4609

MYC

MRTL|bHLHe39|c-Myc;v-myc myelocytomatosis viral oncogene homolog (avian);MYC;v-myc myelocytomatosis viral oncogene homolog (avian)

avian myelocytomatosis viral oncogene homolog|class E basic helix-loop-helix protein 39|myc proto-oncogene protein|myc-related translation/localization regulatory factor|proto-oncogene c-Myc|transcription factor p64|v-myc avian myelocytomatosis viral onco

8q24.21

protein-coding

To identify prognostic factors in medulloblastoma, a common malignant brain tumor of childhood, expression of the oncogene c-myc was examined at the mRNA level byin situ hybridization. c-myc mRNA expression was observed in 30 of 72 tumors (42%). The c-myc gene copy number was determined by quantitative PCR from genomic DNA of paraffin-embedded tumors. c-myc gene amplification was present in 5 of 62cases (8.3%). Therefore, c-myc amplification was obviously not the cause of c-myc mRNA expression in most samples. Kaplan-Meier estimation revealed a significant correlation between c-myc mRNA expression and survival (total mean follow-up 4.6+/- 3.6 years, log-rank p = 0.02). Multivariate logistic regression analysis including sex, age, histological type, degree of surgical resection and expression of synaptophysin, GFAP and c-myc, was carried out on 54 patients who received both radiotherapy and chemotherapy. The analysis identified expression of c-myc as an independent predictive factor of death from disease.#CI- Copyright 2000 Wiley-Liss, Inc.

Hepatoblastoma (HBL) is the most common malignant liver tumor in young children.Recent reports have shown that the beta-catenin gene was frequently mutated or deleted in HBLS: To elucidate the role of beta-catenin abnormalities in HBLs, wesearched for mutations of beta-catenin and APC as well as expression of the target genes, cyclin D1, c-myc, and fibronectin, in 68 primary HBLS: The mutation analysis revealed that 44 (65%) tumors carried missense mutations or deletions of beta-catenin, ALL of which were somatic and targeted to the exon 3 encoding the amino acid residues involved in its degradation. However, no loss of function mutation of the APC gene was detected by the yeast functional assay. Of interest, beta-catenin mutation was significantly correlated with overexpression of the target genes, cyclin D1 and fibronectin, but not with that of c-myc in HBLs as measured by quantitative real-time reverse transcription-PCR. The immunohistochemical studies in 15 HBLs demonstrated that the nuclear/cytoplasmicaccumulation of beta-catenin was positive in 13 tumors, 9 of which had the deletion or mutation of the gene. The significant correlation between the beta-catenin gene abnormality and the positive staining of cyclin D1 was also confirmed. Furthermore, the nuclear accumulation of beta-catenin was strongly associated with the poorly differentiated tumor cell components as well as with the positive staining of cyclin D1 within the tumor. Thus, our present results suggested that the gain of function mutation of beta-catenin played a crucial role in the malignant progression of HBL in vivo.

In the present study we review ENT tumor pathology in childhood. Only the most salient aspects are emphasized and the variety of entities reviewed was restricted. Molecular biology techniques reveal infection by human papilloma virus (types 6 and 11) in 50 % of papillomas, while immunohistochemical techniques are less effective in papilloma virus detection. The myofibroblastic nature of nasal angiofibroma has been demonstrated and its incidence is 25 timesmore frequent in patients with familial polyposis of the colon. Overexpression of p53 occurs in the initial stages of nasopharyngeal carcinoma, while overexpression of c-myc is correlated with an unfavorable prognosis. Recently, olfactory neuroblastoma has been shown not to express the protein product of theMIC-2 gene (antibody 12E7), thus the hypothesis that it could be a member of theEwing tumor family (neuroectodermal peripheral tumors) has not been confirmed, although it is a primitive neural tumor. The head and neck rhabdomyosarcoma withthe best prognosis is that located in the orbit, and cytogenetic studies have shown chromosomic translocation t(2;13) in 50 % of these childhood tumors when they are of the alveolar-type, while trisomy of chromosome 2 or 20 is more characteristic of the embryonic-type. Currently, any classifying features of ENTlymphomas must be based on the Revised European-American Classification of Lymphoid Neoplasms (REAL). Papillary and medullary carcinomas are the most common histological types of thyroid carcinoma in childhood. Alterations in ret/PTC play a significant role in the pathogenesis of both.

The authors report a case of the rhabdoid predisposition syndrome (RPS) secondary to a germline hSNF5/INI1 mutation, whose brain tumor was originally unclassifiedbut finally diagnosed as an atypical teratoid/rhabdoid tumor (AT/RT) by molecular analysis. A 7-month-old infant presented with hydrocephalus secondary to a huge pineal tumor and subsequently developed a renal rhabdoid tumor. The histology ofthe brain tumor was initially undetermined; however, an AT/RT was strongly suspected because of her clinical course. mutational screening of the hSNF5/INI1gene by heteroduplex and direct sequence analysis detected a missense mutation at codon 53 (CGA --> TGA, arginine --> stop) in both tumors, as well as in normal tissue of the kidney. Polymerase chain reaction (PCR)-based microsatellite analysis showed in both tumors allelic loss on chromosome arm 22q to which the hSNF5/INI1 gene maps. c-myc amplification was examined by differential PCR but not detected. Histologic review of the brain tumor by immunohistochemistry confirmed focal expression of epithelial membrane antigen and smooth muscle actin. These findings suggest that the brain tumor was really an AT/RT as a component of RPS secondary to a germline hSNF5/INI1 mutation. The present mutation has never been reported in the literature.

Eleven pediatric brain tumors were studied for the histone H3, Vimentin and MYC gene expression. H3, an S phase cell cycle-related gene (ccr), was found prevalently expressed in tumors with a high mitotic index (MI). Vimentin gene, which contributes to maintaining the cell structure but is also demonstrated to be an early responder gene to growth stimulation was found variously expressed. The different expression of Vimentin gene in the examined samples suggests the active proliferation of the tumor cells. Analysis of MYC gene expression was found increased only in a mesenchymal chondrosarcoma while in other samples MYC mRNA was undetectable. Medulloblastoma, chondrosarcoma, and choroid plexus carcinoma have high S phase H3 gene expression associated with a high MI. Differently an astrocytoma shows a low MI associated with high H3 gene expression. This first preliminary report of H3, Vimentin and MYC gene expression in brain tumors demonstrates that malignant cells are characterized by a different gene expression and different growth potentials.

