Marco Forni

Identification of Tumor-Specific Molecular Signatures in Intracranial Ependymoma and Association With Clinical Characteristics

PURPOSE To delineate clinically relevant molecular signatures of intracranial ependymoma. MATERIALS AND METHODS We analyzed 24 primary intracranial ependymomas. For genomic profiling, microarray-based comparative genomic hybridization (CGH) was used and results were validated by fluorescent in situ hybridization and loss of heterozygosity mapping. We performed gene expression profiling using microarrays, real-time quantitative reverse transcriptase polymerase chain reaction, and methylation analysis of selected genes. We applied class comparison analyses to compare both genomic and expression profiling data with clinical characteristics. RESULTS A variable number of genomic imbalances were detected by array CGH, revealing multiple regions of recurrent gain (including 2q23, 7p21, 12p, 13q21.1, and 20p12) and loss (including 5q31, 6q26, 7q36, 15q21.1, 16q24, 17p13.3, 19p13.2, and 22q13.3). An ependymoma-specific gene expression signature was characterized by the concurrent abnormal expression of developmental and differentiation pathways, including NOTCH and sonic hedgehog signaling. We identified specific differentially imbalanced genomic clones and gene expression signatures significantly associated with tumor location, patient age at disease onset, and retrospective risk for relapse. Integrated genomic and expression profiling allowed us to identify genes of which the expression is deregulated in intracranial ependymoma, such as overexpression of the putative proto-oncogene YAP1 (located at 11q22) and downregulation of the SULT4A1 gene (at 22q13.3). CONCLUSION The present exploratory molecular profiling study allowed us to refine previously reported intervals of genomic imbalance, to identify novel restricted regions of gain and loss, and to identify molecular signatures correlating with various clinical variables. Validation of these results on independent data sets represents the next step before translation into the clinical setting.

Hyperfractionated Versus Conventional Radiotherapy Followed by Chemotherapy in Standard-Risk Medulloblastoma: Results From the Randomized Multicenter HIT-SIOP PNET 4 Trial

PURPOSE To compare event-free survival (EFS), overall survival (OS), pattern of relapse, and hearing loss in children with standard-risk medulloblastoma treated by postoperative hyperfractionated or conventionally fractionated radiotherapy followed by maintenance chemotherapy. PATIENTS AND METHODS In all, 340 children age 4 to 21 years from 122 European centers were postoperatively staged and randomly assigned to treatment with hyperfractionated radiotherapy (HFRT) or standard (conventional) fractionated radiotherapy (STRT) followed by a common chemotherapy regimen consisting of eight cycles of cisplatin, lomustine, and vincristine. RESULTS After a median follow-up of 4.8 years (range, 0.1 to 8.3 years), survival rates were not significantly different between the two treatment arms: 5-year EFS was 77% ± 4% in the STRT group and 78% ± 4% in the HFRT group; corresponding 5-year OS was 87% ± 3% and 85% ± 3%, respectively. A postoperative residual tumor of more than 1.5 cm(2) was the strongest negative prognostic factor. EFS of children with all reference assessments and no large residual tumor was 82% ± 2% at 5 years. Patients with a delay of more than 7 weeks to the start of RT had a worse prognosis. Severe hearing loss was not significantly different for the two treatment arms at follow-up. CONCLUSION In this large randomized European study, which enrolled patients with standard-risk medulloblastoma from more than 100 centers, excellent survival rates were achieved in patients without a large postoperative residual tumor and without RT treatment delays. EFS and OS for HFRT was not superior to STRT, which therefore remains standard of care in this disease.

