations in genes encoding for the subunits of the succinate dehydrogenase (SDH) complex. Germline mutations in SDH complex genes have been described in patients with the Carney-Stratakis syndrome (CSS), a rare inherited condition that predisposes to GIST and paraganglioma. We report two pediatric patients with multifocal GIST, harboring respectively a novel and a previously reported loss-of-function germline variant, in SDHC and SDHB genes.
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http://dx.doi.org/10.1016/j.cancergen.2019.12.002DOI Listing
February 2020

EGFR inhibition for metastasized cutaneous squamous cell carcinoma in dystrophic epidermolysis bullosa.

Orphanet J Rare Dis 2019 12 3;14(1):278. Epub 2019 Dec 3.

Department of Dermatology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Hauptstrasse 7, 79104, Freiburg, Germany.

Dystrophic epidermolysis bullosa (DEB) is a hereditary skin fragility disorder, characterized by trauma-induced blistering followed by soft tissue fibrosis. One of the most feared complications is the early development of aggressive cutaneous squamous cell carcinomas (SCC). For patients with locally advanced or metastasized SCCs treatment with cetuximab, a monoclonal antibody against epidermal growth factor receptor (EGFR), has been proposed and so far, treatment of five DEB patients with cetuximab has been published. With this report, we extend the spectrum of EB patients treated with cetuximab by adding two additional patients. Taking together all DEB cases treated with cetuximab, we propose that cetuximab should be administered as early as possible, since it seems to be more efficient and is accompanied by rather mild adverse effects. We also show that EGFR is frequently expressed in DEB-associated SCCs, although there were noticeable differences in the level of expression, which may influence responsiveness to EGFR-targeting therapies. Although only limited experiences with targeted cancer treatments in EB exist, such reports highlight the treatments' effects in this specific cohort and assist our therapeutic decisions.
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http://dx.doi.org/10.1186/s13023-019-1262-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889571PMC
December 2019

The Contribution of the Adipose Tissue-Liver Axis in Pediatric Patients with Nonalcoholic Fatty Liver Disease after Laparoscopic Sleeve Gastrectomy.

J Pediatr 2020 01 13;216:117-127.e2. Epub 2019 Sep 13.

Hepatology, Gastroenterology and Nutrition Unit - Bambino Gesù Children's Hospital, Rome, Italy; Department of Pediatric - University "La Sapienza", Rome, Italy.

Objective: To evaluate the histopathologic modifications in liver and visceral adipose tissue (VAT), and to correlate these changes with clinical measures, adipokine production, and proinflammatory cytokines in a population of adolescents with obesity with nonalcoholic fatty liver disease (NAFLD) who underwent laparoscopic sleeve gastrectomy (LSG).

Study Design: Twenty adolescents with obesity who underwent LSG and with biopsy-proven NAFLD were included. Patients underwent clinical evaluation and blood tests at baseline and 1 year after the surgical procedure. Liver and VAT specimens were processed for routine histology, immunohistochemistry, and immunofluorescence.

Results: In adolescents with obesity and NAFLD, hepatic histologic alterations were uncorrelated with VAT inflammation. LSG induced in both liver and VAT tissue histopathology amelioration and macrophage profile modification that were correlated with body mass index and improvement in insulin resistance. The adipokine profile in liver and VAT was associated with weight loss and histologic improvement after LSG. Serum proinflammatory cytokines were correlated with liver and VAT histopathology and IL-1β and IL-6 levels were independently predicted by liver necroinflammatory grade.

Conclusions: This study suggests a unique adipose tissue/fatty liver crosstalk in pediatric patients. LSG induces a similar pattern of histologic improvement in the liver and in VAT. Besides VAT, our results strengthen the role of the liver in adipocytokine production and its contribution to systemic inflammation in pediatric patients with NAFLD.
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http://dx.doi.org/10.1016/j.jpeds.2019.07.037DOI Listing
January 2020

Dissecting the mechanisms of bone loss in Gorham-Stout disease.

Bone 2020 01 13;130:115068. Epub 2019 Sep 13.

