Embryonal tumor with abundant neuropil and true rosettes with only one structure suggestive of an ependymoblastic rosette

Embryonal tumor with abundant neuropil and true rosettes (ETANTR) is a very aggressive embryonal central nervous system (CNS) tumor, histologically featuring ependymoblastic rosettes and neuronal differentiation in a neuropil‐like background. 19q13.42 amplification was identified in ETANTR and epndymoblastoma, suggesting that these tumors constitute a single entity, called embryonal tumor with multilayered rosettes (ETMR). Here, we report a case involving a 2‐year‐old boy with a pontine embryonal tumor composed of clusters of poorly differentiated neuroepithelial cells, and smaller neuroblastic/neurocytic cells in a fibrillary and paucicellular neuropil‐like matrix, where clear ependymoblastic rosettes were not detected but only one structure suggestive of an ependymoblastic multilayered rosette was found. Fluorescence in situ hybridazation analysis revealed 19q13.42 amplification, supporting the diagnosis of ETANTR. This report indicates that rare ependymoblasic rosettes found in embryonal tumors, which are otherwise CNS primitive neuroectodermal tumors or medulloblastomas, are significant for considering the examination of 19q13.42 amplification to confirm the diagnosis of ETMR.     

Comprehensive high-resolution genomic profiling and cytogenetics of two pediatric and one adult medulloblastoma

Medulloblastoma (WHO grade IV) is a rare, malignant, invasive, embryonal tumor which mainly occurs in children and represents less than 1% of all adult brain tumors. Systematic comprehensive genetic analyses on medulloblastomas are rare but necessary to provide more detailed information.     

Claudin 6 Is a Positive Marker for Atypical Teratoid/Rhabdoid Tumors

Atypical teratoid/rhabdoid tumors (AT/RTs) are highly aggressive pediatric brain tumors characterized by the presence of rhabdoid cells and negative immunostaining for INI1 (BAF47). Histogenesis is unknown and diagnosis can be challenging because of their extreme morphological and immunophenotypic heterogeneity. Currently no signature markers other than INI1 loss have been identified. To search for possible candidate proteins of interest in AT/RTs, Affymetrix GeneChip® microarrays were utilized to investigate nine AT/RTs vs. 124 other tumor samples. The most distinctive gene identified was claudin 6 ( CLDN6 ), a key component of tight junctions. CLDN6 showed moderate or higher mRNA expression in eight of nine AT/RTs, with little to no expression in 114 of 115 other tumors. Average expression was 38-fold higher in AT/RTs vs. other samples. Immunohistochemical (IHC) staining of 33 tumor specimens found positive membrane staining in seven of seven AT/RTs, and was negative in 26 of 27 other brain tumor samples. Notably, none of the 16 medulloblastomas/primitive neuroectodermal tumors showed IHC staining for CLDN6. IHC staining results closely matched the level of mRNA expression detected by microarray. CLDN6 may be a useful positive marker to help further identify AT/RTs for diagnostic and treatment purposes.     

Analysis of Chromosome 19q13.42 Amplification in Embryonal Brain Tumors with Ependymoblastic Multilayered Rosettes

Recently, it was reported that ependymoblastoma and embryonal tumor with abundant neuropil and true rosettes (ETANTR) show 19q13.42 amplification at a high frequency, suggesting that these tumors may constitute a single entity. As ependymoblastic rosettes are the most prominent features in both subtypes, embryonal tumor with multilayered rosettes (ETMR) was proposed, for which 19q13.42 amplification represents a specific molecular hallmark. However, ependymoblastic rosettes are not specific to ependymoblastoma and ETANTR, and are also found in a few other embryonal tumors as well as immature teratomas, and knowledge on 19q13.42 amplification in these tumors is limited. In this study, we performed fluorescence in situ hybridazation (FISH) analysis and differential polymerase chain reaction (PCR), and detected 19q13.42 amplification in three out of four ETANTR, one ependymoblastoma and one medulloepithelioma with ETANTR components, whereas none of the two atypical teratoid/rhabdoid tumors (AT/RT) with ependymoblastic rosettes nor two immature teratomas with developing neuroectodermal structures showed such amplification, suggesting that medulloepitheliomas would possibly be included in ETMR, and ependymoblastic rosettes in AT/RT do not signify that these tumors constitute ETMR. Also, we found C19MC rather than miR-371-373 was amplified in one ETANTR, suggesting that C19MC miRNA cluster seems to be more closely linked to the pathogenesis of ETMR.     