Recent investigations indicate that Burkitt's lymphoma consists of several subtypes, defined by their clinical and molecular features. Each geographical region so far studied appears to consist of a different mixture of subtypes. Interestingly, there appear to be geographic 'gradients' with respect to the fraction of tumors associated with EBV and the type of 8;14 chromosomal translocation. The rate of EBV association is highest in Equatorial Africa, lowest in North America and intermediate in South America. The fraction of tumors with breakpoints far upstream of the c-myc gene follows a similar pattern. Thesefindings strongly suggest that the subtypes of Burkitt's lymphoma are environmentally determined, and we propose that the pattern of infection (e.g. malaria) to which the young child is exposed influences the tumor subtype distribution by altering the relative and absolute numbers of various B cell precursors at sites of B cell ontogeny (the bone marrow, and possibly mesentery). These B cell precursors are the cells which are susceptible to the specific chromosomal translocations associated with Burkitt's lymphoma. We further propose that immunoglobulin enhancers (recognized and unrecognized) both influence the likelihood of the translocation occurring, and in at least a fraction of cases, contribute to the deregulation of a c-myc. EBV, via EBNA-1, the only invariably expressed latent-gene in Burkitt's lymphoma, probably influences c-myc expression in Burkitt's lymphoma by increasing immunoglobulin enhancer function. Thus, in effect, EBV collaborates with the translocations associated with Burkitt's lymphoma in causing c-myc deregulation. This collaboration is independent of thebreakpoint location. While other molecular abnormalities must be able to contribute to myc deregulation in the same way, EBV association in Burkitt's lymphoma is probably determined by the age at which EBV infection occurs (being more likely when infection occurs in very young children) and perhaps also by other infectious diseases that numerically influence the fraction, and predominant stage of differentiation (and hence translocation breakpoint sites) of immature B cells infected by EBV. The presence of EBV in many such cells greatly increases the incidence rate of Burkitt's lymphoma, since one of the genetic lesions needed to deregulate c-myc is already present.

CONTEXT: B-lineage acute lymphoblastic leukemia (ALL) with a mature phenotype and L1 morphology is a rare condition that may pose a diagnostic and management challenge. OBJECTIVE: To report our experience with 2 such unusual cases of pediatric B-lineage ALL. DESIGN: Morphologic, immunophenotypic, and cytogenetic features of the leukemic blast cells were reviewed in conjunction with clinical and other laboratory findings. RESULTS: The leukemic blast cells in both cases were small to medium with scant basophilic cytoplasm and several small inconspicuous nucleoli, characteristic of L1 lymphoblasts. Immunophenotypically,they were positive for CD19, CD22, and low-density CD20, with expression of surface immunoglobulin lambda light chain. They were negative for immature (CD34and terminal deoxynucleotidyl transferase), myeloid, and T-cell-associated markers. Conventional cytogenetic and fluorescent in situ hybridization studies failed to demonstrate chromosomal translocations involving the c-myc gene. Both patients were treated with children's cancer Group ALL protocols and had good responses. CONCLUSIONS: B-lineage ALL with a mature phenotype, L1 morphology, and absent chromosomal translocations involving the c-myc gene is best classified and managed as precursor B-lymphoblastic leukemia/lymphoma instead of Burkitt leukemia/lymphoma.

Medulloblastoma, a primitive neuroectodermal tumor of the cerebellum, is one of the most common central nervous system malignancies of childhood. Despite aggressive multimodal therapy, including surgery, irradiation, and chemotherapy,5-year survival rates have only approached 50-60%. To identify potential candidate genes that predict for overall survival (OS), we performed a gene expression profiling analysis in 35 newly diagnosed medulloblastoma neoplasms. Subsequently, the nine most promising candidate genes were analyzed by immunohistochemistry and fluorescence in situ hybridization on tumor tissue microarrays representing a series of 180 tumors. We found 54 genes in which expression levels predicted for unfavorable survival in medulloblastoma. In linewith the gene expression profiling analysis, a positive staining for STK15 (P = 0.0006), stathmin 1 (P = 0.001), and cyclin D1 (P = 0.03) was associated with anunfavorable OS, whereas cyclin B1, DAXX, Ki-67, MYC, NRAS, and p53 showed no statistical significant effect. In comparison to clinically defined parameters such as gender, age, metastatic stage, extent of tumor resection, application ofchemotherapy, and tumor grade, positive staining for STK15 was identified as an independent prognostic factor for OS (P = 0.026). Moreover, additional gene copynumbers of MYC (P = 0.003) and STK15 (P = 0.05) predicted for poor survival. Thecombination of gene expression profiling with tissue microarray experiments allowed the identification of a series of candidate genes that predicts for survival in medulloblastoma. Of the results highlighted by the various data analysis procedures, genes associated with cell proliferation (cyclin D1), transcription (MYC), and especially mitosis (stathmin 1, STK15) appear particularly intriguing with respect to medulloblastoma pathomechanism.

In the wake of recent progress in understanding the genetic pathways involved inthe development of brain tumors, a major goal is to correlate molecular data with clinical outcome, survival, and response to treatment modalities. This is of particular importance among the pediatric population. Reliable prognostic factors could potentially permit a tailoring of therapy in that only patients with the most aggressive tumors would receive the most intense treatments. A survey of publications about prognosis-related molecular features among pediatric brain tumors revealed 74 series, of which 46 presented statistically significant outcome-associated parameters as defined by a p value <0.05. Most investigationsrevealing significant prognosis-related features were performed on medulloblastomas (34 publications), followed by astrocytic tumors (6 publications) and ependymomas (5 publications). Promising approaches and molecular markers include gene expression profiles, DNA ploidy, loss of heterozygosity and chromosomal aberrations as detected by CGH and FISH (1q, 17p,17q), as well as oncogenes/ tumor suppressor genes and their proteins (TP53, PTEN, c-erbB2, N-myc, c-myc), growth factor and hormonal receptors (PDGFRA, VEGF, EGFR, HER2, HER4, ErbB-2, hTERT, TrkC), cell cycle genes (p27) and cell adhesionmolecules, as well as factors potentially related to therapeutic resistance (multi-drug resistance, DNA topoisomerase IIalpha, metallothionein, P-glycoprotein, tenascin). This review discusses the predictive potential of molecular markers for clinical outcome and their influence on therapeutic decision-making among children with brain tumors.

mutations of beta-catenin have been identified in the majority of pediatric hepatic malignancies, including hepatoblastoma (HB) and hepatocellular carcinoma(HCC), suggesting its important contribution in hepatic tumorigenesis in this age group. However, the role of beta-catenin/canonical Wnt signaling pathway in the neoplastic growth of cancer cells has not been directly studied. To address beta-catenin's capability in maintaining the malignant phenotype in established pediatric HB and HCC cell lines, HuH-6 and HepG2, harboring mutated and overexpressed beta-catenin, we carried out a series of in vitro analyses througha transfection of short interfering RNAs (siRNAs) to generate a loss-of-functionmodel. HuH-7, another HB cell line derived from a pediatric patient without a stabilizing mutation was used for comparison. RNA interference successfully manipulated the degradation of overexpressed beta-catenin. In ALL cell lines, beta-catenin mRNA was suppressed by 80-90% after 48 h of transfection, and a reduction of its protein expression was demonstrated. In HuH-6 and HepG2, the pre-existing beta-catenin nuclear accumulation disappeared and reductions of beta-catenin downstream target genes, c-myc and cyclinD1, were also evidenced after the treatment. The in vitro proliferation of both cell lines was transiently inhibited. In contrast, the suppression of beta-catenin in HuH-7 didnot lead to a significant change in the expression of target genes or cellular proliferation. Our data indicate that beta-catenin can be considered a specific target for gene therapy in pediatric hepatic tumors with mutations and overexpression of this gene.