Bone Physiopathology Group, Multifactorial Disease and Complex Phenotype Research Area, Bambino Gesù Children's Hospital, Rome, Italy. Electronic address:

Gorham-Stout disease (GSD) is a rare disorder characterized by progressive osteolysis and angiomatous proliferation. Since the mechanisms leading to bone loss in GSD are not completely understood, we performed histological, serum, cellular and molecular analyses of 7 patients. Increased vessels, osteoclast number and osteocyte lacunar area were revealed in patients' bone biopsies. Biochemical analysis of sera showed high levels of ICTP, Sclerostin, VEGF-A and IL-6. In vitro experiments revealed increased osteoclast differentiation and activity, and impaired mineralization ability of osteoblasts. To evaluate the involvement of systemic factors in GSD, control cells were treated with patients' sera and displayed an increase of osteoclastogenesis, bone resorption activity and a reduction of osteoblast function. Interestingly, GSD sera stimulated the vessel formation by endothelial cells EA.hy926. These results suggest that bone cell autonomous alterations with the cooperation of systemic factors are involved in massive bone loss and angiomatous proliferation observed in GSD patients.
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http://dx.doi.org/10.1016/j.bone.2019.115068DOI Listing
January 2020

The Number of Liver Galectin-3 Positive Cells Is Dually Correlated with NAFLD Severity in Children.

Int J Mol Sci 2019 Jul 14;20(14). Epub 2019 Jul 14.

Molecular Genetics of Complex Phenotypes Research Unit, Bambino Gesù Hospital, IRCCS, 00146 Rome, Italy.

Non-alcoholic fatty liver disease (NAFLD) is a complex disease ranging from steatosis to non-alcoholic steatohepatitis (NASH). Galectin-3 (Gal-3), which is a β-galactoside binding protein, has been associated with liver fibrosis, but its role in NAFLD remains elusive. We investigated the expression of Gal-3 in liver resident cells and its potential association with liver damage in 40 children with biopsy-proven NAFLD. We found that several liver cells expressed Gal-3. The number of total Gal-3 positive cells decreased with the severity of disease and the cells were correlated with the presence of steatosis and the diagnosis of NASH. CD68 macrophages expressed Gal-3 but the number CD68/Gal-3 positive cells was significantly reduced in patients diagnosed with steatosis and NASH. Triple CD68/CD206/Gal-3, which represented the subpopulation of M2 macrophages, were mainly present in patients without NASH, and clearly reduced in patients with steatosis and NASH. On the contrary, the number of α-smooth muscle actin (SMA)/Gal-3 positive cells increased with the severity of fibrosis in children with NAFLD. Our data demonstrated that the number of Gal-3 positive cells was associated with tissue damage in different ways, which suggests a dual role of this protein in the pathogenesis of pediatric NAFLD, even if the role of Gal-3 deserves further studies.
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http://dx.doi.org/10.3390/ijms20143460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679049PMC
July 2019

Angioma serpiginosum: a case report and review of the literature.

Ital J Pediatr 2019 Apr 27;45(1):53. Epub 2019 Apr 27.

Dermatology Unit, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio, 4, 00165, Rome, Italy.

Background: Angioma serpiginosum is a rare vascular anomaly whose pathogenesis is still unknown. It is characterized by the onset of vascular reddish macules and papules during childhood, lesions are usually monolateral with a linear serpiginous pattern. It is rarely associated with extracutaneous findings. This entity has not yet been included in the classification of the International Society for the Study of Vascular Anomalies.

Case Presentation: We describe the first Italian report of angioma serpiginosum with a congenital symmetrical presentation. The patient had a further extension of macules during puberty involving both of the soles. No extracutaneous manifestations were present. Diagnosis was confirmed with dermoscopy and light microscopy that revealed the typical clusters of dilated, thickened and PAS+ capillaries in the upper dermis. Moreover, Immunohistochemistry showed positive WT-1 staining. Genetic analysis with next generation sequencing did not detected any mutation.

Conclusions: Our patient presented a peculiar symmetrical and planar extension with a serpiginous linear pattern. The proliferative nature of this condition has been widely discussed in literature. In our case immunohistochemistry was positive for Wilms tumor-1, a new endothelial marker expressed during angiogenesis in reparative processes and endothelial tumors. Clinical evolution, histological and immunohistochemical findings suggest that angioma serpiginosum should be considered as a vascular proliferation. For these reasons we think it should be included in the international classification as a tumor.
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http://dx.doi.org/10.1186/s13052-019-0644-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6487031PMC
April 2019

First Case of Patient With Two Homozygous Mutations in and Genes Presenting With Pyogenic Bacterial Infections, Elevated IgE, and Persistent EBV Viremia.