Medulloblastoma Variants: Age-Dependent Occurrence and Relation to Gorlin Syndrome—A New Clinical Perspective

Purpose: We aimed to test the hypothesis that medulloblastoma (MB) variants show a different age distribution and clinical behavior reflecting their specific biology, and that MB occurring at very young age is associated with cancer predisposition syndromes such as Gorlin syndrome (GS). Experimental Design: We investigated the frequency, age distribution, location, response to treatment, outcome, and association with familial cancer predisposition syndromes in a series of 82 cases of MB in patients ages <14 years diagnosed at the Giannina Gaslini Childrens Hospital, Genoa, between 1987 and 2004. Results: Desmoplastic MB and MB with extensive nodularity (MBEN), were present in 22 of 82 cases (27%) and were more frequent in children ages ≤3 years (13 of 25; 52%). In this age group, MBEN was significantly more frequent than desmoplastic MB and classic MB (P < 0.001) and had a good prognosis. MBEN was associated with GS in 5 of 12 cases. Overall, 8 cases occurred in the context of familial tumor predisposition syndromes (5 GS, 1 each NF1, Li-Fraumeni, and Fragile X) and 7 of these patients were ages ≤3 years at diagnosis. Desmoplastic histology and a more intensive treatment represented independent favorable prognostic factors in multivariate analysis (P = 0.003 and P = 0.0139, respectively). Metastasis was a predictor of bad outcome (P = 0.0001). Conclusions: Our data indicate that biologically different MB entities warrant risk-adapted treatment and that MBEN is strongly associated with GS. Patients, ages ≤3 years, with MB and their families should be investigated for tumor predisposition syndromes such as GS.

Report of a cavernoma of the cauda equina and review of the literature

Subdural intramedullary and extramedullary cavernous angiomas are rare vascular malformations. A case of cavernoma of the cauda equina affecting a 46-year-old man is reported. He presented with low back and sciatic pain. This is the fourth case of cavernoma of the cauda equina in the literature. The available literature on intramedullary (28 cases) and extramedullary (9 cases) cavernomas is reviewed.

miR148b is a major coordinator of breast cancer progression in a relapse-associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS, and CSF1

Breast cancer is often fatal during its metastatic dissemination. To unravel the role of microRNAs (miRs) during malignancy, we analyzed miR expression in 77 primary breast carcinomas and identified 16 relapse‐associated miRs that correlate with survival and/or distinguish tumor subtypes in different datasets. Among them, miR‐148b, down‐regulated in aggressive breast tumors, was found to be a major coordinator of malignancy. In fact, it is able to oppose various steps of tumor progression when overexpressed in cell lines by influencing invasion, survival to anoikis, extravasation, lung metastasis formation, and chemotherapy response. miR‐148b controls malignancy by coordinating a novel pathway involving over 130 genes and, in particular, it directly targets players of the integrin signaling, such as ITGA5, ROCK1, PIK3CA/p110α, and NRAS, as well as CSF1, a growth factor for stroma cells. Our findings reveal the importance of the identified 16 miRs for disease outcome predictions and suggest a critical role for miR‐148b in the control of breast cancer progression.—Cimino, D., De Pittà, C., Orso, F., Zampini, M., Casara, C., Penna, E., Quaglino, E., Forni, M., Damasco, C., Pinatel, E., Ponzone, R., Romualdi, C., Brisken, C., De Bortoli, M., Biglia, N., Provero, P., Lanfranchi, G., Taverna, D. miR148b is a major coordinator of breast cancer progression in a relapse‐associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS, and CSF1. FASEB J. 27, 1223–1235 (2013). www.fasebj.org

Differentiation of Mesenchymal Stem Cells Derived from Pancreatic Islets and Bone Marrow into Islet-Like Cell Phenotype

Background Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself. Methodology/Principal Findings In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin. Conclusions/Significance Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes.