The vast majority of deaths associated with cancer are a consequence of a complex phenotypic behavior, metastasis, by which tumor cells spread from their primary site of origin to regional and distant sites. This process requires the tumor cell to make numerous adjustments, both subtle and dramatic, to successfully reach, survive, and flourish at favorable secondary sites. It has been suggestedthat molecular mechanisms accounting for metastatic behavior can recapitulate those employed during embryogenesis. We have shown that the homeodomain transcription factor Six1, known to be required for normal development of migratory myogenic progenitor cells, is sufficient to promote metastatic spread in a mouse model of the pediatric skeletal muscle cancer rhabdomyosarcoma. Here,we report that Six1 is able to activate the expression of a set of protumorigenic genes (encoding cyclin D1, c-Myc, and Ezrin) that can control cell proliferation, survival, and motility. Although the role of Ezrin in cytoskeletal organization and adhesion has been well studied, the means by which its expression is regulated are poorly understood. We now show that the gene encoding Ezrin is a direct transcriptional target of Six1. Moreover, Ezrin is indispensable for Six1-induced metastasis and highly expressed in a panel of representative pediatric cancers. Our data indicate that Ezrin represents a promising therapeutic target for patients with advanced-stage rhabdomyosarcoma and perhapsother malignancies.

Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, likely results from deregulation of the skeletal myogenesis program. Although associations between PAX3, PAX7, FOXO1A, and RMS tumorigenesis are well recognized, the entire spectrum of genetic factors underlying RMS development and progression is unclear. Using a combined approach of spectral karyotyping, array-based comparative genomic hybridization (CGH), and expression analysis, we examined 10primary RMS tumors, including embryonal, alveolar, and the rare adult pleomorphic variant, to explore the involvement of different genes and genetic pathways in RMS tumorigenesis. A complete karyotype established for each tumor revealed a high aneuploidy level, mostly tetraploidy, with double minutes and additional structural aberrations. Quantitative expression analysis detected the overexpression of the AURKA gene in ALL tumors tested, suggesting a role for this mitotic regulator in the aneuploidy and chromosomal instability observed in RMS.Array-based CGH analysis in primary RMS tumors detected copy number changes of genes involved in multiple genetic pathways, including transcription factors such as MYC-related gene from lung cancer and the cytoskeleton and cell adhesion-encoding genes laminin gamma-2 and p21-activated kinase-1. Our data suggest the involvement of genes encoding cell adhesion, cytoskeletal signaling,and transcriptional and cell cycle components in RMS tumorigenesis.

BACKGROUND: Neuroblastoma is a frequently lethal pediatric cancer in which MYCN genomic amplification is highly correlated with aggressive disease. Deregulated MYC genes require co-operative lesions to foster tumourigenesis and both direct and indirect evidence support activated Ras signaling for this purpose in many cancers. Yet Ras genes and Braf, while often activated in cancer cells, are infrequent targets for activation in neuroblastoma. Recently, the Ras effector PIK3CA was shown to be activated in diverse human cancers. We therefore assessedPIK3CA for mutation in human neuroblastomas, as well as in neuroblastomas arising in transgenic mice with MYCN overexpressed in neural-crest tissues. In this murine model we additionally surveyed for Ras family and Braf mutations as thesehave not been previously reported. METHODS: Sixty-nine human neuroblastomas (42 primary tumors and 27 cell lines) were sequenced for PIK3CA activating mutationswithin the C2, helical and kinase domain "hot spots" where 80% of mutations cluster. Constitutional DNA was sequenced in cases with confirmed alterations toassess for germline or somatic acquisition. Additionally, Ras family members (Hras1, Kras2 and Nras) and the downstream effectors Pik3ca and Braf, were sequenced from twenty-five neuroblastomas arising in neuroblastoma-prone transgenic mice. RESULTS: We identified mutations in the PIK3CA gene in 2 of 69 human neuroblastomas (2.9%). Neither mutation (R524M and E982D) has been studiedto date for effects on lipid kinase activity. Though both occurred in tumors with MYCN amplification the overall rate of PIK3CA mutations in MYCN amplified and single-copy tumors did not differ appreciably (2 of 31 versus 0 of 38, respectively). Further, no activating mutations were identified in a survey of Ras signal transduction genes (including Hras1, Kras2, Nras, Pik3ca, or Braf genes) in twenty-five neuroblastic tumors arising in the MYCN-initiated transgenic mouse model. CONCLUSION: These data suggest that activating mutationsin the Ras/Raf-MAPK/PI3K signaling cascades occur infrequently in neuroblastoma.Further, despite compelling evidence for MYC and RAS cooperation in vitro and invivo to promote tumourigenesis, activation of RAS signal transduction does not constitute a preferred secondary pathway in neuroblastomas with MYCN deregulation in either human tumors or murine models.

Abnormalities of chromosome 10 are frequently observed in the development of medulloblastoma, the most common malignant brain tumor of childhood. To identifycritical genetic loci involved, we performed detailed physical mapping of regions of allelic loss on this chromosome. 18% of cases (5/32 primary tumors, 2/8 cell lines) harbored allelic losses on 10q. Refined mapping identified a 21.7Mb common interval, affecting the region 10q23.3-10q25.3. This region contains three genes, MXI1, SUFU and BTRC, which represent putative medulloblastoma tumor suppressor (TS) genes on the basis of either (i) negative regulation of critical medulloblastoma pathways, or (ii) mutation in other cancer types. We therefore sought evidence of their genetic inactivation in 46 cases, by mutational analysis of their entire coding regions. A MXI1 mutation was identified which abolishes its translation initiation site (A1G; MET1VAL), however no further tumor-specific sequence variations were detected. We next identified and characterised CpG islands associated with 5' regions of the MXI1, SUFU and BTRC genes; analysis ofthese regions for evidence of DNA hypermethylation, alongside expression analysis of their respective transcripts, revealed no evidence to support epigenetic inactivation of any gene. These findings implicate the inactivation of critical TS loci at 10q23.3-25.3 in medulloblastoma, however comprehensive analysis of SUFU, BTRC and MXI1 indicates they are unlikely to represent major targets of these allelic losses. MXI1 mutation appears to play a role in the pathogenesis of a small subset of cases, and suggests an alternative mechanism to MYC amplification for disruption of the MYC/MAD/MAX network in medulloblastoma.

Multiple genetic changes take place during tumor development and progression. These genetic changes result in inactivation of tumor suppressor genes and activation of proto-oncogenes. Frequent genetic changes observed in gliomas are losses of chromosomal regions on 9p, 10q, 13q, 17p and on 22. Loss of 10q is seen in more than 80% of the glioblastoma multiforme (GBM) tumors suggesting the presence of a gene critical for GBM formation on this chromosome. Amplification of epidermal growth factor receptor gene and expression of platelet derived growth factor and fibroblast growth factor genes are also common among gliomas. The most common genetic abnormality found in medulloblastomas is loss of 17p. The C-myc gene is amplified in a few primary tumors, but the incidence of amplification is higher in medulloblastoma derived cell lines. These findings suggest that the same two genetic processes, gene amplification and regional chromosomal loss, which characterize other primitive childhood neuroectodermal tumors such as retinoblastoma and neuroblastoma are also important in medulloblastomas.