Front Immunol 2019 14;10:130. Epub 2019 Feb 14.

University Department of Pediatrics, Unit of Immune and Infectious Diseases, Childrens' Hospital Bambino Gesù, Rome, Italy.

We described for the first time a female patient with the simultaneous presence of two homozygous mutations in and genes presenting with pyogenic bacterial infections, elevated IgE, and persistent EBV viremia. In addition to defective TLR/IL1R-signaling, we described novel functional alterations into the myeloid compartment. In particular, we demonstrated a defective production of reactive oxygen species exclusively in monocytes upon stimulation, the inability of immature mono-derived DCs (iDCs) to differentiate into mature DCs (mDCs) and the incapacity of mono-derived macrophages (MDMs) to resolve BCG infection . Our data do not provide any evidence for digenic inheritance in our patient, but rather for the association of two monogenic disorders. This case illustrates the importance of using next generation sequencing (NGS) to determine the most accurate and early diagnosis in atypical clinical and immunological phenotypes, and with particular concern in consanguineous families. Indeed, besides the increased susceptibility to recurrent invasive pyogenic bacterial infections due to MYD88 deficiency, the identification of mutations underline the risk of developing invasive fungal infections emphasizing the careful monitoring for the occurrence of fungal infection and the opportunity of long-term antifungal prophylaxis.
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http://dx.doi.org/10.3389/fimmu.2019.00130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383679PMC
December 2019

Role of interferon-γ in immune-mediated graft failure after allogeneic hematopoietic stem cell transplantation.

Haematologica 2019 11 21;104(11):2314-2323. Epub 2019 Feb 21.

Bambino Ges Children's Hospital, Department of Pediatric Hematology/Oncology, Cellular and Gene Therapy, Rome, Italy.

Pathophysiology of graft failure (GF) occurring after allogeneic hematopoietic stem cell transplantation (HSCT) still remains elusive. We measured serum levels of several different cytokines/chemokines in 15 children experiencing GF, comparing their values with those of 15 controls who had sustained donor cell engraftment. Already at day +3 after transplantation, patients developing GF had serum levels of interferon (IFN)-γ and CXCL9 (a chemokine specifically induced by IFNγ) significantly higher than those of controls (8859±7502 0 pg/mL, =0.03, and 1514.0±773 233.6±50.1 pg/mlL, =0.0006, respectively). The role played by IFNγ in HSCT-related GF was further supported by the observation that a rat anti-mouse IFNγ-neutralizing monoclonal antibody promotes donor cell engraftment in Ifngr1mice receiving an allograft. In comparison to controls, analysis of bone marrow-infiltrating T lymphocytes in patients experiencing GF documented a predominance of effector memory CD8 cells, which showed markers of activation (overexpression of CD95 and downregulation of CD127) and exhaustion (CD57, CD279, CD223 and CD366). Finally, we obtained successful donor engraftment in 2 out of 3 children with primary hemophagocytic lymphohistiocytosis who, after experiencing GF, were re-transplanted from the same HLA-haploidentical donor under the compassionate use coverage of emapalumab, an anti-IFNγ monoclonal antibody recently approved by the US Food and Drug Administration for treatment of patients with primary hemophagocytic lymphohistiocytosis. Altogether, these results suggest that the IFNγ pathway plays a major role in GF occurring after HSCT. Increased serum levels of IFNγ and CXCL9 represent potential biomarkers useful for early diagnosis of GF and provide the rationale for exploring the therapeutic/preventive role of targeted neutralization of IFNγ.
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http://dx.doi.org/10.3324/haematol.2019.216101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821635PMC
November 2019

Expression profiles of exosomal miRNAs isolated from plasma of patients with desmoplastic small round cell tumor.

Epigenomics 2019 04 20;11(5):489-500. Epub 2018 Dec 20.