Mutation Analysis of CCM1, CCM2 and CCM3 Genes in a Cohort of Italian Patients with Cerebral Cavernous Malformation

Cerebral cavernous malformations (CCMs) are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities. CCMs can occur as sporadic or familial autosomal dominant form. Familial cases are associated with mutations in CCM1[K‐Rev interaction trapped 1 (KRIT1)], CCM2 (MGC4607) and CCM3 (PDCD10) genes. In this study, a three‐gene mutation screening was performed by direct exon sequencing, in a cohort of 95 Italian patients either sporadic or familial, as well as on their at‐risk relatives. Sixteen mutations in 16 unrelated CCM patients were identified, nine mutations are novel: c.413T > C; c.601C > T; c.846 + 2T > G; c.1254delA; c.1255‐4delGTA; c.1681‐1682delTA in CCM1; c.48A > G; c.82‐83insAG in CCM2; and c.396G > A in CCM3 genes. The samples, negative to direct exon sequencing, were investigated by MLPA to search for intragenic deletions or duplications. One deletion in CCM1 exon 18 was detected in a sporadic patient. Among familial cases 67% had a mutation in CCM1, 5.5% in CCM2, and 5.5% in CCM3, whereas in the remaining 22% no mutations were detected, suggesting the existence of either undetectable mutations or other CCM genes. This study represents the first extensive research program for a comprehensive molecular screening of the three known genes in an Italian cohort of CCM patients and their at‐risk relatives.

Endoscopic and histologic findings in the upper gastrointestinal tract of children with coeliac disease

Frequency of mucosal damage to the esophagus, stomach, and duodenum was investigated in 176 children with coeliac disease (CD) during 230 upper GI en-doscopies performed to obtain duodenal biopsy specimens and was compared with findings in 230 age-matched children who underwent endoscopy for upper GI complaints without CD (non-CD patients). To evaluate a possible association with gluten ingestion, we then compared frequency of mucosal damage in patients on a gluten-containing diet and those on a gluten-free Diet (GFD). In children with CD, frequency of esophageal damage seen at endoscopy and of peptic esophagitis shown by histology were significantly lower than in non-CD patients (p < 0.01) due to the very low frequency of mucosal damage in CD children on GFD; however, frequency of columnar metaplasia was significantly higher (p < 0.05). At endoscopy, CD children had a significantly lower frequency of gastric abnormalities, but histology showed a higher prevalence of superficial chronic gastritis (SCG; p < 0.01). SCG was associated with gluten ingestion, since its frequency in CD children on GFD was similar to the frequency in non-CD patients. At endoscopy, frequency of duodenal mucosal damage was similar in CD and non-CD patients. In addition to villous atrophy, histology showed a significantly higher frequency of duodenitis in CD children on a gluten-containing diet (p < 0.001 vs. non-CD patients; p < 0.05 vs. CD children on GFD). Our findings show that the mucosa of the whole upper GI tract can be damaged in CD patients and that the prevalence of some changes is higher with a gluten-containing diet.

Medullospheres from DAOY, UW228 and ONS-76 cells: increased stem cell population and proteomic modifications.

Background Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies. Methodology/Principal Findings The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression. Conclusions/Significance Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB.

Proteomic identification of heat shock protein 27 as a differentiation and prognostic marker in neuroblastoma but not in Ewing’s sarcoma

Neuroblastoma (NB) and Ewing’s sarcoma (ES) cell lines were analysed by two-dimensional gel electrophoresis (2-DE) searching for new diagnostic/prognostic markers. Protein expression profiles displayed a high degree of similarity with the exception of marked heat shock protein (HSP) 27 and less marked HSP60 and HSP70 family up-modulations in NB cells. HSP27, which showed peculiar variability in different NB cell preparations, responded to all trans-retinoic acid treatment in NB cells but not in ES cells at gene and protein expression levels. Immunohistochemistry studies showed different behaviours of HSP27 and HSP70 expression in NB and ES biopsies. HSP27 was less expressed, whereas HSP70 was more expressed in the immature areas of NB. HSP27 expression showed positive and statistically significant correlation with favourable prognosis, and HSP27 expression also negatively correlated with increasing aggressiveness of histological type. In ES, both chaperones were expressed without characteristic patterns. Our results suggest that HSP27, after further clinical validations, could be used as a marker of neuronal differentiation in vivo for the assessment of the biological behaviour of NB and for the risk stratification of patients.