Amplifications of the epidermal growth factor receptor gene (egfr) are common in phyllodes tumors of the breast and are associated with tumor progression

Phyllodes tumors of the breast are rare biphasic tumors with the potential for invasion and metastatic spread. An important role of the epidermal growth factor receptor (EGFR) in phyllodes tumors has been proposed. However, detailed pathogenetic mechanisms remained unclear. We investigated 58 phyllodes tumors of the breast (40 benign, 10 borderline and eight malignant) by means of egfr fluorescence in situ hybridization (FISH) and gene dosage PCR for a regulatory sequence within intron 1 of egfr. Immunohistochemical staining was performed for EGFR, p16, p21, p27, p53, c-myc, Cyclin A, Cyclin D1, Cyclin E, c-kit and Ki67. Immunopositivity for EGFR was detected in 19% of phyllodes tumors (75% of all malignant tumors) in stromal tumor cells but not in the epithelial component. Whole-gene amplifications were seen by FISH in 15.8% (in stromal cells only) and intron 1 amplifications by gene dosage PCR in as much as 41.8% of all phyllodes tumors. Significant correlations were seen between tumor grade on the one hand and EGFR overexpression (P=0.001) and intron 1 amplifications (P<0.05) on the other. EGFR overexpression further correlated positively with immunohistochemical staining for p53, p16, Cyclin A, Cyclin E, Ki67 and c-kit. Presence of intron 1 amplifications correlated with p16 (P<0.01), p21 (P=0.009) and p53 immunoreactivity (P<0.001). Neither EGFR overexpression nor whole-gene amplification was observed in a control series of 167 fibroadenomas and only one of 43 (2.3%) exhibited intron 1 amplification in gene dosage PCR. In conclusion, our results show for the first time that activating mutations in and overexpression of egfr are associated with the progression in grade of phyllodes tumors of the breast. The observed association between intron 1 amplification and overexpression of EGFR provides further insight into regulation mechanisms of EGFR overexpression.     

Retention of imprinting of the human apoptosis-related gene TSSC3 in human brain tumors

Genomic imprinting is the result of a gamete-specific modification leading to parental origin-specific gene expression in somatic cells of the offspring. Several embryonal tumors show loss of imprinting of genes clustered in human chromosome 11p15.5, an important tumor suppressor gene region, harboring several normally imprinted genes. TSSC3, a gene homologous to mouse TDAG51, implicated in Fas-mediated apoptosis, is also located in this region between hNAP2 and p57 (KIP2). TSSC3 is the first apoptosis-related gene found to be imprinted in placenta, liver and fetal tissues where it is expressed from the maternal allele in normal human development. This study investigated the imprinting status of TSSC3 in human normal, adult brain and in human neuroblastomas, medulloblastomas and glioblastomas. A polymorphism in exon 1 at position 54 was used to analyze the allelic expression of the TSSC3 gene by a primer oligo base extension (PROBE) assay using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We found that the TSSC3 gene is not imprinted in human normal, adult brain and blood. In contrast, strong allelic bias resembling imprinting could be detected in most examined tumor specimens. The results demonstrate for the first time that the tumors under investigation are associated with a retention of imprinting of a potential growth inhibitory gene.     

APC mutations in sporadic medulloblastomas

The cerebellar medulloblastoma (WHO Grade IV) is a highly malignant, invasive embryonal tumor with preferential manifestation in children. Several molecular alterations appear to be involved, including isochromosome 17q and the p53, PTCH, and beta-catenin gene mutations. In this study, 46 sporadic medulloblastomas were screened for the presence of mutations in genes of the Wnt signaling pathway (APC and beta-catenin). Single-strand conformational polymorphism (SSCP) analysis followed by direct DNA sequencing revealed 3 miscoding APC mutations in 2 (4.3%) medulloblastomas. One case contained a GCA-->GTA mutation at codon 1296 (Ala-->Val), and another case had double point mutations at codons 1472 (GTA-->ATA, Val-->Ile) and 1495 (AGT-->GGT, Ser-->Gly). Miscoding beta-catenin mutations were detected in 4 tumors (8.7%). Three of these were located at codon 33 (TCT -->TTT, Ser-->Phe) and another at codon 37 (TCT-->GCT, Ser-->Ala). Adenomatous polyposis coli (APC) gene and beta-catenin mutations were mutually exclusive and occurred in a total of 6 of 46 cases (13%). Although germline APC mutations are a well established cause of familial colon and brain tumors (Turcot syndrome), this study provides the first evidence that APC mutations are also operative in a subset of sporadic medulloblastomas.     

MYCC and MYCN oncogene amplification in medulloblastoma. A fluorescence in situ hybridization study on paraffin sections from the Children's Oncology Group