Department of Pediatric Hematology/Oncology, Bambino Gesù Children's Hospital, IRCCS, Piazza di Sant' Onofrio, 4, 00165 Rome, Italy.

Aim: Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive mesenchymal tumor, lacking biomarkers for diagnosis, treatment stratification and prognosis. We investigated the exosomal miRNA profile in plasma samples collected from DSRCT patients, evaluating their potential as circulating biomarkers for this tumor.

Patients & Methods: We isolated exosomes from plasma of three DSRCT adolescents and four age-matched healthy controls; expression of circulating miRNAs was quantified by qPCR.

Results: We identified 55 miRNAs significantly modulated compared with healthy controls. Among these miRNAs, 14 were highly dysregulated in at least one patient and 5 were expressed in all patients.

Conclusion: To our knowledge, this is the first report describing exosomal miRNAs as promising biomarkers to characterize disease status in DSRCT patients.
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http://dx.doi.org/10.2217/epi-2018-0179DOI Listing
April 2019

Burkitt lymphoma in a patient with Kabuki syndrome carrying a novel KMT2D mutation.

Am J Med Genet A 2019 01 20;179(1):113-117. Epub 2018 Dec 20.

Department of Pediatric Hematology/Oncology and Cellular and Gene Therapy, Bambino Gesù Children's Hospital, Rome, Italy.

Kabuki syndrome (KS) is an extremely rare genetic disorder, mainly caused by germline mutations at specific epigenetic modifier genes, including KMT2D. Because the tumor suppressor gene KMT2D is also frequently altered in many cancer types, it has been suggested that KS may predispose to the development of cancer. However, KS being a rare disorder, few data are available on the incidence of cancer in KS patients. Here, we report the case of a 5-year-old boy affected by KS who developed Burkitt lymphoma (BL). Genetic analysis revealed the presence of a novel heterozygous mutation in the splice site of the intron 4 of KMT2D gene in both peripheral blood-extracted DNA and tumour cells. In addition, the tumour sample of the patient was positive for the classical somatic chromosomal translocation t(8;14) involving the c-MYC gene frequently identified in BL. We propose that the mutated KMT2D gene contributes to the development of both KS and BL observed in our patient and we suggest that strict surveillance must be performed in KS patients.
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http://dx.doi.org/10.1002/ajmg.a.60674DOI Listing
January 2019

Macrophage Markers Are Poorly Associated With Liver Histology in Children With Nonalcoholic Fatty Liver Disease.

J Pediatr Gastroenterol Nutr 2018 11;67(5):635-642

Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus N.

Objectives: We have previously demonstrated associations between the macrophage activation marker soluble (s)CD163 and histology of nonalcoholic fatty liver disease (NAFLD) in adults, and elevated sCD163 levels in children with obesity with NAFLD. Macrophage activation has, however, not been investigated in children with biopsy-proven NAFLD, which was the objective of the present study.

Methods: We used in-house enzyme-linked immunosorbent assays to measure sCD163 and the novel macrophage marker soluble mannose receptor (sMR) in a cross-sectional (n = 155) pediatric NAFLD cohort, and a cohort of NAFLD children (n = 36) undergoing a randomized trial by the probiotic VSL#3. We included 56 healthy nonobese children for comparison.

Results: Levels of sCD163 and sMR were higher in both of the NAFLD cohorts compared with controls (P < 0.001). In the cross-sectional cohort, sCD163 only showed trends toward association with ballooning (rho = 0.14, P = 0.08) and portal inflammation (rho = 0.17, P = 0.08). sMR showed similar associations with liver histology. In the VSL#3 cohort, sCD163 correlated inversely with steatosis (rho = -0.35, P = 0.04), and lobular (rho = -0.57, P < 0.001) and portal inflammation (rho = -0.38, P = 0.02); sMR was not associated with any histological scores. Neither sCD163 nor sMR changed significantly during intervention, and without association with NAFLD resolution.

Conclusions: The macrophage activation markers sCD163 and sMR showed poor associations with liver histology in 2 different cohorts of children with biopsy-proven NAFLD, and none of the markers decreased during successful intervention. These results are in contrast with studies of adult NAFLD and may suggest a possibility of different roles for macrophages in the pathogenesis of adult and pediatric NAFLD.
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http://dx.doi.org/10.1097/MPG.0000000000002111DOI Listing
November 2018

Next-Generation Sequencing Reveals A JAGN1 Mutation in a Syndromic Child With Intermittent Neutropenia.