Medullomyoblastoma (MMB) is a rare cerebellar childhood tumor containing both myoblastic and primitive neuroectodermal components. Similar to the scenario in classical medulloblastoma, which contains the primitive neuroectodermal component only, gain of sequences from the long arm of chromosome 17 (17q) and gain of theMYC gene in 8q have been implicated in the pathogenesis of MMB. Because karyotypic analysis has not previously been performed for MMB, the mechanisms behind genomic imbalances in this tumor have remained unknown. Several other central aspects of this tumor, such as histocytogenetic origin, clinical characteristics, tumor behavior and prognosis, also remain unknown. We here report neuropathological and cytogenetic features of an MMB in a 3-year-old boy.Chromosome banding analysis and multicolor karyotyping revealed a hyperdiploid karyotype including an unbalanced 1; 17 translocation and isochromosome 17q formation, both leading to gain of 17q. There were also two extra copies of chromosome 8, leading to gain of the MYC oncogene, trisomies 5 and 13, and monosomy 9. Clonal chromosome changes were present in both the myoblastic and neuroectodermal components. Our findings support the notion that MMB and classical medulloblastoma arise through similar genetic mechanisms and that the two main tissue components in MMB are clonally related.

BACKGROUND: T-lymphoblastic lymphoma (T-LBL) accounts for 25-30% of childhood non-Hodgkin's lymphoma and is closely related to T-lymphoblastic leukemia (T-ALL). Recently, we demonstrated distinct differences in gene expression between childhood T-LBL and T-ALL, but molecular pathogenesis and relevant protein expression patterns in T-LBL remain poorly understood. PROCEDURE: children with T-LBL with disseminated disease were registered and treated on COGprotocol 5971. Paraffin-embedded tumor tissue was obtained at diagnosis for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) studies. We determined the pattern and intensity of staining for c-Myc, Skp2, Mib-1, p53,TCL-1, bcl-2, and bcl-6 proteins by IHC and c-Myc, p53, bcl-2, bcl-6, and TCR alpha/delta molecular alterations by FISH in 22 pediatric T-LBL cases. RESULTS: The majority of T-LBL samples expressed Mib-1 (59%) and c-Myc (77%) proteins in greater than 50% of the cells, but Skp2 (14%), p53 (14%), and bcl-2 (23%) expression was less common. FISH studies demonstrated 18% gains and 10% losses in c-Myc, 16% gains in p53, 12% gains and 6% losses in bcl-2, and 6% gains and 19% losses in bcl-6 with little direct correlation between the IHC and FISH studies.CONCLUSIONS: childhood T-LBL is a highly proliferative tumor associated with enhanced expression of c-Myc protein, but without detectable c-Myc molecular alterations. FISH studies did not identify consistent etiologies of molecular dysregulation, and future studies with other molecular approaches may be required to elucidate the molecular pathogenesis of childhood T-LBL.#CI- (c) 2008 Wiley-Liss, Inc.

Hepatoblastoma, the most common pediatric liver cancer, is tightly linked to excessive Wnt/beta-catenin signaling. Here, we used microarray analysis to identify two tumor subclasses resembling distinct phases of liver development and a discriminating 16-gene signature. beta-catenin activated different transcriptional programs in the two tumor types, with distinctive expression of hepatic stem/progenitor markers in immature tumors. This highly proliferating subclass was typified by gains of chromosomes 8q and 2p and upregulated Myc signaling. Myc-induced hepatoblastoma-like tumors in mice strikingly resembled the human immature subtype, and Myc downregulation in hepatoblastoma cells impaired tumorigenesis in vivo. Remarkably, the 16-gene signature discriminated invasive and metastatic hepatoblastomas and predicted prognosis with high accuracy.

Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcomas. Theyresemble developing skeletal muscle and are histologically divided into two mainsubtypes; alveolar and embryonal RMS. Characteristic genomic aberrations, including the PAX3- and PAX7-FOXO1 fusion genes in alveolar cases, have led to increased understanding of their molecular biology. Here, we determined the effect of genomic copy number on gene expression levels through array comparative genomic hybridization (CGH) analysis of 13 RMS cell lines, confirmed by multiplex ligation-dependent probe amplification copy number analyses, combined with theircorresponding expression profiles. Genes altered at the transcriptional level bygenomic imbalances were identified and the effect on expression was proportionalto the level of genomic imbalance. Extrapolating to a public expression profiling dataset for 132 primary RMS identified features common to the cell lines and primary samples and associations with subtypes and fusion gene status. Genes identified such as CDK4 and MYCN are known to be amplified, overexpressed, and involved in RMS tumorigenesis. Of the many genes identified, those with likely functional relevance included CENPF, DTL, MYC, EYA2, and FGFR1. Copy number and expression of FGFR1 was validated in additional primary material and found amplified in 6 out of 196 cases and overexpressed relative to skeletal muscle and myoblasts, with significantly higher expression levels in the embryonal comparedwith alveolar subtypes. This illustrates the ability to identify genes of potential significance in tumor development through combining genomic and transcriptomic profiles from representative cell lines with publicly available expression profiling data from primary tumors.

PURPOSE: The molecular pathogenesis of pediatric ependymoma remains unclear. Ourstudy was designed to identify genetic changes implicated in ependymoma progression. PATIENTS AND METHODS: We characterized 59 ependymoma samples (33 atdiagnosis and 26 at relapse) using array-comparative genomic hybridization (aCGH). Specific chromosomal imbalances were confirmed by fluorescent in situ hybridization, and candidate genes were assessed by real-time quantitative polymerase chain reaction (qPCR), immunohistochemistry, sequencing, and in vitrofunctional studies. RESULTS: aCGH analysis revealed a significant increase in genomic imbalances on relapse compared with diagnosis, such as gain of 9qter and1q (54% v 21% and 12% v 0%, respectively) and loss of 6q (27% v 6%). Supervised tumor classification showed that gain of 9qter was associated with tumor recurrence, age older than 3 years, and posterior fossa location. Using a candidate-gene strategy, we found an overexpression of two potential oncogenes at the locus 9qter: Tenascin-C and Notch1. Moreover, Notch pathway analysis (qPCR) revealed overexpression of Notch ligands, receptors, and target genes (Hes-1, Hey2, and c-Myc), and downregulation of Notch repressor Fbxw7. We confirmed by immunohistochemistry the overexpression of Tenascin-C and Hes-1. We detected Notch1 missense mutations in 8.3% of the tumors (only in the posterior fossa location and in case of 9q33-34 gain). Furthermore, inhibition of Notch pathway with a gamma-secretase inhibitor impaired the growth of ependymoma stem cell cultures. CONCLUSION: The activation of the Notch pathway and Tenascin-C seem tobe important events in ependymoma progression and may represent future targets for therapy. We report, to our knowledge for the first time, recurrent oncogenicmutations in pediatric posterior fossa ependymomas.

