Manuela Badiali

large-cell Medulloblastomas : A Distinct Variant with Highly Aggressive Behavior

&NA; We present four cases of infantile cerebellar neoplasms composed of cells with large vesicular nuclei with prominent nucleoli. All four cases were strongly immunoreactive for synaptophysin, and one case showed immunoreactivity for neurofilaments. Filter hybridization for N‐myc and c‐myc oncogenes showed a 27‐fold c‐myc amplification in one case. The cytogenetic analysis in this case showed Double‐Minutes and isochromosome 17q. An intracerebral xenograft in nude mice obtained from one such tumor showed a similar morphology to that of the original tumor as well as strong immunoreactivity for synaptophysin and neurofilaments. All the neoplasms were characterized by highly aggressive behavior leading to early cerebrospinal fluid dissemination despite radiotherapy and chemotherapy. We conclude that large‐cell medulloblastoma represents a distinct and more aggressive variant of medulloblastoma that requires more aggressive therapy.

N-myc and c-myc oncogenes amplification in medulloblastomas. Evidence of particularly aggressive behavior of a tumor with c-myc amplification.

N-myc and c-myc amplification was investigated in 27 medulloblastomas. DNA was extracted from 19 formalin fixed and paraffin embedded tumors and from fresh frozen tumor tissue in 8 other cases. The results showed no evidence of amplification of N-myc oncogene and only 1 case had a 27 fold amplification of c-myc. Cytogenetically, this neoplasm presented numerous double minute chromosomes (DMs). Moreover, it had an unusual rapidly aggressive course with massive cerebrospinal fluid dissemination unresponsive to intrathecal chemotherapy. Our results indicate a low incidence of N-myc and c-myc gene amplification in medulloblastomas, suggesting that the oncogenic mechanism in these neoplasms is not closely related to DNA gene amplification. C-myc amplification, although not frequently observed, may however provide a growth advantage for medulloblastoma cells in vivo, favoring their rapid dissemination. Medulloblastomas with c-myc activation may represent a subgroup of tumors with a more aggressive behavior.

KIAA1549-BRAF Fusions and IDH Mutations Can Coexist in Diffuse Gliomas of Adults

KIAA1549‐BRAF fusion gene and isocitrate dehydrogenase (IDH) mutations are considered two mutually exclusive genetic events in pilocytic astrocytomas and diffuse gliomas, respectively. We investigated the presence of the KIAA1549‐BRAF fusion gene in conjunction with IDH mutations and 1p/19q loss in 185 adult diffuse gliomas. Moreover BRAFv600E mutation was also screened. The KIAA1549‐BRAF fusion gene was evaluated by reverse‐transcription polymerase chain reaction (RT‐PCR) and sequencing. We found IDH mutations in 125 out 175 cases (71.4%). There were KIAA1549‐BRAF fusion gene in 17 out of 180 (9.4%) cases and BRAFv600E in 2 out of 133 (1.5%) cases. In 11 of these 17 cases, both IDH mutations and the KIAA1549‐BRAF fusion were present, as independent molecular events. Moreover, 6 of 17 cases showed co‐presence of 1p/19q loss, IDH mutations and KIAA1549‐BRAF fusion. Among the 17 cases with KIAA1549‐BRAF fusion gene 15 (88.2%) were oligodendroglial neoplasms. Similarly, the two cases with BRAFv600E mutation were both oligodendroglioma and one had IDH mutations and 1p/19q co‐deletion. Our results suggest that in a small fraction of diffuse gliomas, KIAA1549‐BRAF fusion gene and BRAFv600E mutation may be responsible for deregulation of the Ras‐RAF‐ERK signaling pathway. Such alterations are more frequent in oligodendroglial neoplasm and may be co‐present with IDH mutations and 1p/19q loss.

p53 gene mutations in medulloblastoma. Immunohistochemistry, gel shift analysis, and sequencing.