J Pediatr Hematol Oncol 2019 05;41(4):e266-e269

University Department of Pediatrics, Unit of Immune and Infectious Diseases, Childrens' Hospital Bambino Gesù.

Background: Jagunal homolog 1 (JAGN1) gene was identified as a novel responsible for severe congenital neutropenia. The protein encoded by this gene is required for neutrophil differentiation, survival and function in microbial activity. JAGN1-deficient human neutrophils are characterized by alterations in trafficking within the endoplasmic reticulum and golgi compartments because of ultrastructural defects in endoplasmic reticulum and susceptibility to apoptosis.

Observations: We report a patient exhibiting an intermittent neutropenia, for which a next-generation sequencing revealed a homozygous mutation in the JAGN1 gene.

Conclusions: The patient extends the clinical variability associated to JAGN1 mutations, and this case highlights the importance of genetic investigations in patients with suspected neutropenia.
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http://dx.doi.org/10.1097/MPH.0000000000001256DOI Listing
May 2019

Clinical impact of miR-223 expression in pediatric T-Cell lymphoblastic lymphoma.

Oncotarget 2017 Dec 11;8(64):107886-107898. Epub 2017 Nov 11.

Department of Women's and Children's Health, Clinic of Pediatric Hemato-Oncology, University of Padova, 35128 Padova, Italy.

Although probability of event-free survival in pediatric lymphoblastic T-cell lymphoma (T-LBL) is about 75%, survival in relapsed patients is very poor, so the identification of new molecular markers is crucial for treatment optimization. Here, we demonstrated that the over-expression of promotes tumor T-LBL cell growth, migration and invasion . We found out that SIK1, an anti-metastatic protein, is a direct target of and consequently is significantly reduced in -overexpressing tumor cells. We measured expression levels at diagnosis in tumor biopsies from 67 T-LBL pediatric patients for whom complete clinical and follow up data were available, and we found that high expression (above the median value) is associated with worse prognosis (PFS 66% vs 94%, P=0.0036). In addition, the multivariate analysis, conducted taking into account expression level and other molecular and clinical characteristics, showed that only high level of is an independent factor for worse prognosis. represents a promising marker for treatment stratification in pediatric patients with T-LBL and we provide the first evidence of potential role as oncomir by SIK1 repression.
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http://dx.doi.org/10.18632/oncotarget.22386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746112PMC
December 2017

Quantitative Multiplexed Imaging Analysis Reveals a Strong Association between Immunogen-Specific B Cell Responses and Tonsillar Germinal Center Immune Dynamics in Children after Influenza Vaccination.

J Immunol 2018 01 13;200(2):538-550. Epub 2017 Dec 13.

Tissue Analysis Core, Immunology Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;

Generation of Ag-specific humoral responses requires the orchestrated development and function of highly specialized immune cells in secondary lymphoid organs. We used a multiparametric approach combining flow cytometry, confocal microscopy, and histocytometry to analyze, for the first time to our knowledge in children, tonsils from seasonal influenza-vaccinated children. We used these novel imaging assays to address the mucosal immune dynamics in tonsils investigating the spatial positioning, frequency, and phenotype of immune cells after vaccination. Vaccination was associated with a significantly higher frequency of follicular helper CD4 T cells compared with the unvaccinated control group. The imaging analysis revealed that potential suppressor (FOXP3) CD4 T cells are mainly located in extrafollicular areas. Furthermore, a significantly reduced frequency of both follicular and extrafollicular FOXP3 CD4 T cells was found in the vaccine group compared with the control group. Levels of circulating CXCL13 were higher in those vaccinated compared with controls, mirroring an increased germinal center reactivity in the tonsils. Notably, a strong correlation was found between the frequency of tonsillar T follicular helper cells and tonsillar Ag-specific Ab-secreting cells. These data demonstrate that influenza vaccination promotes the prevalence of relevant immune cells in tonsillar follicles and support the use of tonsils as lymphoid sites for the study of germinal center reactions after vaccination in children.
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http://dx.doi.org/10.4049/jimmunol.1701312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760299PMC
January 2018

Liver zonation in children with non-alcoholic fatty liver disease: Associations with dietary fructose and uric acid concentrations.