Neuroblastoma is derived from neural crest precursor components of the peripheral sympathetic nervous system and accounts for more than 15% of ALL pediatric cancer deaths. A clearer understanding of the molecular basis of neuroblastoma is required for novel therapeutic approaches to improve morbidity and mortality. Neuroblastoma is uniformly p53 wild type at diagnosis and must overcome p53-mediated tumor suppression during pathogenesis. Amplification of the MYCN oncogene correlates with the most clinically aggressive form of the cancer, and MDM2, a primary inhibitor of the p53 tumor suppressor, is a direct transcriptional target of, and positively regulated by, both MYCN and MYCC. We hypothesize that MDM2 contributes to MYCN-driven tumorigenesis helping to ameliorate p53-dependent apoptotic oncogenic stress during tumor initiation and progression. To study the interaction of MYCN and MDM2, we generated an Mdm2 haploinsufficient transgenic animal model of neuroblastoma. In Mdm2(+/-)MYCN transgenics, tumor latency and animal survival are remarkably extended, whereas tumor incidence and growth are reduced. Analysis of the Mdm2/p53 pathway revealsremarkable p53 stabilization counter-balanced by epigenetic silencing of the p19(Arf) gene in the Mdm2 haploinsufficient tumors. In human neuroblastoma xenograft models, conditional small interfering RNA-mediated knockdown of MDM2 in cells expressing wild-type p53 dramatically suppresses tumor growth in a p53-dependent manner. In summary, we provided evidence for a crucial role for direct inhibition of p53 by MDM2 and suppression of the p19(ARF)/p53 axis in neuroblastoma tumorigenesis, supporting the development of therapies targeting these pathways.

The loss of genetic material from specific chromosomal locations has been identified for a number of pediatric tumors. This loss has been taken as evidence for the importance of tumor suppressor genes at these loci in the genesis of these tumors. One of these pediatric tumors, the primitive neuroectodermal tumorof the central nervous system, has not been well studied. In this report, an analysis of primitive neuroectodermal tumors for allelic deletions on chromosomes 1p, 7q, 10, 11p, 13q, and 17p has been performed. One of ten tumors was found tohave increased copies of c-myc. Three different patients were found to reduce tohomozygosity at one of three different locations. Significantly, however, three of nine informative patients showed a reduction to homozygosity on chromosome 17p. Thus, primitive neuroectodermal tumor is one of a growing number of tumor types in which deletions in the short arm of chromosome 17 might be important inoncogenesis.

OBJECTIVES: Atypical teratoid/rhabdoid tumor (AT/RT) and medulloblastoma (MB) are the most malignant primary brain tumors in early childhood. AT/RT is frequently misdiagnosed as primitive neuroectodermal tumor/medulloblastoma. The biological features and clinical outcomes of AT/RT and MB are extremely different. In this study, we used microarray as a platform to distinguish these two tumors with thedefinitive diagnostic marker as well as the profiling of expression genes. METHODS: In order to clarify the pathogenesis and find the biological markers for AT/RT, we established a derivative AT/RT primary cell culture. The differential profiling between AT/RT and MB were analyzed by using microarray method. RESULTS: With the use of the microarray method, we demonstrated that 15 genes were significantly changed (at least 5-fold in upregulation and 1/5-fold in downregulation) between AT/RT and MB in tissues and cell lines. The quantitativereverse transcription-polymerase chain reaction analyses further confirmed that mRNA expression levels of SERPINI1 and osteopontin were highly expressed in AT/RT cells and tissues than those in MB. Importantly, our microarray result suggestedthat AT/RT presents the stemness-like pattern and expression profiling of embryonic stem cells as well as high mRNA expressions of Oct-4, Nanog, Sox-2, and c-Myc. CONCLUSIONS: Our study demonstrated the differential gene expression profiling between AT/RT and MB. Based on the microarray findings, AT/RTs presentembryonic stem-like gene recapitulation and further provide novel insights into their underlying biology.

OBJECTIVE: To study the clinicopathologic features, immunohistochemical findings, EBV and c-myc gene status of intra-abdominal non-Hodgkin B-cell lymphoma occurring in children. METHODS: Seventy-four cases of pediatric intra-abdominal non-Hodgkin B-cell lymphoma were retrieved from the archival file. The cases were classified according to the 2008 WHO classification. Tissue microarray includingtumor tissues from ALL the 74 cases was produced. Immunohistochemical study (SP method) for CD20, CD3, CD79a, CD10, bcl-6, MUM1, bcl-2, CD43, CD38 and Ki-67 wasperformed. In-situ hybridization for Epstein-Barr virus-encoded RNA (EBER) and fluorescence in-situ hybridization for c-myc gene were also carried out. RESULTS: Amongst the 74 cases studied, 65 of them (87.8%) were Burkitt lymphoma (BL), 4 cases (5.4%) were diffuse large B-cell lymphoma (DLBCL) and the remaining 5 cases (6.8%) showed features in-between DLBCL and BL (DLBCL/BL). The patients often presented with abdominal pain, abdominal masses, ileus and intussusception. The ileocecal bowel wall and mesenteric lymph nodes were commonly involved. The lymphoma cells were of high histologic grade and suggested an aggressive clinical behavior. The staining for CD20 and CD79a were positive in ALL of the cases, while CD3 was negative. The positive rates of CD10, bcl-6, bcl-2, MUM1, CD43, CD38 and EBER in BL were 96.9% (63 cases), 95.4% (62 cases), 0 (0 case), 23.1% (15 cases), 70.8% (46 cases), 96.9% (63 cases) and 41.5% (27 cases), respectively. Fifty-four cases carried translocation of c-myc gene. As for DLBCL, the positive cases of CD10, bcl-6, bcl-2, MUM1, CD43, CD38 and EBER were 3 cases, 2 cases, 3 cases, 2 cases, 2 cases, 2 cases and 0 case, respectively. One of these cases showed c-myc gene translocation. Amongst the 4 cases of DLBCL, 2 of them belonged to germinal center B-cell-like subtype, while the remaining 2 cases were of non-germinal center B-cell-like subtype. The expression rates of CD10, bcl-6, bcl-2, MUM1, CD43, CD38 and EBER in DLBCL/BL were 5/5, 4/5, 0, 3/5, 5/5, 3/5 and 0, respectively. Three of the cases were positive for c-myc gene translocation. CONCLUSIONS: The majority of pediatric intra-abdominal non-Hodgkin B-cell lymphoma belonged to BL. They have characteristic clinical presentation and sites of predilection and are often associated with an aggressive clinical behavior. Thorough morphologic assessment, immunohistochemistry and in-situ hybridization play an important role in subtyping this group of lymphoid malignancy.

Myc proteins (c-myc, Mycn and Mycl) target proliferative and apoptotic pathways vital for progression in cancer. Amplification of the MYCN gene has emerged as one of the clearest indicators of aggressive and chemotherapy-refractory diseasein children with neuroblastoma, the most common extracranial solid tumor of childhood. Phosphorylation and ubiquitin-mediated modulation of Myc protein influence stability and represent potential targets for therapeutic intervention. Phosphorylation of Myc proteins is controlled in-part by the receptor tyrosine kinase/phosphatidylinositol 3-kinase/Akt/mTOR signaling, with additional contributions from Aurora A kinase. Myc proteins regulate apoptosis in part through interactions with the p53/Mdm2/Arf signaling pathway. mutation in p53 iscommonly observed in patients with relapsed neuroblastoma, contributing to both biology and therapeutic resistance. This review examines Myc function and regulation in neuroblastoma, and discusses emerging therapies that target Mycn.