Medulloblastoma (MB), the most common malignant tumor of the CNS in children, bears a loss of the short arm of chromosome 17 in almost half of the cases. The tumor suppressor gene p53 is located on this chromosome and its role in the pathogenesis of this primitive tumor is controversial. Twenty-two MBs were analyzed by single-strand conformation polymorphism (SSCP) of polymerase chain reaction-amplified conserved exons. Fragments displaying a gel mobility shift were subsequently analyzed by direct sequencing. Immunohistochemistry for p53 was performed in all cases; three had cytogenetic analysis. Two cases (9%) were found to harbor a mutation: one homozygous and one heterozygous. The latter showed focal p53 immunostaining. None of the cases with chromosome 17p abnormality by cytogenetic analysis were found to have a mutation in the remaining allele. Loss of heterozygosity (LOH) of 17p, however, was found in four cases (one by SSCP and three by cytogenetic analysis). Together with the homozygous deletion in one case, the overall incidence of p53 allelic involvement in MB is 23% Although LOH for the p53 gene may confer a selective advantage to tumor cells harboring mutations with dominant negative oncogenic effect, the infrequent occurrence of p53 mutations in face of frequent LOH for this gene supports the previously formulated hypothesis of a novel tumor-related locus distal to p53 on chromosome 17p.

Age-dependent prognostic significance of N-myc amplification in neuroblastoma: The Italian experience

From the Italian Neuroblastic Research Program: Section of Epidemiological Genetics & Eco-Oncogenetics (R. S., P. S., V. F.) and Peripheral Section of Population Genetics (R. S., P. S., V. F.), Natl. Cancer Research Institute (IST), Genova; Pediatric Clinic (M. B.), University of Bologna; Pediatric Clinic (C. D.), University of Rome; Pediatric Clinic (A. I.), University of Naples; Pediatric Oncology Division (B. D. B.), G. Gaslini Childrens Hospital, Genova; and Research Group of Molecular Diagnostic (G. P. T.), G. Gaslini Childrens Hospital, Genova, Italy.

KIAA1549: BRAF fusion gene in pediatric brain tumors of various histogenesis

The KIAA1549:BRAF fusion gene is considered a driver genetic event in pilocytic astrocytoma. We investigated a series of 69 pediatric brain neoplasms of diverse histogenesis and grade using the RT‐PCR and sequencing. We detected the KIAA1549:BRAF fusion gene in five of 34 non‐PA tumors (14.7%), that is, one glioblastoma, one anaplastic astrocytoma, one anaplastic pleomorphic xanthoastrocytoma, 1 ependymoma, and 1 Atypical Teratoid Rhabdoid Tumor. Our study showed that the K–B, although uncommon, it can be detected in non‐PA tumors of various histogenesis and grading. Pediatr Blood Cancer 2015;62:724–727.

Establishment of a human medulloblastoma cell line (BO-101) demonstrating skeletal muscle differentiation.

A permanent cell line, BO-101, was derived from a classic vermian medulloblastoma in a 9-year-old child. This line grew in vitro in adherent cultures and grew in athymic mice as serially transplantable intracranial and subcutaneous xenografts. Intracranial neoplasms grew as masses of small cells, which focally showed large cells with intense immunoreactivity for desmin, myoglobin and α-striated actin. The rhabdomyoblastic nature of these cells was confirmed ultrastructurally. The primary neoplasm showed immunoreactivity for synaptophysin, neuron-specific enolase and vimentin. A large panel of monoclonal antibodies and antisera against neuronal and glial antigens failed to show glial and neuronal immunoreactivity in the cell culture and xenografts. Despite the marked genotypic and phenotypic differences, the original neoplasm and the cell line share a common chromosomal marker del (12) (p 13.1). The BO-101 line differs phenotypically and genotypically from previously established medulloblastoma cell lines and further supports the heterogeneous biologic proprieties of the cell populations that constitute these neoplasms.