Liver Int 2018 06 31;38(6):1102-1109. Epub 2018 Jan 31.

Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton, UK.

Background & Aims: As dietary components are delivered directly to the periportal zone of the liver lobule, there is the potential for greater injury in this zone (zone 1) compared to the perivenous zone (zone 3). We investigated the associations between dietary fructose consumption and uric acid concentrations and differential zonal injury in periportal and perivenous zones.

Methods: A total of 271 children's histological images were scored in 5 periportal and 5 perivenous zones for steatosis, ballooning, inflammation and fibrosis severity. Dietary fructose consumption (g/d) was assessed and uric acid measured in serum. Logistic regression was undertaken to test associations between both high fructose consumption and hyperuricaemia, and histological disease in periportal and perivenous zones.

Results: Children with a mean age of 12.5 years were included in the study. Inflammation (mean ± SD) was increased in the periportal vs perivenous zones (0.78 ± 0.43 vs 0.41 ± 0.48, P = .041). There were non-significant trends towards greater steatosis, ballooning and fibrosis in the periportal zone. In the fully adjusted models, high fructose intake was associated with disease in both zones. Example for periportal and perivenous zones, respectively, steatosis 1.56 (1.12, 2.49) and 1.21 (1.09, 2.73); inflammation 4.29 (2.31, 5.88) and 3.69 (2.14, 4.56); and fibrosis 2.72 (1.43, 3.76) and 1.96 (1.24, 2.37). Hyperuricaemia (uric acid ≥5.9 mg/dL) was associated with inflammation in the periportal zone 1.71 (1.17, 2.35); and was associated with steatosis and fibrosis in both zones; for example, for periportal and perivenous zones, respectively, steatosis 2.98 (1.65, 3.23) and 1.14 (1.05, 1.99); and fibrosis, 2.65 (1.35, 2.99) and 1.31 (1.13, 2.17).

Conclusions: High fructose consumption is associated with disease severity in both lobular zones and hyperuricaemia may be associated with more severe disease in the periportal zone.
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http://dx.doi.org/10.1111/liv.13661DOI Listing
June 2018

Intraspinal mesenchymal chondrosarcoma: report of a pediatric case and literature review.

Tumori 2017 Nov 15;103(Suppl. 1):e66-e72. Epub 2017 Nov 15.

Department of hematology/Oncology and Stem Cell Transplantation, Bambino Gesù Children's Hospital, IRCCS, Rome - Italy.

Purpose: Mesenchymal chondrosarcoma (MCS) is an aggressive variant of chondrosarcoma and is a rare tumor, particularly within the pediatric population. Commonly, MCS originates in the bone, but it can also arise in extraskeletal sites, such as the brain and the intraspinal area. Due to the rarity of this tumor, there are no guidelines for its optimal treatment.

Methods: We report a case of intradural extramedullary MCS, located at the T11-T12 level, in a 14-year-old male. The tumor was documented by magnetic resonance imaging and treated with gross total resection (GTR) without adjuvant treatment. We further reviewed the relevant pediatric literature and discussed the management and outcome of intracranial and intraspinal MCS.

Results: The patient's follow-up showed no evidence of disease 2 years from diagnosis. A total of 51 cases of intracranial and intraspinal MCS have been reported (24 intraspinal and 27 intracranial). Recurrence has been described in only 4 patients with intraspinal MSC, and among them 3 received adjuvant chemotherapy and radiotherapy. GTR seems to reduce the risk of recurrence and, due to a higher cancer-mortality rate for these patients, adjuvant chemotherapy and radiotherapy are recommended in case aggressive surgery is not possible.

Conclusions: According to our single experience, we would suggest that adjuvant therapy might be unnecessary in cases where a localized MCS undergoes GTR. Chemotherapy and radiotherapy should be recommended when GTR cannot be obtained. Further studies are needed to investigate a standard treatment approach for this rare tumor.
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http://dx.doi.org/10.5301/tj.5000689DOI Listing
November 2017