Medulloblastoma (MB) is the most common malignant brain tumor in children. It isknown that overexpression and/or amplification of the MYC oncogene is associatedwith poor clinical outcome, but the molecular mechanisms and the MYC downstream effectors in MB remain still elusive. Besides contributing to elucidate how progression of MB takes place, most importantly, the identification of novel MYC-target genes will suggest novel candidates for targeted therapy in MB. A group of 209 MYC-responsive genes was obtained from a complementary DNA microarray analysis of a MB-derived cell line, following MYC overexpression and silencing. Among the MYC-responsive genes, we identified the members of the bonemorphogenetic protein (BMP) signaling pathway, which have a crucial role during the development of the cerebellum. In particular, the gene BMP7 was identified as a direct target of MYC. A positive correlation between MYC and BMP7 expression was documented by analyzing two distinct sets of primary MB samples. Functional studies in vitro using a small-molecule inhibitor of the BMP/SMAD signaling pathway reproduced the effect of the small interfering RNA-mediated silencing ofBMP7. Both approaches led to a block of proliferation in a panel of MB cells andto inhibition of SMAD phosphorylation. Altogether, our findings indicate that high MYC levels drive BMP7 overexpression, promoting cell survival in MB cells. This observation suggests the potential relevance of targeting the BMP/SMAD pathway as a novel therapeutic approach for the treatment of childhood MB.

Advances in understanding the molecular basis of primitive neuroectodermal tumors of the central nervous system (CNS-PNET) biology are critical to improve patientoutcome. Recently, new data on their molecular features have been reported, suggesting that supratentorial PNET (s-PNET) in adult patients may represent a specific tumor entity among CNS-PNETs. In this study, we analyzed the clinicopathologic and molecular features of 12 cases of s-PNET in adult patients. The follow-up analysis showed that these tumors have an aggressive clinical behavior. At the histopathologic level, they resembled their pediatric counterpart, showing a variable spectrum of neuronal differentiation. These cases did not show astrocytic differentiation; therefore, they did not qualify for thedifferential diagnosis of glioblastoma variants. The tumors were also screened for mutation of TP53, IDH1, IDH2, and beta-catenin, using single strand conformation polymorphism-based and sequencing assays, and were analyzed for c-myc/N-myc gene copy numbers with a quantitative polymerase chain reaction-based method. The strand conformation polymorphism-based mutational analysis showed that 5 tumors harbored TP53 mutations. In 2 cases, a mutation at codon 132 of the IDH1 gene was also found. No mutations of the beta-catenin or IDH2 genes were identified. No cases presented c-myc or N-myc amplifications. Only 1 case presented overexpression of epidermal growth factor receptor. In conclusion, ourdata show a high incidence of TP53 mutations in this group of tumors and show, in comparison with pediatric s-PNET, the absence of amplification of the c-myc/N-myc genes, indicating that s-PNET in adult patients may represent a specific subset of tumors among CNS-PNETs.

The most common microdeletion in humans involves the 22q11 region. Congenital anomalies associated with 22q11 loss include cardiac and facial defects. Less frequent is the co-presentation of malignant rhabdoid tumors that are highly aggressive childhood malignancies typically found in renal or extra-renal soft tissues and central nervous system. A newborn patient presented with multiple congenital anomalies consistent with 22q11 deletion syndrome including cleft lipand palate, ear tags and ventricular septal defects co-presenting with an axillary rhabdoid tumor. Comparative genomic hybridization revealed a 2.8 Mb germline deletion in the 22q11.2 region containing genes required for normal fetal development and the SMARCB1 tumor suppressor gene. Analysis of tumor DNA revealed a somatic deletion of exon 7 in the second allele of SMARCB1. expression of SMARCB1 was absent, while tumor markers including MYC, GFAP, and CLAUDIN-6 were upregulated. The presence of tandem oriented BCRL modules located within interspersed low copy repeat elements throughout the 22q11 distal region may predispose this area for microdeletions through nonalleleic homologous recombination.#CI- Copyright (c) 2011 Wiley-Liss, Inc.

Both OTX2 and MYC are important oncogenes in medulloblastoma, the most common malignant brain tumor in childhood. Much is known about MYC binding to promoter regions, but OTX2 binding is hardly investigated. We used ChIP-on-chip data to analyze the binding patterns of both transcription factors in D425 medulloblastoma cells. When combining the data for ALL promoter regions in the genome, OTX2 binding showed a remarkable bi-modal distribution pattern with peaks around -250 bp upstream and +650 bp downstream of the transcription start sites (TSSs). Indeed, 40.2% of ALL OTX2-bound TSSs had more than one significant OTX2-binding peak. This OTX2-binding pattern was very different from the TSS-centered single peak binding pattern observed for MYC and other known transcription factors. However, in individual promoter regions, OTX2 and MYC have a strong tendency to bind in proximity of each other. OTX2-binding sequences aredepleted near TSSs in the genome, providing an explanation for the observed bi-modal distribution of OTX2 binding. This contrasts to the enrichment of E-boxsequences at TSSs. Both OTX2 and MYC binding independently correlated with higher gene expression. Interestingly, genes of promoter regions with multiple OTX2 binding as well as MYC binding showed the highest expression levels in D425 cells and in primary medulloblastomas. Genes within this class of promoter regions were enriched for medulloblastoma and stem cell specific genes. Our data suggest an important functional interaction between OTX2 and MYC in regulating gene expression in medulloblastoma.

pediatric medulloblastoma is considered a highly heterogeneous disease and a newstrategy of risk stratification to optimize therapeutic outcomes is required. Weaimed to investigate a new risk-stratification approach based on expression profiles of medulloblastoma cohorts. We analyzed gene expression profiles of 30 primary medulloblastomas and detected strong evidence that poor survival outcomewas significantly associated with mRNA expression profiles of 17p loss. However,it was not supported in independent cohorts from previously published data (n = 100). We speculated that this discrepancy might come from complex conditions of two important prognostic determinants: loss of tumor suppressors (chromosome 17p) and high expression of oncogenes c-myc (MYCC) or N-myc (MYCN). When patients were stratified into 5 or 7 subgroups based on simultaneous consideration of these 2 factors while defining the Wnt group as independent, obviously different survival expectancies were detected between the subgroups. For instance, predicted 5-yearsurvival probabilities ranged from 19% to 81% in the 5 subgroups. We also found that age became a significant prognostic marker after adjusting for 17p, MYCC, and MYCN status. Diminished survival in age <3 years was more substantial in subgroups with high expression of MYCC, MYCN, or 17p loss but not in other subgroups, indicating that poor survival outcome might be synergistically affected by these 3 factors. Here we suggest a more tailored subgrouping system based on expression profiles of chromosome 17p, MYCC, and MYCN, which could provide the basis for a novel risk-stratification strategy in pediatric medulloblastoma.