p53 Gene Expression in Medulloblastoma by Quantitative Polymerase Chain Reaction

The frequent cytogenetic abnormality—isochromosome 17q [i(17)q]—observed in medulloblastomas (MB) may result in altered expression of the oncosuppressor gene p53 that is located on 17p. p53 expression was therefore evaluated in five MBs and in one MB cell line derived from one of these tumors. Expression levels of p53 utilizing serially diluted unfractionated RNA from tumors and the cell line were assessed both by dot-blot and by reverse transcription (RT) followed by the polymerase chain reaction (PCR). The quality of RNA, efficiency of reaction, and transcript quantitation were determined by simultaneous transcription and amplification of a similarly sized fragment of the α-tubulin gene. All MBs showed low levels of expression of p53 compared to those found in normal tissues. p53 messenger RNA (mRNA) was significantly increased (two- to threefold) in the MB cell line compared to its tumor of origin and to the other MBs. Immunoperoxidase studies performed with monoclonal antibodies to the p53 protein product showed focal nuclear expression in one of five of the original tumors while most cells grown in vitro and in the xenograft were positive. The results indicate that (a) p53 mRNA was present in all MBs tested, thereby excluding the possibility of homozygous deletions of 17p, even though one tumor had i(17)q and another had a rearranged 17p; (b) p53 mRNA was not present in MBs in quantities significant enough to suspect inactivation by a combination of loss of heterozygosity and allelic point mutation; (c) p53 mRNA was overexpressed only in the MB cell line and xenograft suggesting an acquired p53 mutation conferring a growth advantage to selected clones of MB cells; and (d) RT-PCR is a rapid and simple nonisotopic technique that utilizes minimal quantities of total RNA for the assessment of gene expression.

Proven Epstein–Barr encephalitis with negative EBV‐DNA load in cerebrospinal fluid after allogeneic hematopoietic stem cell transplantation in a child with acute lymphoblastic leukemia

We report a case of EBV encephalitis in a seven‐yr‐old child with Ph+ ALL. Two months after an allogeneic HSCT from his HLA mismatched mother, the patient showed an altered sensorium, generalized seizures, and a left hemiparesis. Brain MRI demonstrated multiple lesions highly suggestive for viral encephalitis. Blood and CSF PCR analyses were negative for the most common viruses involved in immunocompromised patients including EBV. A cerebral biopsy was performed, which showed intense gliosis and perivascular lymphocytic cuffing. PCR analysis performed on brain tissue was positive only for the EBV genome, while extensive investigations for other viral infections were negative. The patients neurological symptoms rapidly worsened and he died two months later. This case report suggests that in patients presenting neurological and radiological signs of encephalitis after an HSCT, an EBV involvement should be considered, even in the absence of CSF and blood PCR virus detection.

Integrated DNA methylation analysis identifies topographical and tumoral biomarkers in pilocytic astrocytomas

Pilocytic astrocytoma (PA) is the most common glioma in pediatric patients and occurs in different locations. Chromosomal alterations are mostly located at chromosome 7q34 comprising the BRAF oncogene with consequent activation of the mitogen-activated protein kinase pathway. Although genetic and epigenetic alterations characterizing PA from different localizations have been reported, the role of epigenetic alterations in PA development is still not clear. The aim of this study was to investigate whether distinctive methylation patterns may define biologically relevant groups of PAs. Integrated DNA methylation analysis was performed on 20 PAs and 4 normal brain samples by Illumina Infinium HumanMethylation27 BeadChips. We identified distinct methylation profiles characterizing PAs from different locations (infratentorial vs supratentorial) and tumors with onset before and after 3 years of age. These results suggest that PA may be related to the specific brain site where the tumor arises from region-specific cells of origin. We identified and validated in silico the methylation alterations of some CpG islands. Furthermore, we evaluated the expression levels of selected differentially methylated genes and identified two biomarkers, one, IRX2, related to the tumor localization and the other, TOX2, as tumoral biomarker.