CONTEXT: Brain tumors are the most common solid tumor in childhood, and medulloblastoma is the most common malignant brain tumor in this age group. Cytogenetic abnormalities that have been described in childhood medulloblastoma include loss of 17p, amplification of MYCC (c-myc), amplification of MYCN (N-myc), and isochromosome 17q. Data on these tumors indicate that the frequency of MYCC amplification is 5% to 10%. Fluorescence in situ hybridization is a powerful tool for investigating these features on archival material. OBJECTIVES: To determine if intratumoral heterogeneity exists for MYCC and MYCN in medulloblastomas and if tumors with amplified MYCC or MYCN exhibit consistent histologic patterns. DESIGN: In this fluorescence in situ hybridization study, we investigated the frequency and prognostic significance of MYCC and MYCN amplification in 77 medulloblastomas derived from the Children's Oncology Group. RESULTS: MYCC amplification occurred in only 4 (5.2%) of 77 tumors. The 4 patients died of clinically aggressive neoplasms within 7 months of diagnosis. Similarly, 4 of 77 patients' tumors were found to exhibit MYCN amplification, but survival data are incomplete at present, therefore prognostic significance cannot be characterized. CONCLUSIONS: These data establish the frequency of MYCC amplification in a large cohort of children with medulloblastoma and further suggest that MYCC amplification may be a marker of poor prognosis. Intratumoral heterogeneity was identified for these oncogenes in that 1 patient's tumor exhibited evidence of both MYCN and MYCC amplification, and this patient experienced a shortened survival time.     

hTERT-逆转录病毒载体构建及其介导的脐血间质干细胞基因转移研究

目的：构建hTERT-逆转录病毒载体，向脐血间质干细胞（MSCs）中导入hTERT基因，观察该基因对细胞端粒酶活性的影响，能否延长细胞的寿命以及转基因细胞的分化能力。方法: PCR法扩增出hTERT全部片段，定向克隆入pLNCX2载体。将病毒质粒导入包装细胞内，筛选阳性产毒细胞，测定病毒滴度。取病毒上清感染脐血MSCs，筛选转基因细胞。检测转染前后hTERT在mRNA水平的表达，端粒酶活性的变化，hTERT转入后对细胞增殖的影响。用5-氮杂胞苷将转基因细胞向心肌方向诱导，检测心肌特异抗体的表达。结果:用BglⅡ和NotⅠ双酶切构建质粒，证明PLNCX2-hTERT构建成功，转基因细胞的hTERT基因在mRNA水平得到表达，细胞端粒酶阳性。生长曲线显示转基因细胞增殖速度快于未转染细胞。向心肌细胞诱导后，心肌特异性抗体 α-sarcomeric actin和connexin-43均有表达。结论:在逆转录病毒介导下，外源性hTERT基因转入脐血MSCs后得到表达，激活细胞端粒酶的活性，有效延长了细胞的寿命，不影响其分化功能。     

Genetic alterations in microRNAs in medulloblastomas

MicroRNAs (miRNAs) regulate a variety of cellular processes via the regulation of multiple target genes. We screened 48 medulloblastomas for mutation, deletion and amplification of nine miRNA genes that were selected on the basis of the presence of potential target sequences within the 3′‐untranslated region of the MYCC mRNA. Differential PCR revealed deletions in miR‐186 (15%), miR‐135a‐1 (33%), miR‐548d‐1 (42%), miR‐548d‐2 (21%) and miR‐512‐2 (33%) genes, whereas deletion or amplification was detected in miR‐135b (23%) and miR‐135a‐2 (15%). In miR‐33b, deletion, amplification or a mutation at the precursor miRNA were detected in 10% of medulloblastomas. Overall, 35/48 (73%) medulloblastomas had at least one alteration. Real‐time RT‐PCR revealed MYCC overexpression in 11 of 37 (30%) medulloblastomas, and there was a correlation between MYCC overexpression and miR‐512‐2 gene deletion (P = 0.0084). Antisense‐based knockdown of miR‐512‐5p (mature sequence of miR‐512‐2) resulted in significant upregulation of MYCC expression in HeLa and A549 cells, while forced overexpression of miR‐512‐2 in medulloblastoma/PNET cell lines DAOY, UW‐228‐2, PFSK resulted in the downregulation of MYCC protein. Furthermore, the results of luciferase reporter assays suggested that miR‐512‐2 targets the MYCC gene. These results suggest that alterations in the miRNA genes may be an alternative mechanism leading to MYCC overexpression in medulloblastomas.     

Metaphase and array comparative genomic hybridization: unique copy number changes and gene amplification of medulloblastomas in South America

Tumors of the central nervous system are the second most frequent malignancy of childhood, accounting for the majority of cancer-related deaths in this age group. Among these tumors, medulloblastomas (MB) remain in need of further genomic characterization toward understanding of pathogenesis and outcome predictors. Eight pediatric embryonal brain tumors were analyzed: five MB (one being desmoplastic), one PNET, one medulloepithelioma, and one ependymoblastoma. Analyses identified genomic imbalances, including the gain of 16p and the nonsyntenic coamplification of MYCN and TERT loci. More detailed FISH analysis showed that coamplification of MYCN and TERT in one of the MBs manifested as dispersed nuclear speckling, consistent with the presence of double minute chromosomes. There was considerable cell-to-cell copy number heterogeneity present, but it was clear that both genes were amplified concordantly. The amplification of oncogenes seems to play an important role in the pathogenesis of MB, and the association between MYCN and TERT amplifications and poor prognosis has not been well recognized. The uncharacteristic pattern of genomic imbalances detected in MB tumors may be a reflection of the characteristics of these tumors occurring in South America.