Anaplastic lymphoma kinase (ALK) and MYC are oncogenes often dysregulated in pediatric lymphomas. NPM-ALK/t(2;5)(p23;q35) is a genetic hallmark of ALK anaplastic large cell lymphoma (ALCL). MYC gene translocations are frequently detected in high-grade B-cell lymphomas. ALKALCL cases with concurrent MYC translocation are exceedingly rare and are more aggressive and chemoresistent compared with other ALKALCL. We report a patient who presented with ALKALCL possessing coexistent MYC rearrangement, massive tumor dissemination, and early widespread relapse. This case underscores the importance of recognition of closecorrelation between dual ALK and MYC rearrangements and the characteristic clinical features in this unusual ALCL variant.

The recessive autosomal hereditary disease, xeroderma pigmentosum (XP), is characterized by a high incidence of tumors in sun-exposed skin. The defect in early steps of excision repair of XP cells leads to hypermutability towards UV-mimicking agents. DNA from eight XP tumors were screened for activated transforming genes using 3T3 transfection. In two skin tumors isolated from a XPchild, an activated N-ras oncogene was detected. Synthetic oligonucleotide probes were used to characterize the mutation in the ras gene. Both tumors were found to be mutated in the 61st N-ras codon from gln to his. The mutation was accompaniedby an increase in the level of N-ras specific mRNA and in one transformant, by the alteration of the p21 protein. In the same tumors, c-myc amplification and over transcription, and Ha-ras gene rearrangement and amplification were also detected. Analysis of other XP tumors with eleven different oncogene probes revealed an amplification of the Ha-ras gene in 6 out of 10 cases. The normal skin fibroblasts from XP patients show normal pattern levels of N-ras, c-myc andHa-ras sequences. The hypothesis is proposed that the presence of several oncogene alterations in the same tumor could be due to the high amount of UV-induced DNA lesions found in the exposed skin cells, in the absence of efficient repair.

DNA was extracted from formalin-fixed and paraffin-embedded tissues of 85 patients with pediatric malignant solid tumors which had been resected at surgery or obtained at autopsy during a 24-year period. The tumors examined included 25 rhabdomyosarcomas, 12 Wilms' tumors, 10 hepatoblastomas and 37 neuroblastoma group tumors. Neuroblastoma group tumors were subclassified into 25 neuroblastomas and 12 ganglioneuroblastomas among which 6 composite ganglioneuroblastomas were included. Sample blocks were selected from both tumors and normal tissues in the majority of cases. We were able to reliably detect N- and c-myc gene amplification in tumor DNA by dot blot-hybridization. The N-myc gene showed approximately from 3- to 500-fold amplification in 19 of 33 cases ofstage IV neuroblastoma group tumor. ALL of these 33 patients had been intensively treated with chemotherapy and/or radiotherapy. The c-myc was amplified 8-fold in1 case of rhabdomyosarcoma, but neither N-myc nor c-myc was amplified in any cases of Wilms' tumor or hepatoblastoma. We retrospectively examined the association among N-myc gene amplification, prognosis, and histologic subtype in33 patients with stage IV neuroblastoma group tumors. The survival of the patients with N-myc gene amplification was shorter than that of the patients without amplification of N-myc (p less than 0.05). There was no significant difference in prognosis between the 2 histologic subtypes; neuroblastoma and ganglioneuroblastoma, and the cases of tumors with amplified N-myc showed shorter survivals for each subtype (p less than 0.05). In every case of neuroblastoma group tumor, the copy number of the N-myc gene was the same among primary site and multiple metastatic tumors, even when the lesions showed differences in histologic subtype like neuroblastoma and ganglioneuroblastoma.

DNA was extracted from formalin-fixed and paraffin-embedded tissues of 85 patients with pediatric malignant solid tumors which had been resected at surgery or obtained at autopsy during a 24-year period. The tumors examined included 25 rhabdomyosarcomas, 12 Wilms' tumors, 10 hepatoblastomas and 37 neuroblastoma group tumors. Neuroblastoma group tumors were subclassified into 25 neuroblastomas and 12 ganglioneuroblastomas among which 6 composite ganglioneuroblastomas were included. Sample blocks were selected from both tumors and normal tissues in the majority of cases. We were able to reliably detect N- and c-myc gene amplification in tumor DNA by dot blot-hybridization. The N-myc gene showed approximately from 3- to 500-fold amplification in 19 of 33 cases ofstage IV neuroblastoma group tumor. ALL of these 33 patients had been intensively treated with chemotherapy and/or radiotherapy. The c-myc was amplified 8-fold in1 case of rhabdomyosarcoma, but neither N-myc nor c-myc was amplified in any cases of Wilms' tumor or hepatoblastoma. We retrospectively examined the association among N-myc gene amplification, prognosis, and histologic subtype in33 patients with stage IV neuroblastoma group tumors. The survival of the patients with N-myc gene amplification was shorter than that of the patients without amplification of N-myc (p less than 0.05). There was no significant difference in prognosis between the 2 histologic subtypes; neuroblastoma and ganglioneuroblastoma, and the cases of tumors with amplified N-myc showed shorter survivals for each subtype (p less than 0.05). In every case of neuroblastoma group tumor, the copy number of the N-myc gene was the same among primary site and multiple metastatic tumors, even when the lesions showed differences in histologic subtype like neuroblastoma and ganglioneuroblastoma.

DNA was extracted from formalin-fixed and paraffin-embedded tissues of 85 patients with pediatric malignant solid tumors which had been resected at surgery or obtained at autopsy during a 24-year period. The tumors examined included 25 rhabdomyosarcomas, 12 Wilms' tumors, 10 hepatoblastomas and 37 neuroblastoma group tumors. Neuroblastoma group tumors were subclassified into 25 neuroblastomas and 12 ganglioneuroblastomas among which 6 composite ganglioneuroblastomas were included. Sample blocks were selected from both tumors and normal tissues in the majority of cases. We were able to reliably detect N- and c-myc gene amplification in tumor DNA by dot blot-hybridization. The N-myc gene showed approximately from 3- to 500-fold amplification in 19 of 33 cases ofstage IV neuroblastoma group tumor. ALL of these 33 patients had been intensively treated with chemotherapy and/or radiotherapy. The c-myc was amplified 8-fold in1 case of rhabdomyosarcoma, but neither N-myc nor c-myc was amplified in any cases of Wilms' tumor or hepatoblastoma. We retrospectively examined the association among N-myc gene amplification, prognosis, and histologic subtype in33 patients with stage IV neuroblastoma group tumors. The survival of the patients with N-myc gene amplification was shorter than that of the patients without amplification of N-myc (p less than 0.05). There was no significant difference in prognosis between the 2 histologic subtypes; neuroblastoma and ganglioneuroblastoma, and the cases of tumors with amplified N-myc showed shorter survivals for each subtype (p less than 0.05). In every case of neuroblastoma group tumor, the copy number of the N-myc gene was the same among primary site and multiple metastatic tumors, even when the lesions showed differences in histologic subtype like neuroblastoma and ganglioneuroblastoma.

DNA was extracted from formalin-fixed and paraffin-embedded tissues of 85 patients with pediatric malignant solid tumors which had been resected at surgery or obtained at autopsy during a 24-year period. The tumors examined included 25 rhabdomyosarcomas, 12 Wilms' tumors, 10 hepatoblastomas and 37 neuroblastoma group tumors. Neuroblastoma group tumors were subclassified into 25 neuroblastomas and 12 ganglioneuroblastomas among which 6 composite ganglioneuroblastomas were included. Sample blocks were selected from both tumors and normal tissues in the majority of cases. We were able to reliably detect N- and c-myc gene amplification in tumor DNA by dot blot-hybridization. The N-myc gene showed approximately from 3- to 500-fold amplification in 19 of 33 cases ofstage IV neuroblastoma group tumor. ALL of these 33 patients had been intensively treated with chemotherapy and/or radiotherapy. The c-myc was amplified 8-fold in1 case of rhabdomyosarcoma, but neither N-myc nor c-myc was amplified in any cases of Wilms' tumor or hepatoblastoma. We retrospectively examined the association among N-myc gene amplification, prognosis, and histologic subtype in33 patients with stage IV neuroblastoma group tumors. The survival of the patients with N-myc gene amplification was shorter than that of the patients without amplification of N-myc (p less than 0.05). There was no significant difference in prognosis between the 2 histologic subtypes; neuroblastoma and ganglioneuroblastoma, and the cases of tumors with amplified N-myc showed shorter survivals for each subtype (p less than 0.05). In every case of neuroblastoma group tumor, the copy number of the N-myc gene was the same among primary site and multiple metastatic tumors, even when the lesions showed differences in histologic subtype like neuroblastoma and ganglioneuroblastoma.

Hepatoblastoma is the most common pediatric liver cancer, and complete resectionis required for long-term survival. There are few reported hepatoblastoma cell lines available for study. In order to develop in vitro and animal models, the authors isolated an additional cell line (HB1) from a human hepatoblastoma and here report its characteristics in culture. Cells were analyzed for growth rate,clonigenicity, and tumorigenicity, using electron microscopy. Immunocytochemistry and Northern analysis for the expression of specific surface markers and genes of interest were also performed. HB1 cells required fetal calf serum but grew well in culture (population doubling time of 2 days) and had an undifferentiated appearance under electron microscopy. Epidermal growth factor (EGF) and hydrocortisone stimulated growth with or without serum, but not autocrine growthstimulation via the epidermal growth factor receptor was detected. Proteins produced by HB1 cells under normal culture conditions included alpha-fetoprotein, cytokeratins 8 and 18, and lactate dehydrogenase (with an isozyme subunit composition similar to that of liver). HB1 cells showed chronic, high expressionof c-myc and Ha-ras oncogenes but no N-myc and apparently normal RB gene expression. This cell line shows characteristics in culture consistent with malignant, hepatic epithelial cells and is apparently EGF dependent. It may provide a model system for the future development of alternative therapies.

Medulloblastoma (MB) represents the most frequent malignant brain tumor of childhood but only a few cell lines and animal models of this primitive neuroectodermal tumor (PNET) have thus far been established. Using specific cellculture conditions, we were able to derive four human MB cell lines (MHH-MED-1-4) as well as a cell line from a spinal PNET (MHH-PNET-5). The four MB cell lines grew in suspension as floating cell aggregates or as slightly adherent cells. They consisted of undifferentiated cells that did not express markers of late neuronal or glial lineages such as neurofilaments or glial fibrillary acidic protein. They also lacked expression of major histocompatibility complex class Ior II antigens on the cell surface. ALL four MB lines were positive for vimentinand neuron-specific enolase, whereas synaptophysin, neural cell adhesion molecule, galactocerebroside, GD2, GD3, and the A2B5 antigen were expressed inconsistently. In contrast, MHH-PNET-5 grew as adherent monolayer and expressedmajor histocompatibility complex class I antigen. By cytogenetic analysis, the lines were near diploid with clonal aberrations. The MB lines showed no losses of chromosome arm 17p by either cytogenetic or microsatellite analyses. The cell line MHH-MED-2 exhibited double minute chromosomes, amplification of the c-myc gene, and overexpression of c-myc mRNA and protein. N-myc, p53, and Rb protein expression were unaltered. ALL four continuously passaged MB cell lines and the MHH-PNET-5 line were xenotransplanted s.c. into athymic mice; three of four MB lines and the spinal PNET line gave rise to tumors. These cell lines will be useful tools for biological and preclinical studies on PNETs.

PURPOSE: The purpose of this study was to determine the incidence of c-Myc protein expression in medulloblastoma/primitive neuroectodermal tumor (MB/PNET) and to identify mechanisms in addition to c-myc gene amplification that lead to increased protein expression. METHODS: We analyzed c-myc gene copy number, mRNA level and protein expression in a panel of MB/PNET cell lines. C-Myc protein levels were assessed in tumor specimens and cell lines using immunohistochemicalstaining with a c-Myc-specific monoclonal antibody. RESULTS: Southern analysis confirmed c-myc gene amplification in the D425 MED cell line and re-arrangement of one allele in D283 MED, which was analyzed further and appeared to represent a small deletion 3' of exon 3. C-myc transcript levels were dramatically elevated in both lines. Using a c-myc probe, fluorescence in situ hybridization (FISH) showed c-myc present in 3 tandem copies at 8q24 in D283 MED and multiple copies as double minutes in D425 MED. Immunohistochemistry showed c-Myc protein expression in 9 of 10 tumors and ALL cell lines, regardless of gene amplification status or level of mRNA expression. CONCLUSIONS: c-Myc protein expression is common in MB/PNET tumor specimens and cell lines. Elevated protein levels are observed in the absence of amplification, suggesting that multiple mechanisms ofc-myc dysregulation may be involved in MB/PNET. These studies support a role forc-Myc in the development of this common childhood tumor.

Malignant rhabdoid tumor (MRT) is a rare, enigmatic childhood cancer characterized by extreme aggressiveness and resistance to chemotherapy. To understand better the origin of the tumor and the mechanisms by which it develops and resists treatment, five cell lines were established from patients presentingwith MRT (two renal and three extrarenal tumors). ALL of the cell lines display the light microscopic and ultrastructural features, as well as the variable immunohistochemical profile, characteristic of MRT. ALL are capable of forming tumors in nude mice. Three of the cell lines have detectable abnormalities of chromosome 22: one a t(22, 22) unbalanced translocation and two others a loss ofheterozygosity of polymerase chain reaction-based microsatellite markers. Northern blot analysis showed that overexpression of the c-myc message was a consistent characteristic of the five MRTs evaluated. Although mutations of the p53 gene were not detectable by sequence analysis, ALL of the cell lines showed nuclear accumulation of the p53 protein by an immunocytochemical analysis in a minority of the cells. This result suggests that dysfunction in a p53-dependent apoptotic pathway might play a role in the multiple drug resistance phenotype ofthese tumors.